Philippe M. Campeau

Key Points AD STAT1 GOF is the most common genetic cause of inherited CMC and is not restricted to a specific age or ethnic group. STAT1 GOF underlies a variety of infectious and autoimmune features, as well as carcinomas and aneurysms associated with a poor outcome.

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy. Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

The administration of ex vivo culture-expanded mesenchymal stromal cells (MSCs) has been shown to reverse symptomatic neuroinflammation observed in experimental autoimmune encephalomyelitis (EAE). The mechanism by which this therapeutic effect occurs remains unknown. In an effort to decipher MSC mode of action, we found that MSC conditioned medium inhibits EAE-derived CD4 T cell activation by suppressing STAT3 phosphorylation via MSC-derived CCL2. Further analysis demonstrates that the effect is dependent on MSC-driven matrix metalloproteinase proteolytic processing of CCL2 to an antagonistic derivative. We also show that antagonistic CCL2 suppresses phosphorylation of AKT and leads to a reciprocal increased phosphorylation of ERK associated with an up-regulation of B7.H1 in CD4 T cells derived from EAE mice. CD4 T cell infiltration of the spinal cord of MSC-treated group was robustly decreased along with reduced plasma levels of IL-17 and TNF-alpha levels and in vitro from restimulated splenocytes. The key role of MSC-derived CCL2 was confirmed by the observed loss of function of CCL2(-/-) MSCs in EAE mice. In summary, this is the first report of MSCs modulating EAE biology via the paracrine conversion of CCL2 from agonist to antagonist of CD4 Th17 cell function.

This report identifies human skeletal diseases associated with mutations in WNT1. In 10 family members with dominantly inherited, early-onset osteoporosis, we identified a heterozygous missense mutation in WNT1, c.652T→G (p.Cys218Gly). In a separate family with 2 siblings affected by recessive osteogenesis imperfecta, we identified a homozygous nonsense mutation, c.884C→A, p.Ser295*. In vitro, aberrant forms of the WNT1 protein showed impaired capacity to induce canonical WNT signaling, their target genes, and mineralization. In mice, Wnt1 was clearly expressed in bone marrow, especially in B-cell lineage and hematopoietic progenitors; lineage tracing identified the expression of the gene in a subset of osteocytes, suggesting the presence of altered cross-talk in WNT signaling between the hematopoietic and osteoblastic lineage cells in these diseases.

<h3>Importance</h3> Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality. <h3>Objective</h3> To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly. <h3>Design, Setting, and Participants</h3> Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Children’s Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase–AKT (serine/threonine kinase)–mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations. <h3>Main Outcomes and Measures</h3> Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders. <h3>Results</h3> Low-level mosaic mutations of<i>MTOR</i>were identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation of<i>MTOR</i>(p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation of<i>MTOR</i>(p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all<i>MTOR</i>mutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size. <h3>Conclusions and Relevance</h3> In this study, mutations of<i>MTOR</i>were associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations of<i>MTOR</i>and pigmentary mosaicism in skin.

During bone homeostasis, osteoblast and osteoclast differentiation is coupled and regulated by multiple signaling pathways and their downstream transcription factors. Here, we show that microRNA 34 (miR-34) is significantly induced by BMP2 during osteoblast differentiation. In vivo, osteoblast-specific gain of miR-34c in mice leads to an age-dependent osteoporosis due to the defective mineralization and proliferation of osteoblasts and increased osteoclastogenesis. In osteoblasts, miR-34c targets multiple components of the Notch signaling pathway, including Notch1, Notch2 and Jag1 in a direct manner, and influences osteoclast differentiation in a non-cell-autonomous fashion. Taken together, our results demonstrate that miR-34c is critical during osteoblastogenesis in part by regulating Notch signaling in bone homeostasis. Furthermore, miR-34c-mediated post-transcriptional regulation of Notch signaling in osteoblasts is one possible mechanism to modulate the proliferative effect of Notch in the committed osteoblast progenitors which may be important in the pathogenesis of osteosarcomas. Therefore, understanding the functional interaction of miR-34 and Notch signaling in normal bone development and in bone cancer could potentially lead to therapies modulating miR-34 signaling.

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called “episignatures”). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders. Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called “episignatures”). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders.

Zimmermann-Laband syndrome (ZLS) is a developmental disorder characterized by facial dysmorphism with gingival enlargement, intellectual disability, hypoplasia or aplasia of nails and terminal phalanges, and hypertrichosis. We report that heterozygous missense mutations in KCNH1 account for a considerable proportion of ZLS. KCNH1 encodes the voltage-gated K(+) channel Eag1 (Kv10.1). Patch-clamp recordings showed strong negative shifts in voltage-dependent activation for all but one KCNH1 channel mutant (Gly469Arg). Coexpression of Gly469Arg with wild-type KCNH1 resulted in heterotetrameric channels with reduced conductance at positive potentials but pronounced conductance at negative potentials. These data support a gain-of-function effect for all ZLS-associated KCNH1 mutants. We also identified a recurrent de novo missense change in ATP6V1B2, encoding the B2 subunit of the multimeric vacuolar H(+) ATPase, in two individuals with ZLS. Structural analysis predicts a perturbing effect of the mutation on complex assembly. Our findings demonstrate that KCNH1 mutations cause ZLS and document genetic heterogeneity for this disorder.

Efforts to identify the genetic underpinnings of rare undiagnosed diseases increasingly involve the use of next-generation sequencing and comparative genomic hybridization methods. These efforts are limited by a lack of knowledge regarding gene function, and an inability to predict the impact of genetic variation on the encoded protein function. Diagnostic challenges posed by undiagnosed diseases have solutions in model organism research, which provides a wealth of detailed biological information. Model organism geneticists are by necessity experts in particular genes, gene families, specific organs, and biological functions. Here, we review the current state of research into undiagnosed diseases, highlighting large efforts in North America and internationally, including the Undiagnosed Diseases Network (UDN) (Supplemental Material, File S1) and UDN International (UDNI), the Centers for Mendelian Genomics (CMG), and the Canadian Rare Diseases Models and Mechanisms Network (RDMM). We discuss how merging human genetics with model organism research guides experimental studies to solve these medical mysteries, gain new insights into disease pathogenesis, and uncover new therapeutic strategies.

Pediatric developmental syndromes present with systemic, complex, and often overlapping clinical features that are not infrequently a consequence of Mendelian inheritance of mutations in genes involved in DNA methylation, establishment of histone modifications, and chromatin remodeling (the “epigenetic machinery”). The mechanistic cross-talk between histone modification and DNA methylation suggests that these syndromes might be expected to display specific DNA methylation signatures that are a reflection of those primary errors associated with chromatin dysregulation. Given the interrelated functions of these chromatin regulatory proteins, we sought to identify DNA methylation epi-signatures that could provide syndrome-specific biomarkers to complement standard clinical diagnostics. In the present study, we examined peripheral blood samples from a large cohort of individuals encompassing 14 Mendelian disorders displaying mutations in the genes encoding proteins of the epigenetic machinery. We demonstrated that specific but partially overlapping DNA methylation signatures are associated with many of these conditions. The degree of overlap among these epi-signatures is minimal, further suggesting that, consistent with the initial event, the downstream changes are unique to every syndrome. In addition, by combining these epi-signatures, we have demonstrated that a machine learning tool can be built to concurrently screen for multiple syndromes with high sensitivity and specificity, and we highlight the utility of this tool in solving ambiguous case subjects presenting with variants of unknown significance, along with its ability to generate accurate predictions for subjects presenting with the overlapping clinical and molecular features associated with the disruption of the epigenetic machinery. Pediatric developmental syndromes present with systemic, complex, and often overlapping clinical features that are not infrequently a consequence of Mendelian inheritance of mutations in genes involved in DNA methylation, establishment of histone modifications, and chromatin remodeling (the “epigenetic machinery”). The mechanistic cross-talk between histone modification and DNA methylation suggests that these syndromes might be expected to display specific DNA methylation signatures that are a reflection of those primary errors associated with chromatin dysregulation. Given the interrelated functions of these chromatin regulatory proteins, we sought to identify DNA methylation epi-signatures that could provide syndrome-specific biomarkers to complement standard clinical diagnostics. In the present study, we examined peripheral blood samples from a large cohort of individuals encompassing 14 Mendelian disorders displaying mutations in the genes encoding proteins of the epigenetic machinery. We demonstrated that specific but partially overlapping DNA methylation signatures are associated with many of these conditions. The degree of overlap among these epi-signatures is minimal, further suggesting that, consistent with the initial event, the downstream changes are unique to every syndrome. In addition, by combining these epi-signatures, we have demonstrated that a machine learning tool can be built to concurrently screen for multiple syndromes with high sensitivity and specificity, and we highlight the utility of this tool in solving ambiguous case subjects presenting with variants of unknown significance, along with its ability to generate accurate predictions for subjects presenting with the overlapping clinical and molecular features associated with the disruption of the epigenetic machinery.

De novo germline mutations in the RNA helicase DDX3X account for 1%-3% of unexplained intellectual disability (ID) cases in females and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here, we use human and mouse genetics and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n = 107), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuron generation. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity, induce ectopic RNA-protein granules in neural progenitors and neurons, and impair translation. Together, these results uncover key mechanisms underlying DDX3X syndrome and highlight aberrant RNA metabolism in the pathogenesis of neurodevelopmental disease.

Abstract The “Nosology of genetic skeletal disorders” has undergone its 11th revision and now contains 771 entries associated with 552 genes reflecting advances in molecular delineation of new disorders thanks to advances in DNA sequencing technology. The most significant change as compared to previous versions is the adoption of the dyadic naming system, systematically associating a phenotypic entity with the gene it arises from. We consider this a significant step forward as dyadic naming is more informative and less prone to errors than the traditional use of list numberings and eponyms. Despite the adoption of dyadic naming, efforts have been made to maintain strong ties to the MIM catalog and its historical data. As with the previous versions, the list of disorders and genes in the Nosology may be useful in considering the differential diagnosis in the clinic, directing bioinformatic analysis of next‐generation sequencing results, and providing a basis for novel advances in biology and medicine.

Overlapping clinical phenotypes and an expanding breadth and complexity of genomic associations are a growing challenge in the diagnosis and clinical management of Mendelian disorders. The functional consequences and clinical impacts of genomic variation may involve unique, disorder-specific, genomic DNA methylation episignatures. In this study, we describe 19 novel episignature disorders and compare the findings alongside 38 previously established episignatures for a total of 57 episignatures associated with 65 genetic syndromes. We demonstrate increasing resolution and specificity ranging from protein complex, gene, sub-gene, protein domain, and even single nucleotide-level Mendelian episignatures. We show the power of multiclass modeling to develop highly accurate and disease-specific diagnostic classifiers. This study significantly expands the number and spectrum of disorders with detectable DNA methylation episignatures, improves the clinical diagnostic capabilities through the resolution of unsolved cases and the reclassification of variants of unknown clinical significance, and provides further insight into the molecular etiology of Mendelian conditions.

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.

Cornelia de Lange syndrome (CdLS) is a multisystem disorder with distinctive facial appearance, intellectual disability and growth failure as prominent features. Most individuals with typical CdLS have de novo heterozygous loss-of-function mutations in NIPBL with mosaic individuals representing a significant proportion. Mutations in other cohesin components, SMC1A, SMC3, HDAC8 and RAD21 cause less typical CdLS.We screened 163 affected individuals for coding region mutations in the known genes, 90 for genomic rearrangements, 19 for deep intronic variants in NIPBL and 5 had whole-exome sequencing.Pathogenic mutations [including mosaic changes] were identified in: NIPBL 46 [3] (28.2%); SMC1A 5 [1] (3.1%); SMC3 5 [1] (3.1%); HDAC8 6 [0] (3.6%) and RAD21 1 [0] (0.6%). One individual had a de novo 1.3 Mb deletion of 1p36.3. Another had a 520 kb duplication of 12q13.13 encompassing ESPL1, encoding separase, an enzyme that cleaves the cohesin ring. Three de novo mutations were identified in ANKRD11 demonstrating a phenotypic overlap with KBG syndrome. To estimate the number of undetected mosaic cases we used recursive partitioning to identify discriminating features in the NIPBL-positive subgroup. Filtering of the mutation-negative group on these features classified at least 18% as 'NIPBL-like'. A computer composition of the average face of this NIPBL-like subgroup was also more typical in appearance than that of all others in the mutation-negative group supporting the existence of undetected mosaic cases.Future diagnostic testing in 'mutation-negative' CdLS thus merits deeper sequencing of multiple DNA samples derived from different tissues.

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations.

Infantile myofibromatosis (IM) is the most common benign fibrous tumor of soft tissues affecting young children. By using whole-exome sequencing, RNA sequencing, and targeted sequencing, we investigated germline and tumor DNA in individuals from four distinct families with the familial form of IM and in five simplex IM cases with no previous family history of this disease. We identified a germline mutation c.1681C>T (p.Arg561Cys) in platelet-derived growth factor receptor β (PDGFRB) in all 11 affected individuals with familial IM, although none of the five individuals with nonfamilial IM had mutations in this gene. We further identified a second heterozygous mutation in PDGFRB in two myofibromas from one of the affected familial cases, indicative of a potential second hit in this gene in the tumor. PDGFR-β promotes growth of mesenchymal cells, including blood vessels and smooth muscles, which are affected in IM. Our findings indicate p.Arg561Cys substitution in PDGFR-β as a cause of the dominant form of this disease. They provide a rationale for further investigations of this specific mutation and gene to assess the benefits of targeted therapies against PDGFR-β in aggressive life-threatening familial forms of the disease. Infantile myofibromatosis (IM) is the most common benign fibrous tumor of soft tissues affecting young children. By using whole-exome sequencing, RNA sequencing, and targeted sequencing, we investigated germline and tumor DNA in individuals from four distinct families with the familial form of IM and in five simplex IM cases with no previous family history of this disease. We identified a germline mutation c.1681C>T (p.Arg561Cys) in platelet-derived growth factor receptor β (PDGFRB) in all 11 affected individuals with familial IM, although none of the five individuals with nonfamilial IM had mutations in this gene. We further identified a second heterozygous mutation in PDGFRB in two myofibromas from one of the affected familial cases, indicative of a potential second hit in this gene in the tumor. PDGFR-β promotes growth of mesenchymal cells, including blood vessels and smooth muscles, which are affected in IM. Our findings indicate p.Arg561Cys substitution in PDGFR-β as a cause of the dominant form of this disease. They provide a rationale for further investigations of this specific mutation and gene to assess the benefits of targeted therapies against PDGFR-β in aggressive life-threatening familial forms of the disease. Infantile myofibromatosis (IM) (MIM 228550) is the most common benign tumor of soft tissue of infancy and childhood.1Wiswell T.E. Davis J. Cunningham B.E. Solenberger R. Thomas P.J. Infantile myofibromatosis: the most common fibrous tumor of infancy.J. Pediatr. Surg. 1988; 23: 315-318Abstract Full Text PDF PubMed Scopus (224) Google Scholar First described by Stout,2Stout A.P. Juvenile fibromatoses.Cancer. 1954; 7: 953-978Crossref PubMed Scopus (515) Google Scholar IM is characterized by solitary or multiple nodules in the skin, muscle, subcutaneous tissues, bone, and occasionally viscera. IM is simplex or occurs with an autosomal-dominant (AD) mode of inheritance.3Jennings T.A. Duray P.H. Collins F.S. Sabetta J. Enzinger F.M. Infantile myofibromatosis. Evidence for an autosomal-dominant disorder.Am. J. Surg. Pathol. 1984; 8: 529-538Crossref PubMed Scopus (120) Google Scholar, 4Zand D.J. Huff D. Everman D. Russell K. Saitta S. McDonald-McGinn D. Zackai E.H. Autosomal dominant inheritance of infantile myofibromatosis.Am. J. Med. Genet. A. 2004; 126A: 261-266Crossref PubMed Scopus (48) Google Scholar Myofibromas are usually present at birth or develop shortly thereafter, with 90% of cases occurring before the age of 2 years.5Chung E.B. Enzinger F.M. Infantile myofibromatosis.Cancer. 1981; 48: 1807-1818Crossref PubMed Scopus (534) Google Scholar Solitary and multicentric IMs that do not involve the viscera tend to spontaneously regress and their recurrence is relatively low. However, multicentric IM with visceral involvement has a poor outcome, with a mortality rate greater than 70% despite aggressive therapies.6Auriti C. Kieran M.W. Deb G. Devito R. Pasquini L. Danhaive O. Remission of infantile generalized myofibromatosis after interferon alpha therapy.J. Pediatr. Hematol. Oncol. 2008; 30: 179-181Crossref PubMed Scopus (13) Google Scholar, 7Azzam R. Abboud M. Muwakkit S. Khoury N. Saab R. First-line therapy of generalized infantile myofibromatosis with low-dose vinblastine and methotrexate.Pediatr. Blood Cancer. 2009; 52: 308Crossref PubMed Scopus (20) Google Scholar The molecular etiology of the disease remains unknown. To determine the genetic defect(s) underlying IM and whether the causes of familial and simplex IM are similar, we studied 11 individuals from 4 IM-affected families and 5 simplex cases. The clinical features and genotypes of the individuals investigated in this study are presented in Table S1 (available online) and the pedigrees of the four families are shown in Figure 1. The studies were approved by the Institutional Review Boards of Columbia University, the Baylor College of Medicine, McGill University Health Centre Research Institute, and the Children’s Hospital of Eastern Ontario. Blood and tumor samples were obtained with informed consent from the patients and their parents according to Canadian and US laws. Genomic DNA was isolated from blood and from frozen and paraffin-embedded tissues. Total RNA was extracted from tumor tissue excised from the abdominal wall of individual III-1 of family 2 (Figure 1). We first focused on familial cases and performed next-generation sequencing on DNA and RNA extracted from a discovery set of IM-affected familial cases. Whole-exome sequencing (WES) was performed on germline DNA from two affected siblings from a family of Chinese origin (family 1, Figure 1). The brother carried the typical solitary form, and the sister was treated for a visceral type with multiple myofibromas of the orbit and supranasal region. Exomes were captured with the Illumina TruSeq kit and were sequenced on an Illumina Hiseq 2000 with 100 bp paired-end reads. Reads were aligned against the reference human genome (UCSC Genome Browser hg19) with BWA,8Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26648) Google Scholar variants called and annotated as previously described.9Schwartzentruber J. Korshunov A. Liu X.-Y. Jones D.T.W. Pfaff E. Jacob K. Sturm D. Fontebasso A.M. Quang D.-A.K. Tönjes M. et al.Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.Nature. 2012; 482: 226-231Crossref PubMed Scopus (1707) Google Scholar Given the rarity of the disease, we eliminated variants with minor allele frequency (MAF) greater than 1% in the 1000 Genomes10Abecasis G.R. Altshuler D. Auton A. Brooks L.D. Durbin R.M. Gibbs R.A. Hurles M.E. McVean G.A. 1000 Genomes Project ConsortiumA map of human genome variation from population-scale sequencing.Nature. 2010; 467: 1061-1073Crossref PubMed Scopus (5937) Google Scholar and NHLBI GO Exome Sequencing Project databases or greater than 5% in approximately 500 exomes previously sequenced at our center. We also performed RNA-seq on an abdominal wall myofibroma from the child (III-1) of an affected mother-child pair of European ancestry in family 2 (Figure 1). Both of these affected individuals suffer from multiple myofibromas of the head, neck, and abdominal wall, which were either surgically resected or spontaneously regressed. In brief, mRNAs were enriched from total RNA with poly(A) selection followed by library preparation by Illumina TruSeq RNA prep kit and sequencing on Illumina HiSeq 2000 with single-end 100 bp reads. The pass filter reads were then mapped to the reference human genome (NCBI build 37) by TopHat11Trapnell C. Pachter L. Salzberg S.L. TopHat: discovering splice junctions with RNA-Seq.Bioinformatics. 2009; 25: 1105-1111Crossref PubMed Scopus (8995) Google Scholar (v.1.3.3). For each read, up to two mismatches and ten multiple hits were allowed during the mapping. Variants were called with SAMtools (v.0.1.17), mpileup, and bcftools, filtered by mapping quality ≥ 5, read depth ≥ 5, and base quality ≥ 17. Functional annotations were obtained by SeattleSeq Annotation 134 (NCBI and CCDS 2011) and ANNOVAR.12Wang K. Li M. Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7872) Google Scholar The RNA-seq data revealed a total of 28,141 SNVs and 923 short indels in 6,838 genes. Assuming AD inheritance, variants on the X and Y chromosomes and mitochondrial genome were excluded. We retained only nonsynonymous and splice variants and eliminated variants with MAF greater than 1% in the 1000 Genomes database.10Abecasis G.R. Altshuler D. Auton A. Brooks L.D. Durbin R.M. Gibbs R.A. Hurles M.E. McVean G.A. 1000 Genomes Project ConsortiumA map of human genome variation from population-scale sequencing.Nature. 2010; 467: 1061-1073Crossref PubMed Scopus (5937) Google Scholar We also removed variants predicted to be tolerated, benign, or unknown by both SIFT13Sim N.-L. Kumar P. Hu J. Henikoff S. Schneider G. Ng P.C. SIFT web server: predicting effects of amino acid substitutions on proteins.Nucleic Acids Res. 2012; 40: W452-W457Crossref PubMed Scopus (1345) Google Scholar and PolyPhen-2.14Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat. Methods. 2010; 7: 248-249Crossref PubMed Scopus (9290) Google Scholar After the filtering procedures, there were 385 SNVs and 43 indels in the remaining 338 genes. In both families, we identified the same heterozygous c.1681C>T (p.Arg561Cys) variant in PDGFRB (MIM 173410; RefSeq accession number NM_002609.3), which encodes a receptor tyrosine kinase (RTK). The missense variant was predicted to be damaging by all prediction algorithms and affects a highly conserved amino acid residue. This mutation was absent from our control in-house exome database, dbSNP135, the 1000 Genomes project,10Abecasis G.R. Altshuler D. Auton A. Brooks L.D. Durbin R.M. Gibbs R.A. Hurles M.E. McVean G.A. 1000 Genomes Project ConsortiumA map of human genome variation from population-scale sequencing.Nature. 2010; 467: 1061-1073Crossref PubMed Scopus (5937) Google Scholar and NHLBI Exome Sequencing Project. Sanger sequencing (Table S2) confirmed the heterozygous mutation in all affected members of both families 1 and 2. Because the mutation was absent in family 2 members I-1 and I-2, we determined that it was acquired de novo in II-2. In the same individual, we identified a second variant, c.1998C>A (p.Asn666Lys), that is predicted to be damaging. This variant was identified in a second gingival myofibroma but was absent from a neck myofibroma and from blood, indicating that the variant is somatic. Neither of these two genetic alterations was present in nonaffected family members (Figure 1) or in the 43 somatic missense mutations of PDGFRB listed in the Catalogue Of Somatic Mutations In Cancer.15Forbes S.A. Bindal N. Bamford S. Cole C. Kok C.Y. Beare D. Jia M. Shepherd R. Leung K. Menzies A. et al.COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer.Nucleic Acids Res. 2011; 39: D945-D950Crossref PubMed Scopus (1812) Google Scholar We then screened for mutations in PDGFRB by using Sanger sequencing in an additional two separate families affected with IM (Figure 1, families 3 and 4) and in five simplex IM cases, including three with visceral IM (Table S1). Family 3 is of French-Canadian origin and the two affected siblings (III-2 and III-3) had multiple myofibromas of the skin on the face and upper arms that spontaneously regressed at the age of 4 years, similar to the father who also had myofibroma that spontaneously resolved. Family 4 is of European ancestry and was previously reported.16Smith A. Orchard D. Infantile myofibromatosis: two families supporting autosomal dominant inheritance.Australas. J. Dermatol. 2011; 52: 214-217Crossref PubMed Scopus (8) Google Scholar The two affected brothers (II-1 and II-3) were diagnosed with multiple myofibromas at the young ages of 3 and 11 weeks, respectively, with no clinical evidence of visceral involvement. The mother had swelling in the left side of her neck at 7 months of age, which was found to be fibromatosis and subsequently excised. The five simplex cases included in this study were taken from resected myofibromas from children at the following ages and locations: at birth (cheek), 34 days (pelvic myofibroma, reported as case 2 in a previous study17Wilson M. Emil S. Cowan K. Kalechstein S. Puligandla P. Nguyen V.-H. Laberge J.-M. Chou S. Infantile myofibromas obstructing opposite ends of the gastrointestinal tract.J. Pediatr. Surg. 2013; 48: 449-453Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar), 13 days (lesion from right hemidiaphragm), 5 days (visceral myofibroma), and 1 month (femur and lung). Matched blood was available in two cases and was also analyzed for PDFGRB mutations. All of the affected individuals with familial IM harbored the same missense variant, c.1681C>T, thereby confirming the role of PDGFRB in the pathogenesis of familial IM as well as the de novo occurrence of this mutation in a family member (II-2 in family 2 and II-1 in family 3, Figure 1). None of the five individuals with simplex IM harbored either of the two PDGFRB mutations in myofibromas or in germline DNA in the two cases for which this material was also available. PDGFRB encodes the β polypeptide of the platelet-derived growth factor receptor (PDGFR-β), a RTK and a mitogen for mesenchyme-derived cells, including fibroblasts and smooth muscle,18Andrae J. Gallini R. Betsholtz C. Role of platelet-derived growth factors in physiology and medicine.Genes Dev. 2008; 22: 1276-1312Crossref PubMed Scopus (1695) Google Scholar, 19Fredriksson L. Li H. Eriksson U. The PDGF family: four gene products form five dimeric isoforms.Cytokine Growth Factor Rev. 2004; 15: 197-204Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar which are targeted in IM. Mouse knockout studies showed the critical role of PDGFR-β signaling in the embryonic development of specific subsets of smooth muscle cells,20Levéen P. Pekny M. Gebre-Medhin S. Swolin B. Larsson E. Betsholtz C. Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities.Genes Dev. 1994; 8: 1875-1887Crossref PubMed Scopus (872) Google Scholar, 21Soriano P. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice.Genes Dev. 1994; 8: 1888-1896Crossref PubMed Scopus (798) Google Scholar particularly in the proper recruitment of vascular smooth muscle cells and pericytes to developing blood vessel walls.16Smith A. Orchard D. Infantile myofibromatosis: two families supporting autosomal dominant inheritance.Australas. J. Dermatol. 2011; 52: 214-217Crossref PubMed Scopus (8) Google Scholar, 17Wilson M. Emil S. Cowan K. Kalechstein S. Puligandla P. Nguyen V.-H. Laberge J.-M. Chou S. Infantile myofibromas obstructing opposite ends of the gastrointestinal tract.J. Pediatr. Surg. 2013; 48: 449-453Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 18Andrae J. Gallini R. Betsholtz C. Role of platelet-derived growth factors in physiology and medicine.Genes Dev. 2008; 22: 1276-1312Crossref PubMed Scopus (1695) Google Scholar, 19Fredriksson L. Li H. Eriksson U. The PDGF family: four gene products form five dimeric isoforms.Cytokine Growth Factor Rev. 2004; 15: 197-204Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar Deregulation of PDGF signaling (usually through somatic mutations, overexpression, or fusion with other genes) has been associated with several human disorders and cancers affecting cells of mesenchymal origin (reviewed in Andrae et al.,18Andrae J. Gallini R. Betsholtz C. Role of platelet-derived growth factors in physiology and medicine.Genes Dev. 2008; 22: 1276-1312Crossref PubMed Scopus (1695) Google Scholar Trojanowska,22Trojanowska M. Role of PDGF in fibrotic diseases and systemic sclerosis.Rheumatology (Oxford). 2008; 47: v2-v4Crossref PubMed Scopus (67) Google Scholar and George23George D. Targeting PDGF receptors in cancer—rationales and proof of concept clinical trials.Adv. Exp. Med. Biol. 2003; 532: 141-151Crossref PubMed Scopus (64) Google Scholar), and more recently, p.Leu658Pro and p.Arg987Trp PDGFRB germline variants have been associated with idiopathic basal ganglia calcification (MIM 615007).24Nicolas G. Pottier C. Maltête D. Coutant S. Rovelet-Lecrux A. Legallic S. Rousseau S. Vaschalde Y. Guyant-Maréchal L. Augustin J. et al.Mutation of the PDGFRB gene as a cause of idiopathic basal ganglia calcification.Neurology. 2013; 80: 181-187Crossref PubMed Scopus (198) Google Scholar The germline mutation in PDFGRB we identify herein has not been previously reported. To gain insight into its potential effects on receptor activity, we constructed a homology model of the structure of the cytoplasmic domain of human PDGFR-β. The sequence of the PDGFR-β cytoplasmic domain (residues 554–1106) was obtained from the UniProt database and converted to PIR format for use in the Modeler program suite.25Sali A. Blundell T.L. Comparative protein modelling by satisfaction of spatial restraints.J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10514) Google Scholar A search for related sequences of known structure identified the autoinhibited form of human KIT kinase (PDB ID 1T45) as a useful template upon which to model the structure of PDGFR-β. KIT and PDGFR-β are members of the same receptor tyrosine kinase family (class III) and possess a high degree of sequence identity in the cytoplasmic region (∼57%). Because only the extracellular domain of PDGFR-β has been previously modeled, the human KIT kinase was used for this modeling, based on extensive homology with PDGFRs.26Qiu F.H. Ray P. Brown K. Barker P.E. Jhanwar S. Ruddle F.H. Besmer P. Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family—oncogenic activation of v-kit involves deletion of extracellular domain and C terminus.EMBO J. 1988; 7: 1003-1011Crossref PubMed Scopus (568) Google Scholar The sequence of the PDGFR-β cytoplasmic domain was aligned to that of KIT and several three-dimensional models of PDGFR-β were generated with Modeler according to this target-template alignment. The resulting structural models were reasonable, as suggested by the scores from Modeler and by visual inspection with the program PyMOL.27Schrödinger, L. (2010). The {PyMOL} Molecular Graphics System, Version∼1.3r1.Google Scholar The model includes the cytoplasmic juxtamembrane (JM) domain and the two lobes of the kinase domain. The structures of all models were highly similar, except in the area of the kinase insert segment (approximately residues 700–795). Because the KIT template structure lacks the kinase insert segment found in PDGFR-β, modeling of these residues is less reliable. However, the c.1681C>T (p.Arg561Cys) and c.1998C>A (p.Asn666Lys) alterations are situated far from the site of the kinase insert and consequently are not impacted by uncertainty for that segment of the PDGFR-β model. To assess the role of residues Arg561 and Asn666 and the potential impact of the observed mutations for the function of the PDGFR-β JM and kinase domains, we compared the PDGFR-β model with the structures of the autoinhibited (PDB ID 1T45) and active (PDB ID 1PKG) forms of KIT. Arg561 maps to the JM region of PDGFR-β between the helical transmembrane segment and the kinase domain, lying at the boundary of the JM domain and the short section of polypeptide that links it to the transmembrane helix. The JM domain is autoinhibitory, masking the catalytic cleft when the receptor is not bound by its ligand.28Hubbard S.R. Juxtamembrane autoinhibition in receptor tyrosine kinases.Nat. Rev. Mol. Cell Biol. 2004; 5: 464-471Crossref PubMed Scopus (232) Google Scholar In the model, the side chain of Arg561 forms a salt bridge with residue Glu644 in an adjacent α helix from the C-terminal lobe of the kinase domain (Figure 2C). This interaction would be expected to tether this part of the JM domain to the C-terminal lobe and contribute to the binding of the autoinhibitory JM segment to the kinase domain. A p.Arg561Cys change would abrogate the salt bridge, weakening the binding of the JM domain. This would compromise the autoinhibitory role of the JM domain in preventing receptor firing at baseline. The second somatic change, c.1998C>A (p.Asn666Lys), is located in the N-terminal lobe of the kinase domain, near the binding site for RTK inhibitors such as imatinib and sunitinib. In the PDGFR-β model, the side chain of Asn666 participates in hydrogen-bonding interactions with the backbone of His661. A similar interaction is observed between Asn655 and His650 in the structure of the autoinhibited form of KIT kinase.29Mol C.D. Dougan D.R. Schneider T.R. Skene R.J. Kraus M.L. Scheibe D.N. Snell G.P. Zou H. Sang B.-C. Wilson K.P. Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase.J. Biol. Chem. 2004; 279: 31655-31663Crossref PubMed Scopus (516) Google Scholar Interestingly, the structure of the active form of KIT kinase is different in this area.30Mol C.D. Lim K.B. Sridhar V. Zou H. Chien E.Y.T. Sang B.-C. Nowakowski J. Kassel D.B. Cronin C.N. McRee D.E. Structure of a c-kit product complex reveals the basis for kinase transactivation.J. Biol. Chem. 2003; 278: 31461-31464Crossref PubMed Scopus (206) Google Scholar The side chain of Asn655 is oriented in a different direction and no longer interacts with residue H650 (Figure 2D). The p.Asn666Lys substitution in PDGFR-β would thus abolish the interaction between Asn666 and His661, altering the interactions in this area of the protein and possibly leading to a structure more similar to the active conformation of KIT kinase. PDGFR-β signaling is critical for normal development of mesenchymal tissues.21Soriano P. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice.Genes Dev. 1994; 8: 1888-1896Crossref PubMed Scopus (798) Google Scholar, 22Trojanowska M. Role of PDGF in fibrotic diseases and systemic sclerosis.Rheumatology (Oxford). 2008; 47: v2-v4Crossref PubMed Scopus (67) Google Scholar IM affects infants during their first years of life when expression of PDGFR-β and its ligands are highest. Our findings indicate that a recurrent germline mutation, c.1681C>T (p.Arg561Cys), in PDGFRB is responsible for the autosomal-dominant familial but not for the simplex form of IM. The PDGFRB mutation in familial IM is predicted to decrease autoinhibition of the JM domain at baseline, leading to increased kinase firing and promoting the formation of myofibromas in tissues with high PDGFR-β signaling activity. This may explain the perinatal and early childhood prevalence of this disease and its regression with age. Aberrant signaling through PDGFR-β in the genesis of familial IM is further supported by the presence of a second hit in this RTK in visceral myofibroma. Several drugs effectively inhibit the kinase activity of PDGFR-β, and our data support further investigations into these PDGFRB mutations to assess the potential use of these inhibitors in the treatment of life-threatening forms of the IM, where limited effective therapeutic options are currently available. We would like to thank the study participants and their families; without their participation, this work would not have been possible. T.G. is supported by an award from the Canadian Gene Cure Foundation. N.J. is the recipient of a Chercheur Clinician Senior award from Fonds de la Recherché en santé au Quebec (FRSQ). Research funding includes National Institutes of Health grants PO1 HD22657 and PO1 HD070394 and The Rolanette and Berdon Lawrence Bone Disease Program of Texas (to B.H.L.) and NIH grants U54 HG006542 and U54 HG003273 (to R.A.G.). J.T.L. is supported by Ruth L. Kirschstein National Research Service Award F30 MH098571-01. P.M.C. is supported by a CIHR clinician-scientist training award. Part of this work was completed as a rare disease studied by the FORGE Canada Consortium, funded by the Government of Canada through Genome Canada, the Canadian Institutes of Health Research, and the Ontario Genomics Institute (OGI-049). Additional funding to FORGE was provided by Genome Quebec, Genome British Columbia, and the McLaughlin Centre. We thank Shalini N. Jhangiani for coordination of exome sequencing and Alyssa Tran for clinical research support. We also wish to acknowledge the contribution of the high-throughput sequencing platform of the McGill University and Génome Québec Innovation Centre, Montréal, Canada. Download .pdf (.04 MB) Help with pdf files Document S1. Tables S1 and S2 The URLs for data presented herein are as follows:1000 Genomes, http://browser.1000genomes.orgANNOVAR, http://www.openbioinformatics.org/annovar/NCBI Gene, http://www.ncbi.nlm.nih.gov/geneNHLBI Exome Sequencing Project (ESP) Exome Variant Server, http://evs.gs.washington.edu/EVS/Online Mendelian Inheritance in Man (OMIM), http://www.omim.org/PolyPhen-2, http://www.genetics.bwh.harvard.edu/pph2/SeattleSeq Annotation 137, http://snp.gs.washington.edu/SeattleSeqAnnotation137/SIFT, http://sift.bii.a-star.edu.sg/UCSC Genome Browser, http://genome.ucsc.eduUniProt, http://www.uniprot.org/ Mutations in PDGFRB Cause Autosomal-Dominant Infantile MyofibromatosisMartignetti et al.The American Journal of Human GeneticsMay 23, 2013In BriefInfantile myofibromatosis (IM) is a disorder of mesenchymal proliferation characterized by the development of nonmetastasizing tumors in the skin, muscle, bone, and viscera. Occurrence within families across multiple generations is suggestive of an autosomal-dominant (AD) inheritance pattern, but autosomal-recessive (AR) modes of inheritance have also been proposed. We performed whole-exome sequencing (WES) in members of nine unrelated families clinically diagnosed with AD IM to identify the genetic origin of the disorder. Full-Text PDF Open Archive

The more than 1.5 billion people who live in South Asia are correctly viewed not as a single large population but as many small endogamous groups. We assembled genome-wide data from over 2,800 individuals from over 260 distinct South Asian groups. We identified 81 unique groups, 14 of which had estimated census sizes of more than 1 million, that descend from founder events more extreme than those in Ashkenazi Jews and Finns, both of which have high rates of recessive disease due to founder events. We identified multiple examples of recessive diseases in South Asia that are the result of such founder events. This study highlights an underappreciated opportunity for decreasing disease burden among South Asians through discovery of and testing for recessive disease-associated genes.

Yunis-Varón syndrome (YVS) is an autosomal-recessive disorder with cleidocranial dysplasia, digital anomalies, and severe neurological involvement. Enlarged vacuoles are found in neurons, muscle, and cartilage. By whole-exome sequencing, we identified frameshift and missense mutations of FIG4 in affected individuals from three unrelated families. FIG4 encodes a phosphoinositide phosphatase required for regulation of PI(3,5)P2 levels, and thus endosomal trafficking and autophagy. In a functional assay, both missense substitutions failed to correct the vacuolar phenotype of Fig4-null mouse fibroblasts. Homozygous Fig4-null mice exhibit features of YVS, including neurodegeneration and enlarged vacuoles in neurons. We demonstrate that Fig4-null mice also have small skeletons with reduced trabecular bone volume and cortical thickness and that cultured osteoblasts accumulate large vacuoles. Our findings demonstrate that homozygosity or compound heterozygosity for null mutations of FIG4 is responsible for YVS, the most severe known human phenotype caused by defective phosphoinositide metabolism. In contrast, in Charcot-Marie-Tooth disease type 4J (also caused by FIG4 mutations), one of the FIG4 alleles is hypomorphic and disease is limited to the peripheral nervous system. This genotype-phenotype correlation demonstrates that absence of FIG4 activity leads to central nervous system dysfunction and extensive skeletal anomalies. Our results describe a role for PI(3,5)P2 signaling in skeletal development and maintenance. Yunis-Varón syndrome (YVS) is an autosomal-recessive disorder with cleidocranial dysplasia, digital anomalies, and severe neurological involvement. Enlarged vacuoles are found in neurons, muscle, and cartilage. By whole-exome sequencing, we identified frameshift and missense mutations of FIG4 in affected individuals from three unrelated families. FIG4 encodes a phosphoinositide phosphatase required for regulation of PI(3,5)P2 levels, and thus endosomal trafficking and autophagy. In a functional assay, both missense substitutions failed to correct the vacuolar phenotype of Fig4-null mouse fibroblasts. Homozygous Fig4-null mice exhibit features of YVS, including neurodegeneration and enlarged vacuoles in neurons. We demonstrate that Fig4-null mice also have small skeletons with reduced trabecular bone volume and cortical thickness and that cultured osteoblasts accumulate large vacuoles. Our findings demonstrate that homozygosity or compound heterozygosity for null mutations of FIG4 is responsible for YVS, the most severe known human phenotype caused by defective phosphoinositide metabolism. In contrast, in Charcot-Marie-Tooth disease type 4J (also caused by FIG4 mutations), one of the FIG4 alleles is hypomorphic and disease is limited to the peripheral nervous system. This genotype-phenotype correlation demonstrates that absence of FIG4 activity leads to central nervous system dysfunction and extensive skeletal anomalies. Our results describe a role for PI(3,5)P2 signaling in skeletal development and maintenance. Yunis and Varón first described the syndrome that bears their name in 1980, based on three Colombian families with a total of five affected children.1Yunis E. Varón H. Cleidocranial dysostosis, severe micrognathism, bilateral absence of thumbs and first metatarsal bone, and distal aphalangia: a new genetic syndrome.Am. J. Dis. Child. 1980; 134: 649-653Crossref PubMed Scopus (44) Google Scholar Since then, approximately 25 individuals with Yunis-Varón syndrome (YVS) (MIM 216340) have been described.2Hughes H.E. Partington M.W. Brief clinical report: the syndrome of Yunis and Varón—report of a further case.Am. J. Med. Genet. 1983; 14: 539-544Crossref PubMed Scopus (15) Google Scholar, 3Pfeiffer R.A. Diekmann L. Stock H.J. Aplasia of the thumbs and great toes as the outstanding feature of Yunis and Varon syndrome. A new entity. A new observation.Ann. Genet. 1988; 31: 241-243PubMed Google Scholar, 4Hennekam R.C. Vermeulen-Meiners C. Further delineation of the Yunis-Varon syndrome.J. Med. Genet. 1989; 26: 55-58Crossref PubMed Scopus (20) Google Scholar, 5Garrett C. Berry A.C. Simpson R.H. Hall C.M. Yunis-Varon syndrome with severe osteodysplasty.J. Med. Genet. 1990; 27: 114-121Crossref PubMed Scopus (23) Google Scholar, 6Lapeer G.L. Fransman S.L. Hypodontia, impacted permanent teeth, spinal defects, and cardiomegaly in a previously diagnosed case of the Yunis-Varon syndrome.Oral Surg. Oral Med. Oral Pathol. 1992; 73: 456-460Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 7Adès L.C. Morris L.L. Richardson M. Pearson C. Haan E.A. Congenital heart malformation in Yunis-Varón syndrome.J. Med. Genet. 1993; 30: 788-792Crossref PubMed Scopus (19) Google Scholar, 8Dworzak F. Mora M. Borroni C. Cornelio F. Blasevich F. Cappellini A. Tagliavini F. Bertagnolio B. Generalized lysosomal storage in Yunis Varón syndrome.Neuromuscul. Disord. 1995; 5: 423-428Abstract Full Text PDF PubMed Scopus (15) Google Scholar, 9Rabe H. Brune T. Rossi R. Steinhorst V. Jorch G. Horst J. Wittwer B. Yunis-Varon syndrome: the first case of German origin.Clin. Dysmorphol. 1996; 5: 217-222Crossref PubMed Google Scholar, 10Oyer C.E. Tatevosyants N.G. Cortez S.C. Hornstein A. Wallach M. Cleidocranial dysplasia with neonatal death due to central nervous system injury in utero: case report and literature review.Pediatr. Dev. Pathol. 1998; 1: 314-318Crossref PubMed Scopus (9) Google Scholar, 11Christie J. Sacks S. Decorato D. Bergasa N.V. Atrophy of the left lobe of the liver and anomalous hepatic vessel in a patient with Yunis-Varon syndrome.J. Clin. Gastroenterol. 1999; 29: 210-211Crossref PubMed Scopus (9) Google Scholar, 12Walch E. Schmidt M. Brenner R.E. Emons D. Dame C. Pontz B. Wiestler O.D. Bartmann P. Yunis-Varon syndrome: evidence for a lysosomal storage disease.Am. J. Med. Genet. 2000; 95: 157-160Crossref PubMed Scopus (21) Google Scholar, 13Nagai T. [Yunis-Varon syndrome].Ryoikibetsu Shokogun Shirizu. 2001; 34 Part 2: 839-840Google Scholar, 14Sumi M. Kusumoto T. Kondoh T. Moriuchi H. Miyamoto M. Masuzaki H. Ishimaru T. A case of Yunis-Varon syndrome complicated with complete cleft lip and palate.Am. J. Med. Genet. A. 2004; 125A: 92-93Crossref PubMed Google Scholar, 15Bhatia S. Holla R.G. Yunis-Varon syndrome.Indian Pediatr. 2005; 42: 373-375PubMed Google Scholar, 16Kulkarni M.L. Vani H.N. Nagendra K. Mahesh T.K. Kumar A. Haneef S. Mohammed Z. Kulkarni P.M. Yunis Varon syndrome.Indian J. Pediatr. 2006; 73: 353-355Crossref PubMed Scopus (7) Google Scholar, 17Basel-Vanagaite L. Kornreich L. Schiller O. Yacobovich J. Merlob P. Yunis-Varon syndrome: further delineation of the phenotype.Am. J. Med. Genet. A. 2008; 146A: 532-537Crossref PubMed Scopus (13) Google Scholar, 18Corona-Rivera J.R. Romo-Huerta C.O. López-Marure E. Ramos F.J. Estrada-Padilla S.A. Zepeda-Romero L.C. New ocular findings in two sisters with Yunis-Varón syndrome and literature review.Eur. J. Med. Genet. 2011; 54: 76-81Crossref PubMed Scopus (10) Google Scholar, 19Elizondo-Dueñaz R. Rivera-Silva G. Marcos Abdala H. López-Altamirano M. Martínez-Menchaca H.R. [Yunis-Varon syndrome: a case report].Gac. Med. Mex. 2012; 148: 81-82PubMed Google Scholar Frequent features include structural brain abnormalities, sparse and pale hair, and facial dysmorphisms. Skeletal abnormalities include wide fontanelles with calvarial dysostosis, aplasia or hypoplasia of the clavicles and phalanges in the hands and feet, and absence of thumbs and halluces. Pelvic bone dysplasia, absent sternal ossification centers, and fractures are also frequent.17Basel-Vanagaite L. Kornreich L. Schiller O. Yacobovich J. Merlob P. Yunis-Varon syndrome: further delineation of the phenotype.Am. J. Med. Genet. A. 2008; 146A: 532-537Crossref PubMed Scopus (13) Google Scholar Neuropathology shows extensive neuronal loss and diffuse atrophy affecting the cerebellar vermis, corpus callosum, basal ganglia, and frontal lobes. Vacuoles compatible with enlarged lysosomes are seen in neurons, muscle, cartilage, heart, and macrophages.17Basel-Vanagaite L. Kornreich L. Schiller O. Yacobovich J. Merlob P. Yunis-Varon syndrome: further delineation of the phenotype.Am. J. Med. Genet. A. 2008; 146A: 532-537Crossref PubMed Scopus (13) Google Scholar In the urine, multiple abnormal oligosaccharide bands appear, suggesting a dysfunction of lysosomal enzymes,8Dworzak F. Mora M. Borroni C. Cornelio F. Blasevich F. Cappellini A. Tagliavini F. Bertagnolio B. Generalized lysosomal storage in Yunis Varón syndrome.Neuromuscul. Disord. 1995; 5: 423-428Abstract Full Text PDF PubMed Scopus (15) Google Scholar, 12Walch E. Schmidt M. Brenner R.E. Emons D. Dame C. Pontz B. Wiestler O.D. Bartmann P. Yunis-Varon syndrome: evidence for a lysosomal storage disease.Am. J. Med. Genet. 2000; 95: 157-160Crossref PubMed Scopus (21) Google Scholar but no consistent storage material could be identified12Walch E. Schmidt M. Brenner R.E. Emons D. Dame C. Pontz B. Wiestler O.D. Bartmann P. Yunis-Varon syndrome: evidence for a lysosomal storage disease.Am. J. Med. Genet. 2000; 95: 157-160Crossref PubMed Scopus (21) Google Scholar and the enzyme activities of oligosaccharidases were normal.8Dworzak F. Mora M. Borroni C. Cornelio F. Blasevich F. Cappellini A. Tagliavini F. Bertagnolio B. Generalized lysosomal storage in Yunis Varón syndrome.Neuromuscul. Disord. 1995; 5: 423-428Abstract Full Text PDF PubMed Scopus (15) Google Scholar Six families affected by Yunis-Varón syndrome were included in this study. The clinical features of the eight affected individuals are summarized in Table 1. Pictures and radiographs of most affected individuals are available in previously published case reports.5Garrett C. Berry A.C. Simpson R.H. Hall C.M. Yunis-Varon syndrome with severe osteodysplasty.J. Med. Genet. 1990; 27: 114-121Crossref PubMed Scopus (23) Google Scholar, 7Adès L.C. Morris L.L. Richardson M. Pearson C. Haan E.A. Congenital heart malformation in Yunis-Varón syndrome.J. Med. Genet. 1993; 30: 788-792Crossref PubMed Scopus (19) Google Scholar, 8Dworzak F. Mora M. Borroni C. Cornelio F. Blasevich F. Cappellini A. Tagliavini F. Bertagnolio B. Generalized lysosomal storage in Yunis Varón syndrome.Neuromuscul. Disord. 1995; 5: 423-428Abstract Full Text PDF PubMed Scopus (15) Google Scholar, 18Corona-Rivera J.R. Romo-Huerta C.O. López-Marure E. Ramos F.J. Estrada-Padilla S.A. Zepeda-Romero L.C. New ocular findings in two sisters with Yunis-Varón syndrome and literature review.Eur. J. Med. Genet. 2011; 54: 76-81Crossref PubMed Scopus (10) Google Scholar, 20Reutter H. Bagci S. Müller A. Gembruch U. Geipel A. Berg C. Eggermann T. Spengler S. Bartmann P. Rudnik-Schöneborn S. Primary pulmonary hypertension, congenital heart defect, central nervous system malformations, hypo- and aplastic toes: another case of Yunis-Varón syndrome or report of a new entity.Eur. J. Med. Genet. 2012; 55: 27-31Crossref PubMed Scopus (5) Google Scholar The study was conducted according to the guidelines of the institutional review board of the Baylor College of Medicine and informed consent was obtained prior to collection of samples. The inclusion criterion was a high index of suspicion of Yunis-Varón syndrome by a clinical geneticist. Frequent features found in the individuals include sparse scalp hair, protruding eyes, low-set ears, a high arched palate, and micrognatia (Table 1). Skeletal features include wide fontanelles and calvarial dysostosis, digital hypoplasia, especially of the thumbs and halluces, pelvic dysplasia with hip dislocations, and absent or hypoplastic clavicles. Affected individuals were significantly hypotonic and presented global developmental delay and often feeding and swallowing difficulties. Central nervous system anomalies in individuals 1 and 2 consisted of frontal lobe atrophy with pachygyria and hypoplasia of the corpus callosum and cerebellar vermis. In individual 3, autopsy revealed an absent olfactory bulb and tract, an atypical ventricular hamartoma, and neuronal loss with vacuolation in layers 3 and 5 of the cerebral cortex, the cerebellar dentate nucleus, and the olivary bodies. Individual 4 had agenesis of the corpus callosum, and individual 5 had neuronal loss with vacuolation in the cerebral cortex, the thalamus, subthalamic nuclei, globus pallidus, and the cerebellar dentate nucleus. Investigations for peripheral neuropathies had not been performed in part because of the absence of suggestive clinical findings, a fact that might be confounded by the severe central neurological disease in these individuals and the frequent early death. At the autopsy of individual 3, muscle histology was compatible with neurogenic atrophy. Electron microscopy of skin fibroblasts from individual 5 revealed the presence of large vacuoles as well as electron-dense inclusions (Figure 1A). Histological examination of muscle revealed variable fiber size and large vacuoles in many fibers (Figures 1B–1D). Electron microscopy of muscle detected membrane-limited vacuoles derived from sarcolemma and myofibrils, filled with amorphous, granular, or membranous material and partially degraded organelles (Figures 1E and 1F). Most vacuoles were membrane limited, with those at the fiber surface also limited by the basal lamina. Mitochondria were normal in size, number, and morphology.Table 1Clinical Features of Individuals InvestigatedIndividual Number12345678Review of the Literature (n = 17)Familial relationship and reference of first descriptionaffected siblings18Corona-Rivera J.R. Romo-Huerta C.O. López-Marure E. Ramos F.J. Estrada-Padilla S.A. Zepeda-Romero L.C. New ocular findings in two sisters with Yunis-Varón syndrome and literature review.Eur. J. Med. Genet. 2011; 54: 76-81Crossref PubMed Scopus (10) Google Scholaraffected siblings5Garrett C. Berry A.C. Simpson R.H. Hall C.M. Yunis-Varon syndrome with severe osteodysplasty.J. Med. Genet. 1990; 27: 114-121Crossref PubMed Scopus (23) Google Scholarfirst affected child8Dworzak F. Mora M. Borroni C. Cornelio F. Blasevich F. Cappellini A. Tagliavini F. Bertagnolio B. Generalized lysosomal storage in Yunis Varón syndrome.Neuromuscul. Disord. 1995; 5: 423-428Abstract Full Text PDF PubMed Scopus (15) Google Scholarfirst affected child20Reutter H. Bagci S. Müller A. Gembruch U. Geipel A. Berg C. Eggermann T. Spengler S. Bartmann P. Rudnik-Schöneborn S. Primary pulmonary hypertension, congenital heart defect, central nervous system malformations, hypo- and aplastic toes: another case of Yunis-Varón syndrome or report of a new entity.Eur. J. Med. Genet. 2012; 55: 27-31Crossref PubMed Scopus (5) Google Scholarfirst affected child, unaffected brothers7Adès L.C. Morris L.L. Richardson M. Pearson C. Haan E.A. Congenital heart malformation in Yunis-Varón syndrome.J. Med. Genet. 1993; 30: 788-792Crossref PubMed Scopus (19) Google Scholarfirst affected childOrigin and consanguinity in parentsMexican, third cousinsEnglish, nonconsanguineousItalian, nonconsanguineousGerman, nonconsanguineousAnglo-Saxon, nonconsanguineousFrench, nonconsanguineousFIG4 mutationsc.1260_1261delGT (p.Thr422Glnfs*6) from each parentmaternal mutation: c.311G>A (p.Gly104Asp); paternal mutation: c.831_838delTAAATTTG (p.Lys278Trpfs*6)c.524T>C (p.Leu175Pro) from each parentno mutation by Sangerno mutation by Sangerno mutation by SangerGenderFFMMFFMFAge at last follow-up1 yeardeath at 2 monthsdeath at 4 monthsTOPdeath at 4 months9 monthsdeath at 7 years after cardiac surgery1 monthEnlarged vacuoles demonstrated on pathological materialnot on muscle biopsyNA+ (CNS)not on bone histology+ (muscle, CNS, fibroblasts)not on muscle biopsyNANABirth weight (g)2,2002,4703,030250 at 18 weeks2,5803,0352,6303,2502,397 ± 298aData shown are mean ± SD.BW < 3rd percentile−−−−−−−−10/17 (59%)Birth length (cm)45465224 at 18 weeks4751444946 ± 2aData shown are mean ± SD.BL < 3rd percentile−−−−−−−−10/14 (71%)OFC (cm)28303318 at 18 weeks3433313532 ± 3aData shown are mean ± SD.Microcephaly++−−−−+−9/16 (56%)Head and NeckSparse scalp hair+++NA++++17/17 (100%)Wide fontanelle/sutures++++NA−−+16/16 (100%)Protruding eyes+++++−−+10/12 (83%)Anteverted nostrils+−+++−+−12/13 (92%)Short philtrum−−−NA+−−−8/8 (100%)Short upper lip++++++−+13/13 (100%)Labial-gingival retraction++−−+−+−8/10 (80%)High arched palate+++NA++−−9/10 (90%)Micrognathia+++++−++14/14 (100%)Low set/dysplastic ears++++++++18/18 (100%)Loose skin in neck+++−NA+++6/7 (86%)ThoraxCongenital heart defect++−−−+++3/15 (20%)Absent/hypoplastic nipples−−−−−−+−3/11 (27%)Genital anomalies−−+−−−+−5/13 (38%)LimbsAbsent/hypoplastic thumbs+++++−++16/17 (94%)Short pointed fingers+++++−++14/15 (93%)Absent/hypoplastic halluces+++++−−+16/16 (100%)Short pointed toes++++++++15/16 (94%)Absent/hypoplastic nails++++++++12/14 (86%)Single palmar crease++++NA−+−5/7 (71%)Central Nervous SystemStructural brain anomalies (see text)+++++++−6/12 (50%)Hypotonia+++NA++++5/5 (100%)RadiologicalCalvarial dysostosis++++NANA−−15/15 (100%)Craniofacial disproportion++++NA−−−8/8 (100%)Absent/hypoplastic clavicles++−−NA−+−13/17 (76%)Absent sternal ossification center++NANANA−−−7/7 (100%)Pelvic dysplasia+++−NA−++9/10 (90%)Hip dislocation−++NANA−+−5/8 (63%)Thumb aplasia/hypoplasia++++NA−++13/14 (93%)Agenesia/hypoplasia of distal phalanges of the fingers++++NA−++13/14 (93%)Agenesia/hypoplasia of middle phalanges of the fingers++++NA−++10/11 (91%)Number of positive features over features assessed28/3528/3426/3518/2917/2410/3523/3518/35Cases included in the review of the literature are from several researchers.1Yunis E. Varón H. Cleidocranial dysostosis, severe micrognathism, bilateral absence of thumbs and first metatarsal bone, and distal aphalangia: a new genetic syndrome.Am. J. Dis. Child. 1980; 134: 649-653Crossref PubMed Scopus (44) Google Scholar, 2Hughes H.E. Partington M.W. Brief clinical report: the syndrome of Yunis and Varón—report of a further case.Am. J. Med. Genet. 1983; 14: 539-544Crossref PubMed Scopus (15) Google Scholar, 3Pfeiffer R.A. Diekmann L. Stock H.J. Aplasia of the thumbs and great toes as the outstanding feature of Yunis and Varon syndrome. A new entity. A new observation.Ann. Genet. 1988; 31: 241-243PubMed Google Scholar, 4Hennekam R.C. Vermeulen-Meiners C. Further delineation of the Yunis-Varon syndrome.J. Med. Genet. 1989; 26: 55-58Crossref PubMed Scopus (20) Google Scholar, 5Garrett C. Berry A.C. Simpson R.H. Hall C.M. Yunis-Varon syndrome with severe osteodysplasty.J. Med. Genet. 1990; 27: 114-121Crossref PubMed Scopus (23) Google Scholar, 9Rabe H. Brune T. Rossi R. Steinhorst V. Jorch G. Horst J. Wittwer B. Yunis-Varon syndrome: the first case of German origin.Clin. Dysmorphol. 1996; 5: 217-222Crossref PubMed Google Scholar, 11Christie J. Sacks S. Decorato D. Bergasa N.V. Atrophy of the left lobe of the liver and anomalous hepatic vessel in a patient with Yunis-Varon syndrome.J. Clin. Gastroenterol. 1999; 29: 210-211Crossref PubMed Scopus (9) Google Scholar, 12Walch E. Schmidt M. Brenner R.E. Emons D. Dame C. Pontz B. Wiestler O.D. Bartmann P. Yunis-Varon syndrome: evidence for a lysosomal storage disease.Am. J. Med. Genet. 2000; 95: 157-160Crossref PubMed Scopus (21) Google Scholar, 14Sumi M. Kusumoto T. Kondoh T. Moriuchi H. Miyamoto M. Masuzaki H. Ishimaru T. A case of Yunis-Varon syndrome complicated with complete cleft lip and palate.Am. J. Med. Genet. A. 2004; 125A: 92-93Crossref PubMed Google Scholar, 15Bhatia S. Holla R.G. Yunis-Varon syndrome.Indian Pediatr. 2005; 42: 373-375PubMed Google Scholar, 16Kulkarni M.L. Vani H.N. Nagendra K. Mahesh T.K. Kumar A. Haneef S. Mohammed Z. Kulkarni P.M. Yunis Varon syndrome.Indian J. Pediatr. 2006; 73: 353-355Crossref PubMed Scopus (7) Google Scholar, 17Basel-Vanagaite L. Kornreich L. Schiller O. Yacobovich J. Merlob P. Yunis-Varon syndrome: further delineation of the phenotype.Am. J. Med. Genet. A. 2008; 146A: 532-537Crossref PubMed Scopus (13) Google Scholar, 21Partington M.W. Cardiomyopathy added to the Yunis-Varon syndrome.Proc. Greenwood Genetic Center. 1988; 7: 224-225Google Scholar For individual 4, because a termination of pregnancy was performed, several clinical parameters could not be assessed. For individual 5, skeletal radiographs were not available. Features present in individual 8 not fully captured by this table are asthma, tracheomalacia, dysphagia lusoria, ventricular septal defect, and anemia of unknown etiology. Abbreviations are as follows: TOP, termination of pregnancy; F, female; M, male; BW, birth weight; BL, birth length; OFC, occipitofrontal circumference; NA, not assessed. Plus signs (+) and minus signs (−) indicate presence and absence, respectively, of trait.a Data shown are mean ± SD. Open table in a new tab Cases included in the review of the literature are from several researchers.1Yunis E. Varón H. Cleidocranial dysostosis, severe micrognathism, bilateral absence of thumbs and first metatarsal bone, and distal aphalangia: a new genetic syndrome.Am. J. Dis. Child. 1980; 134: 649-653Crossref PubMed Scopus (44) Google Scholar, 2Hughes H.E. Partington M.W. Brief clinical report: the syndrome of Yunis and Varón—report of a further case.Am. J. Med. Genet. 1983; 14: 539-544Crossref PubMed Scopus (15) Google Scholar, 3Pfeiffer R.A. Diekmann L. Stock H.J. Aplasia of the thumbs and great toes as the outstanding feature of Yunis and Varon syndrome. A new entity. A new observation.Ann. Genet. 1988; 31: 241-243PubMed Google Scholar, 4Hennekam R.C. Vermeulen-Meiners C. Further delineation of the Yunis-Varon syndrome.J. Med. Genet. 1989; 26: 55-58Crossref PubMed Scopus (20) Google Scholar, 5Garrett C. Berry A.C. Simpson R.H. Hall C.M. Yunis-Varon syndrome with severe osteodysplasty.J. Med. Genet. 1990; 27: 114-121Crossref PubMed Scopus (23) Google Scholar, 9Rabe H. Brune T. Rossi R. Steinhorst V. Jorch G. Horst J. Wittwer B. Yunis-Varon syndrome: the first case of German origin.Clin. Dysmorphol. 1996; 5: 217-222Crossref PubMed Google Scholar, 11Christie J. Sacks S. Decorato D. Bergasa N.V. Atrophy of the left lobe of the liver and anomalous hepatic vessel in a patient with Yunis-Varon syndrome.J. Clin. Gastroenterol. 1999; 29: 210-211Crossref PubMed Scopus (9) Google Scholar, 12Walch E. Schmidt M. Brenner R.E. Emons D. Dame C. Pontz B. Wiestler O.D. Bartmann P. Yunis-Varon syndrome: evidence for a lysosomal storage disease.Am. J. Med. Genet. 2000; 95: 157-160Crossref PubMed Scopus (21) Google Scholar, 14Sumi M. Kusumoto T. Kondoh T. Moriuchi H. Miyamoto M. Masuzaki H. Ishimaru T. A case of Yunis-Varon syndrome complicated with complete cleft lip and palate.Am. J. Med. Genet. A. 2004; 125A: 92-93Crossref PubMed Google Scholar, 15Bhatia S. Holla R.G. Yunis-Varon syndrome.Indian Pediatr. 2005; 42: 373-375PubMed Google Scholar, 16Kulkarni M.L. Vani H.N. Nagendra K. Mahesh T.K. Kumar A. Haneef S. Mohammed Z. Kulkarni P.M. Yunis Varon syndrome.Indian J. Pediatr. 2006; 73: 353-355Crossref PubMed Scopus (7) Google Scholar, 17Basel-Vanagaite L. Kornreich L. Schiller O. Yacobovich J. Merlob P. Yunis-Varon syndrome: further delineation of the phenotype.Am. J. Med. Genet. A. 2008; 146A: 532-537Crossref PubMed Scopus (13) Google Scholar, 21Partington M.W. Cardiomyopathy added to the Yunis-Varon syndrome.Proc. Greenwood Genetic Center. 1988; 7: 224-225Google Scholar For individual 4, because a termination of pregnancy was performed, several clinical parameters could not be assessed. For individual 5, skeletal radiographs were not available. Features present in individual 8 not fully captured by this table are asthma, tracheomalacia, dysphagia lusoria, ventricular septal defect, and anemia of unknown etiology. Abbreviations are as follows: TOP, termination of pregnancy; F, female; M, male; BW, birth weight; BL, birth length; OFC, occipitofrontal circumference; NA, not assessed. Plus signs (+) and minus signs (−) indicate presence and absence, respectively, of trait. Candidate gene analysis for RUNX2, mutations in which cause classical cleidocranial dysplasia (MIM 119600), failed to detect mutations.22Lee B. Thirunavukkarasu K. Zhou L. Pastore L. Baldini A. Hecht J. Geoffroy V. Ducy P. Karsenty G. Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia.Nat. Genet. 1997; 16: 307-310Crossref PubMed Scopus (492) Google Scholar Exome sequencing was performed as described previously23Campeau P.M. Kim J.C. Lu J.T. Schwartzentruber J.A. Abdul-Rahman O.A. Schlaubitz S. Murdock D.M. Jiang M.M. Lammer E.J. Enns G.M. et al.Mutations in KAT6B, encoding a histone acetyltransferase, cause Genitopatellar syndrome.Am. J. Hum. Genet. 2012; 90: 282-289Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 24Campeau P.M. Lu J.T. Sule G. Jiang M.M. Bae Y. Madan S. Högler W. Shaw N.J. Mumm S. Gibbs R.A. et al.Whole-exome sequencing identifies mutations in the nucleoside transporter gene SLC29A3 in dysosteosclerosis, a form of osteopetrosis.Hum. Mol. Genet. 2012; 21: 4904-4909Crossref PubMed Scopus (72) Google Scholar on individuals 1 and 5 and the parents of individuals 3 and 4 because high-quality DNA required for exome sequencing was not available from individuals 3 and 4. Sequence analysis of genes with rare (MAF < 1%) or novel mutations yielded FIG4 (RefSeq accession number NM_014845.5) as the best candidate because the spontaneous Fig4-null mouse has multiple related phenotypes including cellular vacuolation, neurodegeneration, and dysplasia of the corpus callosum.25Chow C.Y. Zhang Y. Dowling J.J. Jin N. Adamska M. Shiga K. Szigeti K. Shy M.E. Li J. Zhang X. et al.Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J.Nature. 2007; 448: 68-72Crossref PubMed Scopus (386) Google Scholar The FIG4 genotypes of parents and affected offspring in the three families described in Table 1 are shown in Figure 2. In family 1, the parents were third cousins and both were carriers of the same 2 bp frameshift deletion, c.1260_1261delGT, which is predicted to cause protein truncation upstream of the phosphatase catalytic motif CX5RT/S (Figure 3A) and therefore complete loss of function. In family 2, the maternal allele was the missense variant p.Gly104Asp (c.311G>A) and the paternal allele was an 8 bp frameshift deletion (c.831_838delTAAATTTG). After identification of heterozygous mutations in the parents, a pathology specimen demonstrated inheritance of both mutations in individual 3. In family 3, with no reported consanguinity, both parents were heterozygous for the novel missense variant p.Leu175Pro (c.524T>C) and the affected child was homozygous for the variant allele. The missense substitutions in individual 1 (Gly104Asp) and individual 5 (p.Leu175Pro) alter amino acid residues that are highly conserved through evolution (Figure 3B). Glycine 104 is located at the beginning of β sheet 7 within the noncatalytic, protein-binding domain of FIG4 (Figure 3C).27Manford A. Xia T. Saxena A.K. Stefan C. Hu F. Emr S.D. Mao Y. Crystal structure of the yeast Sac1: implications for its phosphoinositide phosphatase function.EMBO J. 2010; 29: 1489-1498Crossref PubMed Scopus (81) Google Scholar Leucine 175 is located in α helix 2 of the same domain and appears to interact directly with residue Glu302, the site of a functionally null substitution in a individual with CMT4J26Nicholson G. Lenk G.M. Reddel S.W. Grant A.E. Towne C.F. Ferguson C.J. Simpson E. Scheuerle A. Yasick M. Hoffman S. et al.Distinctive genetic and clinical features of CMT4J: a severe neuropathy caused by mutations in the PI(3,5)P2 phosphatase FIG4.Brain. 2011; 134: 1959-1971Crossref PubMed Scopus (96) Google Scholar (Figure 3C). Four protein prediction programs assessed these two missense variants as likely to affect FIG4 function (Table S1 available online).Figure 3Location of FIG4 MutationsShow full caption(A) Location of mutations in FIG4 exons; introns not drawn to scale. Above, YVS; below, CMT4J.26Nicholson G. Lenk G.M. Reddel S.W. Grant A.E. Towne C.F. Ferguson C.J. Simpson E. Scheuerle A. Yasick M. Hoffman S. et al.Distinctive genetic and clinical features of CMT4J: a severe neuropathy caused by mutations in the PI(3,5)P2 phosphatase FIG4.Brain. 2011; 134: 1959-1971Crossref PubMed Scopus (96) Google Scholar Protein-interaction domain, blue; catalytic domain, yellow; P loop containing the catalytic CX5R(T/S) motif, orange.(B) Evolutionary conservation of amino acids around the missense mutations. Alignment was performed with ClustalW2. Dots represent identity; dashes represent gaps.(C) Positions of substitutions in the 3-dimensional protein structure (courtesy of Yuxin Mao27Manford A. Xia T. Saxena A.K. Stefan C. Hu F. Emr S.D. Mao Y. Crystal structure of the yeast Sac1: implications for its phosphoinositide phosphatase function.EMBO J. 2010; 29: 1489-1498Crossref PubMed Scopus (81) Google Scholar). Same color scheme as in (A). Abbreviations used to designate amino acids are as follows: G104, p.Gly104; L175, p.Leu175; S176, p.Ser176; S320, p.Ser320; E302, p.Glu302; and R274, p.Arg274. Gly104 is located at the beginning of β sheet 7 within the noncatalytic, protein-binding domain of FIG4.27Manford A. Xia T. Saxena A.K. Stefan C. Hu F. Emr S.D. Mao Y. Crystal structure of the yeast Sac1: implications for its phosphoinositide phosphatase function.EMBO J. 2010; 29: 1489-1498Crossref PubMed Scopus (81) Google Scholar p.Leu175 is located in α helix 2 of the same domain and appears to interact directly with residue p.Glu302, the site of a functionally null mutation in an individual with CMT4J.26Nicholson G. Lenk G.M. Reddel S.W. Grant A.E. Towne C.F. Ferguson C.J. Simpson E. Scheuerle A. Yasick M. Hoffman S. et al.Distinctive genetic and clinical features of CMT4J: a severe neuropathy caused by mutations in the PI(3,5)P2 phosphatase FIG4.Brain. 2011; 134: 195

Cortical-bone fragility is a common feature in osteoporosis that is linked to nonvertebral fractures. Regulation of cortical-bone homeostasis has proved elusive. The study of genetic disorders of the skeleton can yield insights that fuel experimental therapeutic approaches to the treatment of rare disorders and common skeletal ailments.We evaluated four patients with Pyle's disease, a genetic disorder that is characterized by cortical-bone thinning, limb deformity, and fractures; two patients were examined by means of exome sequencing, and two were examined by means of Sanger sequencing. After a candidate gene was identified, we generated a knockout mouse model that manifested the phenotype and studied the mechanisms responsible for altered bone architecture.In all affected patients, we found biallelic truncating mutations in SFRP4, the gene encoding secreted frizzled-related protein 4, a soluble Wnt inhibitor. Mice deficient in Sfrp4, like persons with Pyle's disease, have increased amounts of trabecular bone and unusually thin cortical bone, as a result of differential regulation of Wnt and bone morphogenetic protein (BMP) signaling in these two bone compartments. Treatment of Sfrp4-deficient mice with a soluble Bmp2 receptor (RAP-661) or with antibodies to sclerostin corrected the cortical-bone defect.Our study showed that Pyle's disease was caused by a deficiency of sFRP4, that cortical-bone and trabecular-bone homeostasis were governed by different mechanisms, and that sFRP4-mediated cross-regulation between Wnt and BMP signaling was critical for achieving proper cortical-bone thickness and stability. (Funded by the Swiss National Foundation and the National Institutes of Health.).

Branched-chain amino acid (BCAA) metabolism plays a central role in the pathophysiology of both rare inborn errors of metabolism and the more common multifactorial diseases. Although deficiency of the branched-chain ketoacid dehydrogenase (BCKDC) and associated elevations in the BCAAs and their ketoacids have been recognized as the cause of maple syrup urine disease (MSUD) for decades, treatment options for this disorder have been limited to dietary interventions. In recent years, the discovery of improved leucine tolerance after liver transplantation has resulted in a new therapeutic strategy for this disorder. Likewise, targeting the regulation of the BCKDC activity may be an alternative potential treatment strategy for MSUD. The regulation of the BCKDC by the branched-chain ketoacid dehydrogenase kinase has also been implicated in a new inborn error of metabolism characterized by autism, intellectual disability and seizures. Finally, there is a growing body of literature implicating BCAA metabolism in more common disorders such as the metabolic syndrome, cancer and hepatic disease. This review surveys the knowledge acquired on the topic over the past 50 years and focuses on recent developments in the field of BCAA metabolism.

VPS13 protein family members VPS13A through VPS13C have been associated with various recessive movement disorders. We describe the first disease association of rare recessive VPS13D variants including frameshift, missense, and partial duplication mutations with a novel complex, hyperkinetic neurological disorder. The clinical features include developmental delay, a childhood onset movement disorder (chorea, dystonia, or tremor), and progressive spastic ataxia or paraparesis. Characteristic brain magnetic resonance imaging shows basal ganglia or diffuse white matter T2 hyperintensities as seen in Leigh syndrome and choreoacanthocytosis. Muscle biopsy in 1 case showed mitochondrial aggregates and lipidosis, suggesting mitochondrial dysfunction. These findings underline the importance of the VPS13 complex in neurological diseases and a possible role in mitochondrial function. Ann Neurol 2018;83:1089-1095.

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2β, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we report five individuals with de novo missense substitutions in CHD4 identified through whole-exome sequencing and web-based gene matching. These individuals have overlapping phenotypes including developmental delay, intellectual disability, hearing loss, macrocephaly, distinct facial dysmorphisms, palatal abnormalities, ventriculomegaly, and hypogonadism as well as additional findings such as bone fusions. The variants, c.3380G>A (p.Arg1127Gln), c.3443G>T (p.Trp1148Leu), c.3518G>T (p.Arg1173Leu), and c.3008G>A, (p.Gly1003Asp) (GenBank: NM_001273.3), affect evolutionarily highly conserved residues and are predicted to be deleterious. Previous studies in yeast showed the equivalent Arg1127 and Trp1148 residues to be crucial for SNF2 function. Furthermore, mutations in the same positions were reported in malignant tumors, and a de novo missense substitution in an equivalent arginine residue in the C-terminal helicase domain of SMARCA4 is associated with Coffin Siris syndrome. Cell-based studies of the p.Arg1127Gln and p.Arg1173Leu mutants demonstrate normal localization to the nucleus and HDAC1 interaction. Based on these findings, the mutations potentially alter the complex activity but not its formation. This report provides evidence for the role of CHD4 in human development and expands an increasingly recognized group of Mendelian disorders involving chromatin remodeling and modification.

BackgroundDeafness, onychodystrophy, osteodystrophy, mental retardation, and seizures (DOORS) syndrome is a rare autosomal recessive disorder of unknown cause. We aimed to identify the genetic basis of this syndrome by sequencing most coding exons in affected individuals.MethodsThrough a search of available case studies and communication with collaborators, we identified families that included at least one individual with at least three of the five main features of the DOORS syndrome: deafness, onychodystrophy, osteodystrophy, intellectual disability, and seizures. Participants were recruited from 26 centres in 17 countries. Families described in this study were enrolled between Dec 1, 2010, and March 1, 2013. Collaborating physicians enrolling participants obtained clinical information and DNA samples from the affected child and both parents if possible. We did whole-exome sequencing in affected individuals as they were enrolled, until we identified a candidate gene, and Sanger sequencing to confirm mutations. We did expression studies in human fibroblasts from one individual by real-time PCR and western blot analysis, and in mouse tissues by immunohistochemistry and real-time PCR.Findings26 families were included in the study. We did exome sequencing in the first 17 enrolled families; we screened for TBC1D24 by Sanger sequencing in subsequent families. We identified TBC1D24 mutations in 11 individuals from nine families (by exome sequencing in seven families, and Sanger sequencing in two families). 18 families had individuals with all five main features of DOORS syndrome, and TBC1D24 mutations were identified in half of these families. The seizure types in individuals with TBC1D24 mutations included generalised tonic-clonic, complex partial, focal clonic, and infantile spasms. Of the 18 individuals with DOORS syndrome from 17 families without TBC1D24 mutations, eight did not have seizures and three did not have deafness. In expression studies, some mutations abrogated TBC1D24 mRNA stability. We also detected Tbc1d24 expression in mouse phalangeal chondrocytes and calvaria, which suggests a role of TBC1D24 in skeletogenesis.InterpretationOur findings suggest that mutations in TBC1D24 seem to be an important cause of DOORS syndrome and can cause diverse phenotypes. Thus, individuals with DOORS syndrome without deafness and seizures but with the other features should still be screened for TBC1D24 mutations. More information is needed to understand the cellular roles of TBC1D24 and identify the genes responsible for DOORS phenotypes in individuals who do not have a mutation in TBC1D24.FundingUS National Institutes of Health, the CIHR (Canada), the NIHR (UK), the Wellcome Trust, the Henry Smith Charity, and Action Medical Research.

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.

<h3>Objective:</h3> To evaluate the phenotypic spectrum associated with mutations in <i>TBC1D24</i>. <h3>Methods:</h3> We acquired new clinical, EEG, and neuroimaging data of 11 previously unreported and 37 published patients. <i>TBC1D24</i> mutations, identified through various sequencing methods, can be found online (http://lovd.nl/TBC1D24). <h3>Results:</h3> Forty-eight patients were included (28 men, 20 women, average age 21 years) from 30 independent families. Eighteen patients (38%) had myoclonic epilepsies. The other patients carried diagnoses of focal (25%), multifocal (2%), generalized (4%), and unclassified epilepsy (6%), and early-onset epileptic encephalopathy (25%). Most patients had drug-resistant epilepsy. We detail EEG, neuroimaging, developmental, and cognitive features, treatment responsiveness, and physical examination. In silico evaluation revealed 7 different highly conserved motifs, with the most common pathogenic mutation located in the first. Neuronal outgrowth assays showed that some <i>TBC1D24</i> mutations, associated with the most severe <i>TBC1D24</i>-associated disorders, are not necessarily the most disruptive to this gene function. <h3>Conclusions:</h3> <i>TBC1D24</i>-related epilepsy syndromes show marked phenotypic pleiotropy, with multisystem involvement and severity spectrum ranging from isolated deafness (not studied here), benign myoclonic epilepsy restricted to childhood with complete seizure control and normal intellect, to early-onset epileptic encephalopathy with severe developmental delay and early death. There is no distinct correlation with mutation type or location yet, but patterns are emerging. Given the phenotypic breadth observed, <i>TBC1D24</i> mutation screening is indicated in a wide variety of epilepsies. A <i>TBC1D24</i> consortium was formed to develop further research on this gene and its associated phenotypes.

PurposeCongenital microcephaly (CM) is an important birth defect with long term neurological sequelae. We aimed to perform detailed phenotypic and genomic analysis of patients with Mendelian forms of CM.MethodsClinical phenotyping, targeted or exome sequencing, and autozygome analysis.ResultsWe describe 150 patients (104 families) with 56 Mendelian forms of CM. Our data show little overlap with the genetic causes of postnatal microcephaly. We also show that a broad definition of primary microcephaly —as an autosomal recessive form of nonsyndromic CM with severe postnatal deceleration of occipitofrontal circumference—is highly sensitive but has a limited specificity. In addition, we expand the overlap between primary microcephaly and microcephalic primordial dwarfism both clinically (short stature in >52% of patients with primary microcephaly) and molecularly (e.g., we report the first instance of CEP135-related microcephalic primordial dwarfism). We expand the allelic and locus heterogeneity of CM by reporting 37 novel likely disease-causing variants in 27 disease genes, confirming the candidacy of ANKLE2, YARS, FRMD4A, and THG1L, and proposing the candidacy of BPTF, MAP1B, CCNH, and PPFIBP1.ConclusionOur study refines the phenotype of CM, expands its genetics heterogeneity, and informs the workup of children born with this developmental brain defect.

Pathogenic variants in ARID1B are one of the most frequent causes of intellectual disability (ID) as determined by large-scale exome sequencing studies. Most studies published thus far describe clinically diagnosed Coffin-Siris patients (ARID1B-CSS) and it is unclear whether these data are representative for patients identified through sequencing of unbiased ID cohorts (ARID1B-ID). We therefore sought to determine genotypic and phenotypic differences between ARID1B-ID and ARID1B-CSS. In parallel, we investigated the effect of different methods of phenotype reporting.Clinicians entered clinical data in an extensive web-based survey.79 ARID1B-CSS and 64 ARID1B-ID patients were included. CSS-associated dysmorphic features, such as thick eyebrows, long eyelashes, thick alae nasi, long and/or broad philtrum, small nails and small or absent fifth distal phalanx and hypertrichosis, were observed significantly more often (p < 0.001) in ARID1B-CSS patients. No other significant differences were identified.There are only minor differences between ARID1B-ID and ARID1B-CSS patients. ARID1B-related disorders seem to consist of a spectrum, and patients should be managed similarly. We demonstrated that data collection methods without an explicit option to report the absence of a feature (such as most Human Phenotype Ontology-based methods) tended to underestimate gene-related features.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

Abstract Coffin–Siris and Nicolaides–Baraitser syndromes (CSS and NCBRS) are Mendelian disorders caused by mutations in subunits of the BAF chromatin remodeling complex. We report overlapping peripheral blood DNA methylation epi-signatures in individuals with various subtypes of CSS ( ARID1B , SMARCB1 , and SMARCA4 ) and NCBRS ( SMARCA2 ). We demonstrate that the degree of similarity in the epi-signatures of some CSS subtypes and NCBRS can be greater than that within CSS, indicating a link in the functional basis of the two syndromes. We show that chromosome 6q25 microdeletion syndrome, harboring ARID1B deletions, exhibits a similar CSS/NCBRS methylation profile. Specificity of this epi-signature was confirmed across a wide range of neurodevelopmental conditions including other chromatin remodeling and epigenetic machinery disorders. We demonstrate that a machine-learning model trained on this DNA methylation profile can resolve ambiguous clinical cases, reclassify those with variants of unknown significance, and identify previously undiagnosed subjects through targeted population screening.

Osteocytes are the terminally differentiated cell type of the osteoblastic lineage and have important functions in skeletal homeostasis. Although the transcriptional regulation of osteoblast differentiation has been well characterized, the factors that regulate differentiation of osteocytes from mature osteoblasts are poorly understood. Here we show that miR-23a∼27a∼24-2 (miR-23a cluster) promotes osteocyte differentiation. Osteoblast-specific miR-23a cluster gain-of-function mice have low bone mass associated with decreased osteoblast but increased osteocyte numbers. By contrast, loss-of-function transgenic mice overexpressing microRNA decoys for either miR-23a or miR-27a, but not miR24-2, show decreased osteocyte numbers. Moreover, RNA-sequencing analysis shows altered transforming growth factor-β (TGF-β) signalling. Prdm16, a negative regulator of the TGF-β pathway, is directly repressed by miR-27a with concomitant alteration of sclerostin expression, and pharmacological inhibition of TGF-β rescues the phenotypes observed in the gain-of-function transgenic mice. Taken together, the miR-23a cluster regulates osteocyte differentiation by modulating the TGF-β signalling pathway through targeting of Prdm16.

It is estimated that 0.5% of all mammalian proteins have a glycosylphosphatidylinositol (GPI)‐anchor. GPI‐anchored proteins (GPI‐APs) play key roles, particularly in embryogenesis, neurogenesis, immune response and signal transduction. Due to their involvement in many pathways and developmental events, defects in the genes involved in their synthesis and processing can result in a variety of genetic disorders for which affected individuals display a wide spectrum of features. We compiled the clinical characteristics of 202 individuals with mutations in the GPI biosynthesis and processing pathway through a review of the literature. This review has allowed us to compare the characteristics and the severity of the phenotypes associated with different genes as well as highlight features that are prominent for each. Certain combinations, such as seizures with aplastic/hypoplastic nails or abnormal alkaline phosphatase levels suggest an inherited GPI deficiency, and our review of all clinical findings may orient the management of inherited GPI deficiencies.

Arthrogryposis is a clinical finding that is present either as a feature of a neuromuscular condition or as part of a systemic disease in over 400 Mendelian conditions. The underlying molecular etiology remains largely unknown because of genetic and phenotypic heterogeneity. We applied exome sequencing (ES) in a cohort of 89 families with the clinical sign of arthrogryposis. Additional molecular techniques including array comparative genomic hybridization (aCGH) and Droplet Digital PCR (ddPCR) were performed on individuals who were found to have pathogenic copy number variants (CNVs) and mosaicism, respectively. A molecular diagnosis was established in 65.2% (58/89) of families. Eleven out of 58 families (19.0%) showed evidence for potential involvement of pathogenic variation at more than one locus, probably driven by absence of heterozygosity (AOH) burden due to identity-by-descent (IBD). RYR3, MYOM2, ERGIC1, SPTBN4, and ABCA7 represent genes, identified in two or more families, for which mutations are probably causative for arthrogryposis. We also provide evidence for the involvement of CNVs in the etiology of arthrogryposis and for the idea that both mono-allelic and bi-allelic variants in the same gene cause either similar or distinct syndromes. We were able to identify the molecular etiology in nine out of 20 families who underwent reanalysis. In summary, our data from family-based ES further delineate the molecular etiology of arthrogryposis, yielded several candidate disease-associated genes, and provide evidence for mutational burden in a biological pathway or network. Our study also highlights the importance of reanalysis of individuals with unsolved diagnoses in conjunction with sequencing extended family members.

Down syndrome is the most common cause of cognitive impairment and presents clinically with universally recognizable signs and symptoms. In this study, we focus on exam findings and digital facial analysis technology in individuals with Down syndrome in diverse populations. Photos and clinical information were collected on 65 individuals from 13 countries, 56.9% were male and the average age was 6.6 years (range 1 month to 26 years; SD = 6.6 years). Subjective findings showed that clinical features were different across ethnicities (Africans, Asians, and Latin Americans), including brachycephaly, ear anomalies, clinodactyly, sandal gap, and abundant neck skin, which were all significantly less frequent in Africans ( P &lt; 0.001, P &lt; 0.001, P &lt; 0.001, P &lt; 0.05, and P &lt; 0.05, respectively). Evaluation using a digital facial analysis technology of a larger diverse cohort of newborns to adults (n = 129 cases; n = 132 controls) was able to diagnose Down syndrome with a sensitivity of 0.961, specificity of 0.924, and accuracy of 0.943. Only the angles at medial canthus and ala of the nose were common significant findings amongst different ethnicities (Caucasians, Africans, and Asians) when compared to ethnically matched controls. The Asian group had the least number of significant digital facial biometrics at 4, compared to Caucasians at 8 and Africans at 7. In conclusion, this study displays the wide variety of findings across different geographic populations in Down syndrome and demonstrates the accuracy and promise of digital facial analysis technology in the diagnosis of Down syndrome internationally. © 2016 Wiley Periodicals, Inc.

Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Symptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome. Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Symptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as “GENE-related phenotype descriptor” (e.g., “CFTR-related cystic fibrosis”). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

Abstract PIDD1 encodes p53-Induced Death Domain protein 1, which acts as a sensor surveilling centrosome numbers and p53 activity in mammalian cells. Early results also suggest a role in DNA damage response where PIDD1 may act as a cell-fate switch, through interaction with RIP1 and NEMO/IKKg, activating NF-κB signaling for survival, or as an apoptosis-inducing protein by activating caspase-2. Biallelic truncating mutations in CRADD—the protein bridging PIDD1 and caspase-2—have been reported in intellectual disability (ID), and in a form of lissencephaly. Here, we identified five families with ID from Iran, Pakistan, and India, with four different biallelic mutations in PIDD1 , all disrupting the Death Domain (DD), through which PIDD1 interacts with CRADD or RIP1. Nonsense mutations Gln863* and Arg637* directly disrupt the DD, as does a missense mutation, Arg815Trp. A homozygous splice mutation in the fifth family is predicted to disrupt splicing upstream of the DD, as confirmed using an exon trap. In HEK293 cells, we show that both Gln863* and Arg815Trp mutants fail to co-localize with CRADD, leading to its aggregation and mis-localization, and fail to co-precipitate CRADD. Using genome-edited cell lines, we show that these three PIDD1 mutations all cause loss of PIDDosome function. Pidd1 null mice show decreased anxiety, but no motor abnormalities. Together this indicates that PIDD1 mutations in humans may cause ID (and possibly lissencephaly) either through gain of function or secondarily, due to altered scaffolding properties, while complete loss of PIDD1, as modeled in mice, may be well tolerated or is compensated for.

We identified individuals with variations in ACTL6B, a component of the chromatin remodeling machinery including the BAF complex. Ten individuals harbored bi-allelic mutations and presented with global developmental delay, epileptic encephalopathy, and spasticity, and ten individuals with de novo heterozygous mutations displayed intellectual disability, ambulation deficits, severe language impairment, hypotonia, Rett-like stereotypies, and minor facial dysmorphisms (wide mouth, diastema, bulbous nose). Nine of these ten unrelated individuals had the identical de novo c.1027G>A (p.Gly343Arg) mutation. Human-derived neurons were generated that recaptured ACTL6B expression patterns in development from progenitor cell to post-mitotic neuron, validating the use of this model. Engineered knock-out of ACTL6B in wild-type human neurons resulted in profound deficits in dendrite development, a result recapitulated in two individuals with different bi-allelic mutations, and reversed on clonal genetic repair or exogenous expression of ACTL6B. Whole-transcriptome analyses and whole-genomic profiling of the BAF complex in wild-type and bi-allelic mutant ACTL6B neural progenitor cells and neurons revealed increased genomic binding of the BAF complex in ACTL6B mutants, with corresponding transcriptional changes in several genes including TPPP and FSCN1, suggesting that altered regulation of some cytoskeletal genes contribute to altered dendrite development. Assessment of bi-alleic and heterozygous ACTL6B mutations on an ACTL6B knock-out human background demonstrated that bi-allelic mutations mimic engineered deletion deficits while heterozygous mutations do not, suggesting that the former are loss of function and the latter are gain of function. These results reveal a role for ACTL6B in neurodevelopment and implicate another component of chromatin remodeling machinery in brain disease.

Two lysine acyltransferases govern histone H3 propionylation at lysine 23 in normal and pathological conditions.

Sifrim-Hitz-Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants inCHD4. In this study, we investigated the clinical spectrum of the disorder, genotype-phenotype correlations, and the effect of different missense variants on CHD4 function.We collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains.The majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype-phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains.The CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.

Achondroplasia, the most common skeletal dysplasia, is characterized by a variety of medical, functional and psychosocial challenges across the lifespan. The condition is caused by a common, recurring, gain-of-function mutation in FGFR3, the gene that encodes fibroblast growth factor receptor 3. This mutation leads to impaired endochondral ossification of the human skeleton. The clinical and radiographic hallmarks of achondroplasia make accurate diagnosis possible in most patients. However, marked variability exists in the clinical care pathways and protocols practised by clinicians who manage children and adults with this condition. A group of 55 international experts from 16 countries and 5 continents have developed consensus statements and recommendations that aim to capture the key challenges and optimal management of achondroplasia across each major life stage and sub-specialty area, using a modified Delphi process. The primary purpose of this first International Consensus Statement is to facilitate the improvement and standardization of care for children and adults with achondroplasia worldwide in order to optimize their clinical outcomes and quality of life.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

An expanding range of genetic syndromes are characterized by genome-wide disruptions in DNA methylation profiles referred to as episignatures. Episignatures are distinct, highly sensitive, and specific biomarkers that have recently been applied in clinical diagnosis of genetic syndromes. Episignatures are contained within the broader disorder-specific genome-wide DNA methylation changes, which can share significant overlap among different conditions. In this study, we performed functional genomic assessment and comparison of disorder-specific and overlapping genome-wide DNA methylation changes related to 65 genetic syndromes with previously described episignatures. We demonstrate evidence of disorder-specific and recurring genome-wide differentially methylated probes (DMPs) and regions (DMRs). The overall distribution of DMPs and DMRs across the majority of the neurodevelopmental genetic syndromes analyzed showed substantial enrichment in gene promoters and CpG islands, and under-representation of the more variable intergenic regions. Analysis showed significant enrichment of the DMPs and DMRs in gene pathways and processes related to neurodevelopment, including neurogenesis, synaptic signaling and synaptic transmission. This study expands beyond the diagnostic utility of DNA methylation episignatures by demonstrating correlation between the function of the mutated genes and the consequent genomic DNA methylation profiles as a key functional element in the molecular etiology of genetic neurodevelopmental disorders.

KCNJ8 (NM_004982) encodes the pore forming subunit of one of the ATP-sensitive inwardly rectifying potassium (KATP) channels. KCNJ8 sequence variations are traditionally associated with J-wave syndromes, involving ventricular fibrillation and sudden cardiac death. Recently, the KATP gene ABCC9 (SUR2, NM_020297) has been associated with the multi-organ disorder Cantú syndrome or hypertrichotic osteochondrodysplasia (MIM 239850) (hypertrichosis, macrosomia, osteochondrodysplasia, and cardiomegaly). Here, we report on a patient with a de novo nonsynonymous KCNJ8 SNV (p.V65M) and Cantú syndrome, who tested negative for mutations in ABCC9. The genotype and multi-organ abnormalities of this patient are reviewed. A careful screening of the KATP genes should be performed in all individuals diagnosed with Cantú syndrome and no mutation in ABCC9.

Gaucher disease causes pathologic skeletal changes that are not fully explained. Considering the important role of mesenchymal stromal cells (MSCs) in bone structural development and maintenance, we analyzed the cellular biochemistry of MSCs from an adult patient with Gaucher disease type 1 (N370S/L444P mutations). Gaucher MSCs possessed a low glucocerebrosidase activity and consequently had a 3-fold increase in cellular glucosylceramide. Gaucher MSCs have a typical MSC marker phenotype, normal osteocytic and adipocytic differentiation, growth, exogenous lactosylceramide trafficking, cholesterol content, lysosomal morphology, and total lysosomal content, and a marked increase in COX-2, prostaglandin E2, interleukin-8, and CCL2 production compared with normal controls. Transcriptome analysis on normal MSCs treated with the glucocerebrosidase inhibitor conduritol B epoxide showed an up-regulation of an array of inflammatory mediators, including CCL2, and other differentially regulated pathways. These cells also showed a decrease in sphingosine-1-phosphate. In conclusion, Gaucher disease MSCs display an altered secretome that could contribute to skeletal disease and immune disease manifestations in a manner distinct and additive to Gaucher macrophages themselves.

Newly cost-effective next-generation sequencing has led to an explosion of discoveries of novel genetic mutations that reveal the rampant “promiscuity” of genotype–phenotype relationships. Such discoveries should ultimately revolutionize clinical care.

Genitopatellar syndrome (GPS) and Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) have both recently been shown to be caused by distinct mutations in the histone acetyltransferase KAT6B (a.k.a. MYST4/MORF). All variants are de novo dominant mutations that lead to protein truncation. Mutations leading to GPS occur in the proximal portion of the last exon and lead to the expression of a protein without a C-terminal domain. Mutations leading to SBBYSS occur either throughout the gene, leading to nonsense-mediated decay, or more distally in the last exon. Features present only in GPS are contractures, anomalies of the spine, ribs and pelvis, renal cysts, hydronephrosis, and agenesis of the corpus callosum. Features present only in SBBYSS include long thumbs and long great toes and lacrimal duct abnormalities. Several features occur in both, such as intellectual disability, congenital heart defects, and genital and patellar anomalies. We propose that haploinsufficiency or loss of a function mediated by the C-terminal domain causes the common features, whereas gain-of-function activities would explain the features unique to GPS. Further molecular studies and the compilation of mutations in a database for genotype-phenotype correlations (www.LOVD.nl/KAT6B) might help tease out answers to these questions and understand the developmental programs dysregulated by the different truncations.

Argininosuccinate lyase (ASL) is required for the synthesis and channeling of L-arginine to nitric oxide synthase (NOS) for nitric oxide (NO) production. Congenital ASL deficiency causes argininosuccinic aciduria (ASA), the second most common urea-cycle disorder, and leads to deficiency of both ureagenesis and NO production. Subjects with ASA have been reported to develop long-term complications such as hypertension and neurocognitive deficits despite early initiation of therapy and the absence of documented hyperammonemia. In order to distinguish the relative contributions of the hepatic urea-cycle defect from those of the NO deficiency to the phenotype, we performed liver-directed gene therapy in a mouse model of ASA. Whereas the gene therapy corrected the ureagenesis defect, the systemic hypertension in mice could be corrected by treatment with an exogenous NO source. In an ASA subject with severe hypertension refractory to antihypertensive medications, monotherapy with NO supplements resulted in the long-term control of hypertension and a decrease in cardiac hypertrophy. In addition, the NO therapy was associated with an improvement in some neuropsychological parameters pertaining to verbal memory and nonverbal problem solving. Our data show that ASA, in addition to being a classical urea-cycle disorder, is also a model of congenital human NO deficiency and that ASA subjects could potentially benefit from NO supplementation. Hence, NO supplementation should be investigated for the long-term treatment of this condition.

SMC1A encodes one of the proteins of the cohesin complex. SMC1A variants are known to cause a phenotype resembling Cornelia de Lange syndrome (CdLS). Exome sequencing has allowed recognizing SMC1A variants in individuals with encephalopathy with epilepsy who do not resemble CdLS. We performed an international, interdisciplinary study on 51 individuals with SMC1A variants for physical and behavioral characteristics, and compare results to those in 67 individuals with NIPBL variants. For the Netherlands all known individuals with SMC1A variants were studied, both with and without CdLS phenotype. Individuals with SMC1A variants can resemble CdLS, but manifestations are less marked compared to individuals with NIPBL variants: growth is less disturbed, facial signs are less marked (except for periocular signs and thin upper vermillion), there are no major limb anomalies, and they have a higher level of cognitive and adaptive functioning. Self‐injurious behavior is more frequent and more severe in the NIPBL group. In the Dutch group 5 of 13 individuals (all females) had a phenotype that shows a remarkable resemblance to Rett syndrome: epileptic encephalopathy, severe or profound intellectual disability, stereotypic movements, and (in some) regression. Their missense, nonsense, and frameshift mutations are evenly spread over the gene. We conclude that SMC1A variants can result in a phenotype resembling CdLS and a phenotype resembling Rett syndrome. Resemblances between the SMC1A group and the NIPBL group suggest that a disturbed cohesin function contributes to the phenotype, but differences between these groups may also be explained by other underlying mechanisms such as moonlighting of the cohesin genes.

We report ten individuals of four independent consanguineous families from Turkey, India, Libya, and Pakistan with a variable clinical phenotype that comprises arthrogryposis, spontaneously resolving respiratory insufficiency at birth, muscular atrophy predominantly of the distal lower limbs, scoliosis, and mild distal sensory involvement. Using whole-exome sequencing, SNPchip-based linkage analysis, DNA microarray, and Sanger sequencing, we identified three independent homozygous frameshift mutations and a homozygous deletion of two exons in PIEZO2 that segregated in all affected individuals of the respective family. The mutations are localized in the N-terminal and central region of the gene, leading to nonsense-mediated transcript decay and consequently to lack of PIEZO2 protein. In contrast, heterozygous gain-of-function missense mutations, mainly localized at the C terminus, cause dominant distal arthrogryposis 3 (DA3), distal arthrogryposis 5 (DA5), or Marden-Walker syndrome (MWKS), which encompass contractures of hands and feet, scoliosis, ophthalmoplegia, and ptosis. PIEZO2 encodes a mechanosensitive ion channel that plays a major role in light-touch mechanosensation and has recently been identified as the principal mechanotransduction channel for proprioception. Mice ubiquitously depleted of PIEZO2 are postnatally lethal. However, individuals lacking PIEZO2 develop a not life-threatening, slowly progressive disorder, which is likely due to loss of PIEZO2 protein in afferent neurons leading to disturbed proprioception causing aberrant muscle development and function. Here we report a recessively inherited PIEZO2-related disease and demonstrate that depending on the type of mutation and the mode of inheritance, PIEZO2 causes clinically distinguishable phenotypes.

Intraflagellar transport ( IFT ) is vital for the functioning of primary cilia. Defects in several components of IFT complexes cause a spectrum of ciliopathies with variable involvement of skeleton, brain, eyes, ectoderm and kidneys. We examined a child from a consanguineous family who had short stature, narrow thorax, short hands and feet, postaxial polydactyly of hands, pigmentary retinopathy, small teeth and skeletal dysplasia. The clinical phenotype of the child shows significant overlap with cranioectodermal dysplasia type I (Sensenbrenner syndrome). Whole‐exome sequencing revealed a homozygous nonsense variant p. R142 * in IFT52 encoding an IFT ‐B core complex protein as the probable cause of her condition. This is the first report of a human disease associated with IFT52 .

Noonan syndrome (NS) is a common genetic syndrome associated with gain of function variants in genes in the Ras/MAPK pathway. The phenotype of NS has been well characterized in populations of European descent with less attention given to other groups. In this study, individuals from diverse populations with NS were evaluated clinically and by facial analysis technology. Clinical data and images from 125 individuals with NS were obtained from 20 countries with an average age of 8 years and female composition of 46%. Individuals were grouped into categories of African descent (African), Asian, Latin American, and additional/other. Across these different population groups, NS was phenotypically similar with only 2 of 21 clinical elements showing a statistically significant difference. The most common clinical characteristics found in all population groups included widely spaced eyes and low‐set ears in 80% or greater of participants, short stature in more than 70%, and pulmonary stenosis in roughly half of study individuals. Using facial analysis technology, we compared 161 Caucasian, African, Asian, and Latin American individuals with NS with 161 gender and age matched controls and found that sensitivity was equal to or greater than 94% for all groups, and specificity was equal to or greater than 90%. In summary, we present consistent clinical findings from global populations with NS and additionally demonstrate how facial analysis technology can support clinicians in making accurate NS diagnoses. This work will assist in earlier detection and in increasing recognition of NS throughout the world.

ABSTRACT In a large cohort of osteogenesis imperfecta type V (OI type V) patients (17 individuals from 12 families), we identified the same mutation in the 5′ untranslated region (5′UTR) of the interferon-induced transmembrane protein 5 (IFITM5) gene by whole exome and Sanger sequencing (IFITM5 c.–14C &amp;gt; T) and provide a detailed description of their phenotype. This mutation leads to the creation of a novel start codon adding five residues to IFITM5 and was recently reported in several other OI type V families. The variability of the phenotype was quite large even within families. Whereas some patients presented with the typical calcification of the forearm interosseous membrane, radial head dislocation and hyperplastic callus (HPC) formation following fractures, others had only some of the typical OI type V findings. Thirteen had calcification of interosseous membranes, 14 had radial head dislocations, 10 had HPC, 9 had long bone bowing, 11 could ambulate without assistance, and 1 had mild unilateral mixed hearing loss. The bone mineral density varied greatly, even within families. Our study thus highlights the phenotypic variability of OI type V caused by the IFITM5 mutation.

Autosomal recessive cutis laxa type 2B (ARCL2B; OMIM # 612940) is a segmental progeroid disorder caused by mutations in PYCR1 encoding pyrroline-5-carboxylate reductase 1, which is part of the conserved proline de novo synthesis pathway. Here we describe 33 patients with PYCR1-related ARCL from 27 families with initial diagnoses varying between wrinkly skin syndrome, gerodermia osteodysplastica, De Barsy syndrome or more severe progeria syndromes. Given the difficult differential diagnosis of ARCL syndromes we performed a systematic comparison of clinical features of PYCR1-related ARCL. Intrauterine growth retardation, a characteristic triangular facial gestalt, psychomotor retardation, and hypotonia were the most relevant distinctive hallmarks of ARCL due to proline de novo synthesis defects. Corneal clouding or cataracts, athetoid movements, and finger contractures were rather rare features, but had a high predictive value. In our cohort we identified 20 different PYCR1 mutations of which seven were novel. Most of the mutations accumulated in exons 4 to 6. Missense alterations of highly conserved residues were most frequent followed by splice site changes and a single nonsense mutation. Analysis of genotype-phenotype correlation revealed that patients with mutations in the first two exons had lower average clinical scores and absent or only mild intellectual disability. Structural analyses predicted interference with PYCR1 multimerization for a subset of missense mutations. These findings have implications for the clinics as well as the pathomechanism of PYCR1-related ARCL.

From a GeneMatcher-enabled international collaboration, we identified ten individuals affected by intellectual disability, speech delay, ataxia, and facial dysmorphism and carrying a deleterious EBF3 variant detected by whole-exome sequencing. One 9-bp duplication and one splice-site, five missense, and two nonsense variants in EBF3 were found; the mutations occurred de novo in eight individuals, and the missense variant c.625C>T (p.Arg209Trp) was inherited by two affected siblings from their healthy mother, who is mosaic. EBF3 belongs to the early B cell factor family (also known as Olf, COE, or O/E) and is a transcription factor involved in neuronal differentiation and maturation. Structural assessment predicted that the five amino acid substitutions have damaging effects on DNA binding of EBF3. Transient expression of EBF3 mutant proteins in HEK293T cells revealed mislocalization of all but one mutant in the cytoplasm, as well as nuclear localization. By transactivation assays, all EBF3 mutants showed significantly reduced or no ability to activate transcription of the reporter gene CDKN1A, and in situ subcellular fractionation experiments demonstrated that EBF3 mutant proteins were less tightly associated with chromatin. Finally, in RNA-seq and ChIP-seq experiments, EBF3 acted as a transcriptional regulator, and mutant EBF3 had reduced genome-wide DNA binding and gene-regulatory activity. Our findings demonstrate that variants disrupting EBF3-mediated transcriptional regulation cause intellectual disability and developmental delay and are present in ∼0.1% of individuals with unexplained neurodevelopmental disorders.

SMARCC2 (BAF170) is one of the invariable core subunits of the ATP-dependent chromatin remodeling BAF (BRG1-associated factor) complex and plays a crucial role in embryogenesis and corticogenesis. Pathogenic variants in genes encoding other components of the BAF complex have been associated with intellectual disability syndromes. Despite its significant biological role, variants in SMARCC2 have not been directly associated with human disease previously. Using whole-exome sequencing and a web-based gene-matching program, we identified 15 individuals with variable degrees of neurodevelopmental delay and growth retardation harboring one of 13 heterozygous variants in SMARCC2, most of them novel and proven de novo. The clinical presentation overlaps with intellectual disability syndromes associated with other BAF subunits, such as Coffin-Siris and Nicolaides-Baraitser syndromes and includes prominent speech impairment, hypotonia, feeding difficulties, behavioral abnormalities, and dysmorphic features such as hypertrichosis, thick eyebrows, thin upper lip vermilion, and upturned nose. Nine out of the fifteen individuals harbor variants in the highly conserved SMARCC2 DNA-interacting domains (SANT and SWIRM) and present with a more severe phenotype. Two of these individuals present cardiac abnormalities. Transcriptomic analysis of fibroblasts from affected individuals highlights a group of differentially expressed genes with possible roles in regulation of neuronal development and function, namely H19, SCRG1, RELN, and CACNB4. Our findings suggest a novel SMARCC2-related syndrome that overlaps with neurodevelopmental disorders associated with variants in BAF-complex subunits.

Osteogenesis imperfecta (OI) is the most common skeletal dysplasia that predisposes to recurrent fractures and bone deformities. In spite of significant advances in understanding the genetic basis of OI, there have been no large-scale natural history studies. To better understand the natural history and improve the care of patients, a network of Linked Clinical Research Centers (LCRC) was established. Subjects with OI were enrolled in a longitudinal study, and in this report, we present cross-sectional data on the largest cohort of OI subjects (n = 544). OI type III subjects had higher prevalence of dentinogenesis imperfecta, severe scoliosis, and long bone deformities as compared to those with OI types I and IV. Whereas the mean lumbar spine area bone mineral density (LS aBMD) was low across all OI subtypes, those with more severe forms had lower bone mass. Molecular testing may help predict the subtype in type I collagen-related OI. Analysis of such well-collected and unbiased data in OI can not only help answering questions that are relevant to patient care but also foster hypothesis-driven research, especially in the context of 'phenotypic expansion' driven by next-generation sequencing.

Spondyloepimetaphyseal dysplasias (SEMD) are a group of genetically heterogeneous skeletal disorders characterized by abnormal vertebral bodies and epimetaphyseal abnormalities. We investigated two families with a new SEMD type with one proband each. They showed mild facial dysmorphism, flat vertebral bodies (platyspondyly), large epiphyses, metaphyseal dysplasia, and hallux valgus as common clinical features. By trio-exome sequencing, the homozygous missense variant c.797G>A/p.(Cys266Tyr) in PISD was found in both affected individuals. Based on exome data analyses for homozygous regions, the two patients shared a single homozygous block on chromosome 22 including PISD, indicating their remote consanguinity. PISD encodes phosphatidylserine (PS) decarboxylase that is localized in the inner mitochondrial membrane and catalyzes the decarboxylation of PS to phosphatidylethanolamine (PE) in mammalian cells. PE occurs at high abundance in mitochondrial membranes. Patient-derived fibroblasts showed fragmented mitochondrial morphology. Treatment of patient cells with MG-132 or staurosporine to induce activation of the intrinsic apoptosis pathway revealed significantly decreased cell viability with increased caspase-3 and caspase-7 activation. Remarkably, ethanolamine (Etn) supplementation largely restored cell viability and enhanced apoptosis in MG-132-stressed patient cells. Our data demonstrate that the biallelic hypomorphic PISD variant p.(Cys266Tyr) is associated with a novel SEMD form, which may be treatable with Etn administration.

<h2>Abstract</h2><h3>Purpose</h3> Fetal anomalies represent a poorly studied group of developmental disorders. Our objective was to assess the impact of whole-exome sequencing (WES) on the investigation of these anomalies. <h3>Methods</h3> We performed WES in 101 fetuses or stillborns who presented prenatally with severe anomalies, including renal a/dysgenesis, VACTERL association (vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula, renal anomalies, and limb abnormalities), brain anomalies, suspected ciliopathies, multiple major malformations, and akinesia. <h3>Results</h3> A molecular diagnosis was obtained in 19 cases (19%). In 13 of these cases, the diagnosis was not initially suspected by the clinicians because the phenotype was nonspecific or atypical, corresponding in some cases to the severe end of the spectrum of a known disease (e.g., <i>MNX1-</i>, <i>RYR1-</i>, or <i>TUBB-</i>related disorders). In addition, we identified likely pathogenic variants in genes (<i>DSTYK</i>, <i>ACTB</i>, and <i>HIVEP2</i>) previously associated with phenotypes that were substantially different from those found in our cases. Finally, we identified variants in novel candidate genes that were associated with perinatal lethality, including de novo mutations in <i>GREB1L</i> in two cases with bilateral renal agenesis, which represents a significant enrichment of such mutations in our cohort. <h3>Conclusion</h3> Our study opens a window on the distinctive genetic landscape associated with fetal anomalies and highlights the power—but also the challenges—of WES in prenatal diagnosis.

Williams-Beuren syndrome (WBS) is a common microdeletion syndrome characterized by a 1.5Mb deletion in 7q11.23. The phenotype of WBS has been well described in populations of European descent with not as much attention given to other ethnicities. In this study, individuals with WBS from diverse populations were assessed clinically and by facial analysis technology. Clinical data and images from 137 individuals with WBS were found in 19 countries with an average age of 11 years and female gender of 45%. The most common clinical phenotype elements were periorbital fullness and intellectual disability which were present in greater than 90% of our cohort. Additionally, 75% or greater of all individuals with WBS had malar flattening, long philtrum, wide mouth, and small jaw. Using facial analysis technology, we compared 286 Asian, African, Caucasian, and Latin American individuals with WBS with 286 gender and age matched controls and found that the accuracy to discriminate between WBS and controls was 0.90 when the entire cohort was evaluated concurrently. The test accuracy of the facial recognition technology increased significantly when the cohort was analyzed by specific ethnic population (P-value < 0.001 for all comparisons), with accuracies for Caucasian, African, Asian, and Latin American groups of 0.92, 0.96, 0.92, and 0.93, respectively. In summary, we present consistent clinical findings from global populations with WBS and demonstrate how facial analysis technology can support clinicians in making accurate WBS diagnoses.

The iron-sulfur (Fe-S) cluster (ISC) biogenesis pathway is indispensable for many fundamental biological processes and pathogenic variations in genes encoding several components of the Fe-S biogenesis machinery, such as NFU1, BOLA3, IBA57 and ISCA2 are already implicated in causing four types of multiple mitochondrial dysfunctions syndromes (MMDS). We report on two unrelated families, with two affected children each with early onset neurological deterioration, seizures, extensive white matter abnormalities, cortical migrational abnormalities, lactic acidosis and early demise. Exome sequencing of two affected individuals, one from each family, revealed a homozygous c.259G>A [p.(Glu87Lys)] variant in ISCA1 and Mendelian segregation was confirmed in both families. The ISCA1 variant lies in the only shared region of homozygosity between the two families suggesting the possibility of a founder effect. In silico functional analyses and structural modeling of the protein predict the identified ISCA1 variant to be detrimental to protein stability and function. Notably the phenotype observed in all affected subjects with the ISCA1 pathogenic variant is similar to that previously described in all four types of MMDS. Our findings suggest association of a pathogenic variant in ISCA1 with another MMDS.

Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs∗102] and c.920delG [p.Gly307Alafs∗11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system.

Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX dehydratase (NAXD) is essential for intracellular repair of NAD(P)HX. Here we present a series of infants and children who suffered episodes of febrile illness-induced neurodegeneration or cardiac failure and early death. Whole-exome or whole-genome sequencing identified recessive NAXD variants in each case. Variants were predicted to be potentially deleterious through in silico analysis. Reverse-transcription PCR confirmed altered splicing in one case. Subject fibroblasts showed highly elevated concentrations of the damaged cofactors S-NADHX, R-NADHX and cyclic NADHX. NADHX accumulation was abrogated by lentiviral transduction of subject cells with wild-type NAXD. Subject fibroblasts and muscle biopsies showed impaired mitochondrial function, higher sensitivity to metabolic stress in media containing galactose and azide, but not glucose, and decreased mitochondrial reactive oxygen species production. Recombinant NAXD protein harbouring two missense variants leading to the amino acid changes p.(Gly63Ser) and p.(Arg608Cys) were thermolabile and showed a decrease in Vmax and increase in KM for the ATP-dependent NADHX dehydratase activity. This is the first study to identify pathogenic variants in NAXD and to link deficient NADHX repair with mitochondrial dysfunction. The results show that NAXD deficiency can be classified as a metabolite repair disorder in which accumulation of damaged metabolites likely triggers devastating effects in tissues such as the brain and the heart, eventually leading to early childhood death.

Variants in IQSEC2, escaping X inactivation, cause X-linked intellectual disability with frequent epilepsy in males and females. We aimed to investigate sex-specific differences.We collected the data of 37 unpublished patients (18 males and 19 females) with IQSEC2 pathogenic variants and 5 individuals with variants of unknown significance and reviewed published variants. We compared variant types and phenotypes in males and females and performed an analysis of IQSEC2 isoforms.IQSEC2 pathogenic variants mainly led to premature truncation and were scattered throughout the longest brain-specific isoform, encoding the synaptic IQSEC2/BRAG1 protein. Variants occurred de novo in females but were either de novo (2/3) or inherited (1/3) in males, with missense variants being predominantly inherited. Developmental delay and intellectual disability were overall more severe in males than in females. Likewise, seizures were more frequently observed and intractable, and started earlier in males than in females. No correlation was observed between the age at seizure onset and severity of intellectual disability or resistance to antiepileptic treatments.This study provides a comprehensive overview of IQSEC2-related encephalopathy in males and females, and suggests that an accurate dosage of IQSEC2 at the synapse is crucial during normal brain development.

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

Cornelia de Lange syndrome (CdLS) is a dominant multisystemic malformation syndrome due to mutations in five genes— NIPBL , SMC1A , HDAC8 , SMC3 , and RAD21 . The characteristic facial dysmorphisms include microcephaly, arched eyebrows, synophrys, short nose with depressed bridge and anteverted nares, long philtrum, thin lips, micrognathia, and hypertrichosis. Most affected individuals have intellectual disability, growth deficiency, and upper limb anomalies. This study looked at individuals from diverse populations with both clinical and molecularly confirmed diagnoses of CdLS by facial analysis technology. Clinical data and images from 246 individuals with CdLS were obtained from 15 countries. This cohort included 49% female patients and ages ranged from infancy to 37 years. Individuals were grouped into ancestry categories of African descent, Asian, Latin American, Middle Eastern, and Caucasian. Across these populations, 14 features showed a statistically significant difference. The most common facial features found in all ancestry groups included synophrys, short nose with anteverted nares, and a long philtrum with thin vermillion of the upper lip. Using facial analysis technology we compared 246 individuals with CdLS to 246 gender/age matched controls and found that sensitivity was equal or greater than 95% for all groups. Specificity was equal or greater than 91%. In conclusion, we present consistent clinical findings from global populations with CdLS while demonstrating how facial analysis technology can be a tool to support accurate diagnoses in the clinical setting. This work, along with prior studies in this arena, will assist in earlier detection, recognition, and treatment of CdLS worldwide.

Epigenetic integrity is critical for many eukaryotic cellular processes. An important question is how different epigenetic regulators control development and influence disease. Lysine acetyltransferase 8 (KAT8) is critical for acetylation of histone H4 at lysine 16 (H4K16), an evolutionarily conserved epigenetic mark. It is unclear what roles KAT8 plays in cerebral development and human disease. Here, we report that cerebrum-specific knockout mice displayed cerebral hypoplasia in the neocortex and hippocampus, along with improper neural stem and progenitor cell (NSPC) development. Mutant cerebrocortical neuroepithelia exhibited faulty proliferation, aberrant neurogenesis, massive apoptosis, and scant H4K16 propionylation. Mutant NSPCs formed poor neurospheres, and pharmacological KAT8 inhibition abolished neurosphere formation. Moreover, we describe KAT8 variants in 9 patients with intellectual disability, seizures, autism, dysmorphisms, and other anomalies. The variants altered chromobarrel and catalytic domains of KAT8, thereby impairing nucleosomal H4K16 acetylation. Valproate was effective for treating epilepsy in at least 2 of the individuals. This study uncovers a critical role of KAT8 in cerebral and NSPC development, identifies 9 individuals with KAT8 variants, and links deficient H4K16 acylation directly to intellectual disability, epilepsy, and other developmental anomalies.

Mucopolysaccharidoses (MPS) are monogenic metabolic disorders that significantly affect the skeleton. Eleven enzyme defects in the lysosomal degradation of glycosaminoglycans (GAGs) have been assigned to the known MPS subtypes (I-IX). Arylsulfatase K (ARSK) is a recently characterised lysosomal hydrolase involved in GAG degradation that removes the 2-O-sulfate group from 2-sulfoglucuronate. Knockout of Arsk in mice was consistent with mild storage pathology, but no human phenotype has yet been described.In this study, we report four affected individuals of two unrelated consanguineous families with homozygous variants c.250C>T, p.(Arg84Cys) and c.560T>A, p.(Leu187Ter) in ARSK, respectively. Functional consequences of the two ARSK variants were assessed by mutation-specific ARSK constructs derived by site-directed mutagenesis, which were ectopically expressed in HT1080 cells. Urinary GAG excretion was analysed by dimethylene blue and electrophoresis, as well as liquid chromatography/mass spectrometry (LC-MS)/MS analysis.The phenotypes of the affected individuals include MPS features, such as short stature, coarse facial features and dysostosis multiplex. Reverse phenotyping in two of the four individuals revealed additional cardiac and ophthalmological abnormalities. Mild elevation of dermatan sulfate was detected in the two subjects investigated by LC-MS/MS. Human HT1080 cells expressing the ARSK-Leu187Ter construct exhibited absent protein levels by western blot, and cells with the ARSK-Arg84Cys construct showed markedly reduced enzyme activity in an ARSK-specific enzymatic assay against 2-O-sulfoglucuronate-containing disaccharides as analysed by C18-reversed-phase chromatography followed by MS.Our work provides a detailed clinical and molecular characterisation of a novel subtype of mucopolysaccharidosis, which we suggest to designate subtype X.

Abstract Progressive pseudorheumatoid dysplasia (PPD) is a progressive skeletal syndrome characterized by stiffness, swelling and pain in multiple joints with associated osteoporosis in affected patients. Radiographically, the predominant features resemble a spondyloepiphyseal dysplasia. Mutations in the WISP3 gene are known to cause this autosomal recessive condition. To date, only a limited number of studies have looked into the spectrum of mutations causing PPD. We report on clinical features and WISP3 mutations in a large series of Indian patients with this rare skeletal dysplasia. Families with at least one member showing clinical and radiologic features of PPD were recruited for the study. Symptoms, signs and radiographic findings were documented in 35 patients from 25 unrelated families. Swelling of small joints of hands and contractures are the most common presenting features. Mutation analysis was carried out by bidirectional sequencing of the WISP3 gene in all 35 patients. We summarize the clinical features of 35 patients with PPD and report on 11 different homozygous mutations and one instance of compound heterozygosity. Eight (c.233G&gt;A, c.340T&gt;C, c.348C&gt;A, c.433T&gt;C, c.682T&gt;C, c.802T&gt;G, c.947_951delAATTT, and c.1010G&gt;A) are novel mutations and three (c.156C&gt;A, c.248G&gt;A, and c.739_740delTG) have been reported previously. One missense mutation (c.1010G&gt;A; p.Cys337Tyr) appears to be the most common in our population being seen in 10 unrelated families. This is the largest cohort of patients with PPD in the literature and the first report from India on mutation analysis of WISP3 . We also review all the mutations reported in WISP3 till date. © 2012 Wiley Periodicals, Inc.

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, ( S )-α-chloro-phenylpropionic acid [( S )-CPP]. Crystal structures of the BDK-( S )-CPP complex show that ( S )-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of ( S )-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure.

Laurin‐Sandrow syndrome ( LSS ) is a rare autosomal dominant disorder characterized by polysyndactyly of hands and/or feet, mirror image duplication of the feet, nasal defects, and loss of identity between fibula and tibia. The genetic basis of LSS is currently unknown. LSS shows phenotypic overlap with Haas‐type polysyndactyly ( HTS ) regarding the digital phenotype. Here we report on five unrelated families with overlapping microduplications encompassing the Sonic hedgehog ( SHH ) limb enhancer ZPA regulatory sequence ( ZRS ) on chromosome 7q36. Clinically, the patients show polysyndactyly phenotypes and various types of lower limb malformations ranging from syndactyly to mirror image polydactyly with duplications of the fibulae. We show that larger duplications of the ZRS region (&gt;80 kb) are associated with HTS , whereas smaller duplications (&lt;80 kb) result in the LSS phenotype. On the basis of our data, the latter can be clearly distinguished from HTS by the presence of mirror image polysyndactyly of the feet with duplication of the fibula. Our results expand the clinical phenotype of the ZRS ‐associated syndromes and suggest that smaller duplications (&lt;80 kb) are associated with a more severe phenotype. In addition, we show that these small microduplications within the ZRS region are the underlying genetic cause of Laurin‐Sandrow syndrome.

Shprintzen-Goldberg syndrome (SGS) is a rare, systemic connective tissue disorder characterized by craniofacial, skeletal, and cardiovascular manifestations that show a significant overlap with the features observed in the Marfan (MFS) and Loeys-Dietz syndrome (LDS). A distinguishing observation in SGS patients is the presence of intellectual disability, although not all patients in this series present this finding. Recently, SGS was shown to be due to mutations in the SKI gene, encoding the oncoprotein SKI, a repressor of TGFβ activity. Here, we report eight recurrent and three novel SKI mutations in eleven SGS patients. All were heterozygous missense mutations located in the R-SMAD binding domain, except for one novel in-frame deletion affecting the DHD domain. Adding our new findings to the existing data clearly reveals a mutational hotspot, with 73% (24 out of 33) of the hitherto described unrelated patients having mutations in a stretch of five SKI residues (from p.(Ser31) to p.(Pro35)). This implicates that the initial molecular testing could be focused on mutation analysis of the first half of exon 1 of SKI. As the majority of the known mutations are located in the R-SMAD binding domain of SKI, our study further emphasizes the importance of TGFβ signaling in the pathogenesis of SGS.

To achieve an efficient molecular diagnosis of osteogenesis imperfecta (OI), Ehlers–Danlos syndrome (EDS), and osteopetrosis (OPT), we designed a next-generation sequencing (NGS) platform to sequence 34 genes. We validated this platform on known cases and have successfully identified the causative mutation in most patients without a prior molecular diagnosis. Osteogenesis imperfecta, Ehlers–Danlos syndrome, and osteopetrosis are collectively common inherited skeletal diseases. Evaluation of subjects with these conditions often includes molecular testing which has important counseling and therapeutic and sometimes legal implications. Since several different genes have been implicated in these conditions, Sanger sequencing of each gene can be a prohibitively expensive and time-consuming way to reach a molecular diagnosis. In order to circumvent these problems, we have designed and tested a NGS platform that would allow simultaneous sequencing on a single diagnostic platform of different genes implicated in OI, OPT, EDS, and other inherited conditions, leading to low or high bone mineral density. We used a liquid-phase probe library that captures 602 exons (~100 kb) of 34 selected genes and have applied it to test clinical samples from patients with bone disorders. NGS of the captured exons by Illumina HiSeq 2000 resulted in an average coverage of over 900X. The platform was successfully validated by identifying mutations in six patients with known mutations. Moreover, in four patients with OI or OPT without a prior molecular diagnosis, the assay was able to detect the causative mutations. In conclusion, our NGS panel provides a fast and accurate method to arrive at a molecular diagnosis in most patients with inherited high or low bone mineral density disorders.

Shohat-type spondyloepimetaphyseal dysplasia (SEMD) is a skeletal dysplasia that affects cartilage development. Similar skeletal disorders, such as spondyloepiphyseal dysplasias, are linked to mutations in type II collagen (COL2A1), but the causative gene in SEMD is not known. Here, we have performed whole-exome sequencing to identify a recurrent homozygous c.408+1G>A donor splice site loss-of-function mutation in DDRGK domain containing 1 (DDRGK1) in 4 families affected by SEMD. In zebrafish, ddrgk1 deficiency disrupted craniofacial cartilage development and led to decreased levels of the chondrogenic master transcription factor sox9 and its downstream target, col2a1. Overexpression of sox9 rescued the zebrafish chondrogenic and craniofacial phenotype generated by ddrgk1 knockdown, thus identifying DDRGK1 as a regulator of SOX9. Consistent with these results, Ddrgk1-/- mice displayed delayed limb bud chondrogenic condensation, decreased SOX9 protein expression and Col2a1 transcript levels, and increased apoptosis. Furthermore, we determined that DDRGK1 can directly bind to SOX9 to inhibit its ubiquitination and proteasomal degradation. Taken together, these data indicate that loss of DDRGK1 decreases SOX9 expression and causes a human skeletal dysplasia, identifying a mechanism that regulates chondrogenesis via modulation of SOX9 ubiquitination.

Inherited GPI deficiencies (IGDs) are a subset of congenital disorders of glycosylation that are increasingly recognized as a result of advances in whole-exome sequencing (WES) and whole-genome sequencing (WGS). IGDs cause a series of overlapping phenotypes consisting of seizures, dysmorphic features, multiple congenital malformations, and severe intellectual disability. We present a study of six individuals from three unrelated families in which WES or WGS identified bi-allelic phosphatidylinositol glycan class S (PIGS) biosynthesis mutations. Phenotypes included severe global developmental delay, seizures (partly responding to pyridoxine), hypotonia, weakness, ataxia, and dysmorphic facial features. Two of them had compound-heterozygous variants c.108G>A (p.Trp36∗) and c.101T>C (p.Leu34Pro), and two siblings of another family were homozygous for a deletion and insertion leading to p.Thr439_Lys451delinsArgLeuLeu. The third family had two fetuses with multiple joint contractures consistent with fetal akinesia. They were compound heterozygous for c.923A>G (p.Glu308Gly) and c.468+1G>C, a splicing mutation. Flow-cytometry analyses demonstrated that the individuals with PIGS mutations show a GPI-AP deficiency profile. Expression of the p.Trp36∗ variant in PIGS-deficient HEK293 cells revealed only partial restoration of cell-surface GPI-APs. In terms of both biochemistry and phenotype, loss of function of PIGS shares features with PIGT deficiency and other IGDs. This study contributes to the understanding of the GPI-AP biosynthesis pathway by describing the consequences of PIGS disruption in humans and extending the family of IGDs.

Glycosaminoglycans (GAG) are long, unbranched heteropolymers with repeating disaccharide units that make up the carbohydrate moiety of proteoglycans. Six distinct classes of GAGs are recognized. Their synthesis follows one of three biosynthetic pathways, depending on the type of oligosaccharide linker they contain. Chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin sulfate contain a common tetrasaccharide linker that is O-linked to specific serine residues in core proteins. Keratan sulfate can contain three different linkers, either N-linked to asparagine or O-linked to serine/threonine residues in core proteins. Finally, hyaluronic acid does not contain a linker and is not covalently attached to a core protein. Most inborn errors of GAG biosynthesis are reported in small numbers of patients. To date, in 20 diseases, convincing evidence for pathogenicity has been presented for mutations in a total of 16 genes encoding glycosyltransferases, sulfotransferases, epimerases or transporters. GAG synthesis defects should be suspected in patients with a combination of characteristic clinical features in more than one connective tissue compartment: bone and cartilage (short long bones with or without scoliosis), ligaments (joint laxity/dislocations), and subepithelial (skin, sclerae). Some produce distinct clinical syndromes. The commonest laboratory tests used for this group of diseases are analysis of GAGs, enzyme assays, and molecular testing. In principle, GAG analysis has potential as a general first-line diagnostic test for GAG biosynthesis disorders.

<h3>Background</h3> Of our 1400 exome-studied patients, 67% originate from consanguineous families. ∼80% suffer from variable degree of intellectual disability (ID). The search for disease causing genes using homozygosity mapping was progressing slowly until 2010, then markedly accelerated by the introduction of exome analysis. <h3>Objectives</h3> To identify the disease causing mutation(s) in three patients from two unrelated families who suffered from global developmental delay, severe ID and drug-responsive seizure disorder. <h3>Methods</h3> Exome analysis was performed in DNA of the three patients. The identified <i>PIGC</i> variants were generated and transfected into PIGC-defective mouse cells and the restoration of the surface expression of mouse CD90, CD48 and FLAER was assessed using flow cytometry. The expression of these proteins was also studied on the surface of patients9 leucocytes. <h3>Results</h3> Three <i>PIGC</i> mutations were identified; homozygous p.L189W in one family and compound heterozygosity for p.L212P/p.R21X variants in another. <i>PIGC</i> participates in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor which tethers proteins to plasma membrane. In cells lacking PIGC protein, which were transfected with each of the PIGC variants, we detected a clear reduction of surface expression of GPI-anchored proteins. Furthermore, analyses of patients9 leucocytes showed significant and constant decrease of CD16 surface expression in granulocytes, and moderate decrease of CD14, CD55, CD59 and FLAER levels. <h3>Conclusions</h3> <i>PIGC</i> joins the list of genes in which mutations result in defective biosynthesis of GPI anchoring, manifesting by global developmental delay and seizure disorder. The lack of specific biomarker dictates exome sequencing as the diagnostic procedure of choice in similar patients.

Zimmermann-Laband syndrome (ZLS) is characterized by coarse facial features with gingival enlargement, intellectual disability (ID), hypertrichosis, and hypoplasia or aplasia of nails and terminal phalanges. De novo missense mutations in KCNH1 and KCNK4, encoding K+ channels, have been identified in subjects with ZLS and ZLS-like phenotype, respectively. We report de novo missense variants in KCNN3 in three individuals with typical clinical features of ZLS. KCNN3 (SK3/KCa2.3) constitutes one of three members of the small-conductance Ca2+-activated K+ (SK) channels that are part of a multiprotein complex consisting of the pore-forming channel subunits, the constitutively bound Ca2+ sensor calmodulin, protein kinase CK2, and protein phosphatase 2A. CK2 modulates Ca2+ sensitivity of the channels by phosphorylating SK-bound calmodulin. Patch-clamp whole-cell recordings of KCNN3 channel-expressing CHO cells demonstrated that disease-associated mutations result in gain of function of the mutant channels, characterized by increased Ca2+ sensitivity leading to faster and more complete activation of KCNN3 mutant channels. Pretreatment of cells with the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole revealed basal inhibition of wild-type and mutant KCNN3 channels by CK2. Analogous experiments with the KCNN3 p.Val450Leu mutant previously identified in a family with portal hypertension indicated basal constitutive channel activity and thus a different gain-of-function mechanism compared to the ZLS-associated mutant channels. With the report on de novo KCNK4 mutations in subjects with facial dysmorphism, hypertrichosis, epilepsy, ID, and gingival overgrowth, we propose to combine the phenotypes caused by mutations in KCNH1, KCNK4, and KCNN3 in a group of neurological potassium channelopathies caused by an increase in K+ conductance.

Rothmund-Thomson syndrome (RTS) is an autosomal-recessive disorder characterized by poikiloderma, sparse hair, short stature, and skeletal anomalies. Type 2 RTS, which is defined by the presence of bi-allelic mutations in RECQL4, is characterized by increased cancer susceptibility and skeletal anomalies, whereas the genetic basis of RTS type 1, which is associated with juvenile cataracts, is unknown. We studied ten individuals, from seven families, who had RTS type 1 and identified a deep intronic splicing mutation of the ANAPC1 gene, a component of the anaphase-promoting complex/cyclosome (APC/C), in all affected individuals, either in the homozygous state or in trans with another mutation. Fibroblast studies showed that the intronic mutation causes the activation of a 95 bp pseudoexon, leading to mRNAs with premature termination codons and nonsense-mediated decay, decreased ANAPC1 protein levels, and prolongation of interphase. Interestingly, mice that were heterozygous for a knockout mutation have an increased incidence of cataracts. Our results demonstrate that deficiency in the APC/C is a cause of RTS type 1 and suggest a possible link between the APC/C and RECQL4 helicase because both proteins are involved in DNA repair and replication.

Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of “corner fractures” at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone. Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of “corner fractures” at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone. Spondylometaphyseal dysplasias (SMDs), or bone dysplasias affecting the spine and growth plates, comprise a heterogeneous group of conditions from both a clinical and genetic perspective. Genetic mutations have been identified for several SMDs (in COL2A1 [MIM: 120140], TRPV4 [MIM: 605427], SBDS [MIM: 607444], GPX4 [MIM: 138322], PCYT1A [MIM: 123695], and ACP5 [MIM: 171640]), but rarer forms still escape molecular diagnosis.1Machol K. Jain M. Almannai M. Orand T. Lu J.T. Tran A. Chen Y. Schlesinger A. Gibbs R. Bonafe L. et al.Corner fracture type spondylometaphyseal dysplasia: Overlap with type II collagenopathies.Am. J. Med. Genet. A. 2017; 173: 733-739Crossref PubMed Scopus (6) Google Scholar One such condition is SMD with “corner fractures” (MIM: 184255). First recognized by Sutcliffe in 1966, fewer than 25 individuals or families have been reported.2Sutcliffe J. Metaphyseal dysostosis.Ann. Radiol. (Paris). 1966; 9: R215-R223Google Scholar These individuals generally show developmental coxa vara but no scoliosis (as was the case for several individuals in our cohort without FN1 [MIM: 135600] mutations; see Table S1).3Currarino G. Birch J.G. Herring J.A. Developmental coxa vara associated with spondylometaphyseal dysplasia (DCV/SMD): “SMD-corner fracture type” (DCV/SMD-CF) demonstrated in most reported cases.Pediatr. Radiol. 2000; 30: 14-24Crossref PubMed Scopus (19) Google Scholar, 4Langer Jr., L.O. Brill P.W. Ozonoff M.B. Pauli R.M. Wilson W.G. Alford B.A. Pavlov H. Drake D.G. Spondylometaphyseal dysplasia, corner fracture type: a heritable condition associated with coxa vara.Radiology. 1990; 175: 761-766Crossref PubMed Scopus (33) Google Scholar At the edges of the irregular metaphyses, flake-like, triangular, or curvilinear ossification centers simulate fractures. This specifically affects the distal tibia, the distal radius (ulnar aspect), the proximal humerus, and the proximal femur. The so-called “corner fractures” are unlikely to be true fractures but instead represent irregular ossification at the growth plates and secondary ossification centers.3Currarino G. Birch J.G. Herring J.A. Developmental coxa vara associated with spondylometaphyseal dysplasia (DCV/SMD): “SMD-corner fracture type” (DCV/SMD-CF) demonstrated in most reported cases.Pediatr. Radiol. 2000; 30: 14-24Crossref PubMed Scopus (19) Google Scholar These fractures tend to become larger in older children and disappear after growth has stopped. Corner fractures on radiographs can also be seen in Duetting-type SMD (or SMD type A4 [MIM: 609052]),5Duetting T. Schulze A. Troeger J. Spranger J. A rare form of spondylometaphyseal dysplasia-type A4.Am. J. Med. Genet. 1998; 78: 61-66Crossref PubMed Scopus (7) Google Scholar Schmid (MIM: 156500) and Jansen (MIM: 156400) types of metaphyseal chondrodysplasia, Strudwick-type spondyloepimetaphyseal dysplasia (MIM: 184250),6Walter K. Tansek M. Tobias E.S. Ikegawa S. Coucke P. Hyland J. Mortier G. Iwaya T. Nishimura G. Superti-Furga A. Unger S. COL2A1-related skeletal dysplasias with predominant metaphyseal involvement.Am. J. Med. Genet. A. 2007; 143A: 161-167Crossref PubMed Scopus (30) Google Scholar Blount disease (MIM: 188700), Menkes disease (MIM: 309400), nonaccidental injury, congenital contractures, rickets, and scurvy.7Kozlowski K. Beighton P. Gamut index of skeletal dysplasias: an aid to radiodiagnosis. Springer-Verlag, 1984Crossref Google Scholar Some individuals initially thought to have SMD with corner fractures were later identified to have type 2 collagenopathy.6Walter K. Tansek M. Tobias E.S. Ikegawa S. Coucke P. Hyland J. Mortier G. Iwaya T. Nishimura G. Superti-Furga A. Unger S. COL2A1-related skeletal dysplasias with predominant metaphyseal involvement.Am. J. Med. Genet. A. 2007; 143A: 161-167Crossref PubMed Scopus (30) Google Scholar Fibronectin is found in the human body in both a soluble form (∼300 μg/mL in plasma) and an insoluble form as a principal component of the fibrillar extracellular matrix (ECM) of virtually all tissues.8Mosher D.F. Plasma fibronectin concentration: a risk factor for arterial thrombosis?.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1193-1195Crossref PubMed Scopus (36) Google Scholar, 9Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (616) Google Scholar Fibronectin contains binding sites for integrins, collagens, glycoproteins, and glycosaminoglycans, as well as self-association sites.10Pankov R. Yamada K.M. Fibronectin at a glance.J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1465) Google Scholar It self-assembles in a cell-dependent manner upon binding to integrins and other cell-surface components9Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (616) Google Scholar and initiates the assembly of the ECM.11Sabatier L. Chen D. Fagotto-Kaufmann C. Hubmacher D. McKee M.D. Annis D.S. Mosher D.F. Reinhardt D.P. Fibrillin assembly requires fibronectin.Mol. Biol. Cell. 2009; 20: 846-858Crossref PubMed Scopus (179) Google Scholar, 12Sottile J. Hocking D.C. Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions.Mol. Biol. Cell. 2002; 13: 3546-3559Crossref PubMed Scopus (448) Google Scholar, 13Dallas S.L. Sivakumar P. Jones C.J. Chen Q. Peters D.M. Mosher D.F. Humphries M.J. Kielty C.M. Fibronectin regulates latent transforming growth factor-beta (TGF beta) by controlling matrix assembly of latent TGF beta-binding protein-1.J. Biol. Chem. 2005; 280: 18871-18880Crossref PubMed Scopus (238) Google Scholar We performed exome sequencing in individuals with SMD with corner fractures, who had been identified through the Texas Children’s Skeletal Dysplasia Program, International Skeletal Dysplasia Registry, Baylor-Hopkins Center for Mendelian Genetics, Skeldys emailing list of the International Skeletal Dysplasia Society (ISDS), Shriners Canada Skeletal Dysplasia Clinic, and existing collaborations. Families provided written informed consent for protocols approved by the institutional review board at Baylor College of Medicine or local institutions. The procedures followed were in accordance with the ethical standards of the relevant committees on human experimentation. Details on the exome sequencing libraries and alignment are presented in Table S2. In two of the first few individuals sequenced, who have been previously reported, COL2A1 mutations were identified.1Machol K. Jain M. Almannai M. Orand T. Lu J.T. Tran A. Chen Y. Schlesinger A. Gibbs R. Bonafe L. et al.Corner fracture type spondylometaphyseal dysplasia: Overlap with type II collagenopathies.Am. J. Med. Genet. A. 2017; 173: 733-739Crossref PubMed Scopus (6) Google Scholar In total, 13 individuals were exome sequenced, and a comparison of the rare or undescribed variants shared between these individuals revealed variants in FN1 in three of these affected individuals (two variants segregating with the disease, c.260G>T [p.Cys87Phe] [GenBank: NM_212482.2 and NP_997647.1, respectively] in family 1 and c.718T>G [p.Tyr240Asp] in family 4, and a de novo variant, c.2425_2427del [p.Thr809del] in family 6). The genetic cause of SMD is still undetermined for the other individuals. Subsequently, through the ISDS emailing list, the GeneMatcher tool, and the Shriners Canada Skeletal Dysplasia Clinic,14Sobreira N. Schiettecatte F. Valle D. Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 928-930Crossref PubMed Scopus (830) Google Scholar four other individuals with de novo FN1 variants (c.367T>C [p.Cys123Arg] in families 2 and 7, c.675C>G [p.Cys225Trp] in family 3, and c.778T>G [p.Cys260Gly] in family 5) and SMD with corner fractures were identified. In one of them (individual 3, identified with the ISDS emailing list), only FN1 was sequenced on the basis of the initial findings. Thus, in this cohort, previously unreported FN1 variants were found in 7 of 16 families affected by SMD with corner fractures. The clinical phenotypes of the individuals with FN1 variants are detailed in Table 1, and the variants are presented in Table 2. Figure 1 shows pedigrees, Figure 2 shows photographs, Figure 3 shows selected radiographs, and Figure S1 shows additional radiographs.Table 1Clinical FeaturesFamily 1Family 2Family 3Family 4Family 5Family 6Family 7SMD Corner-Fracture Type or Sutcliffe Type (from Currarino et al.;3Currarino G. Birch J.G. Herring J.A. Developmental coxa vara associated with spondylometaphyseal dysplasia (DCV/SMD): “SMD-corner fracture type” (DCV/SMD-CF) demonstrated in most reported cases.Pediatr. Radiol. 2000; 30: 14-24Crossref PubMed Scopus (19) Google Scholar n = 18)Individual in pedigreeII-2 (mother)III-1 (first child)III-2 (second child)II-1II-1III-2 (child)II-1II-1II-1NAGenderfemalemalemalemalefemalefemalefemalefemalefemaleNAAge at last assessment29 years13 years9 years14 years3 years, 9 months2 years, 1 month5 years, 9 months16 years, 11 months4 yearsNAHeight (cm)147 (−3 SD)113 (−5.7 SD)107 (−4.6 SD)136 (−3.38 SD)92 (−2.5 SD)83.7 (−0.9 SD)97 (−3.3 SD)137 (−4.66 SD)89 (<3.0 SD)NAOvoid vertebral bodiesNA+−−+−+++16/18Scoliosis+ (operated)+ (operated)+ (operated)+−−+ (operated)++1/18Developmental coxa vara−−−−−+−++18/18Irregular metaphysesNA++++++++16/18“Corner fractures”−++++++++15/18Knee anomalies−−genu varum (operated)−genu varum−genu varum (operated)−genu varumone genu varum, one genum valgumChest or rib anomaly (e.g., pectus)pectus carinatumpectus carinatumpectus carinatum−NA−pectus carinatum−−NAOtherhip surgery at 18 years, pregnancy-induced hypertensionborn at term, weight 1,673 g (−4.3 SD), length 38 cm (−2.7 SD), hyponatremia at 1 month (unknown cause), hypoplasia of T12 vertebra and triangular S1, back and leg painborn at term, weight 1,729 g (−3.9 SD), length 39 cm (−2.6 SD), leg painfacial asymmetry and dysmorphisms (dysplastic left ear), missing teeth 34 and 44 (island of compact bone instead), intradural lipoma and megacisterna magna on MRIborn at 33 weeks, weight 1,754 gfemoral roddingborn at 36 weeks, weight 2,060 g (3rd–10th percentile), length 43.5 cm (3rd–10th percentile), OFC 29.5 cm (3rd percentile), normal renal sonography and kidney function, no signs of proteinuria or microalbuminuria, leg painborn at 39 weeks, weight 2,280 g (<3rd percentile), length 45 cm (<3rd percentile), OFC 32 (3rd percentile), hip surgery at 2 years (both sides), multiple corrections to right hip and femur, shortening of right leg by 5 cm, bicuspid aortic valvenormal renal function, no proteinuria, no facial asymmetry or dysmorphismsNAThe following abbreviations are used: OFC, occipitofrontal circumference; and NA, not available. Open table in a new tab Table 2Genetic Description of the FN1 VariantsFamilyGenomic Change (hg19)Coding Change (GenBank: )aAll variants are absent from the ExAC Browser.Protein Change (GenBank: )Inheritance1chr2: g.216299436C>Ac.260G>Tp.Cys87Phedominant2chr2: g.216298095A>Gc.367T>Cp.Cys123Argde novo3chr2: g.216295448G>Cc.675C>Gp.Cys225Trpde novo4chr2: g.216293029A>Cc.718T>Gp.Tyr240Aspdominant5chr2: g.216292969A>Cc.778T>Gp.Cys260Glyde novo6chr2: g.216273022_216273024delc.2425_2427delp.Thr809delde novo7chr2: g.216298095A>Gc.367T>Cp.Cys123Argde novoa All variants are absent from the ExAC Browser. Open table in a new tab Figure 2Photographs of Some of the Individuals with FN1 MutationsShow full caption(A) Photographs in a figure reproduced with permission from Sutton et al.30Sutton V.R. Hyland J.C. Phillips W.A. Schlesinger A.E. Brill P.W. A dominantly inherited spondylometaphyseal dysplasia with “corner fractures” and congenital scoliosis.Am. J. Med. Genet. A. 2005; 133A: 209-212Crossref PubMed Scopus (4) Google Scholar (copyright © 2005 Wiley-Liss, Inc.). On the left are the three affected individuals who have an FN1 mutation and the maternal grandmother. In the middle is the older child at age 5 years, and on the right is the younger child at age 3 years.(B) Affected child from family 2 at age 13 years. Note the short trunk, facial asymmetry, dysplastic left ear, and normal hands and feet.(C) Affected child from family 5 at age 2 years and 11 months. Note the scoliosis, genu varum, and normal hands and feet.(D) Affected child from family 6 at age 8 years. Note the short trunk and scoliotic posture.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Radiographs Showing the “Corner Fractures” and Other Radiological ChangesShow full caption(A) Individual from family 7; note the significant scoliosis.(B) Individual from family 3; note the absence of coxa vara, the presence of irregular metaphyses with corner fractures, and the presence of ovoid vertebral bodies.Additional radiographs from all families are available in Figure S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The following abbreviations are used: OFC, occipitofrontal circumference; and NA, not available. (A) Photographs in a figure reproduced with permission from Sutton et al.30Sutton V.R. Hyland J.C. Phillips W.A. Schlesinger A.E. Brill P.W. A dominantly inherited spondylometaphyseal dysplasia with “corner fractures” and congenital scoliosis.Am. J. Med. Genet. A. 2005; 133A: 209-212Crossref PubMed Scopus (4) Google Scholar (copyright © 2005 Wiley-Liss, Inc.). On the left are the three affected individuals who have an FN1 mutation and the maternal grandmother. In the middle is the older child at age 5 years, and on the right is the younger child at age 3 years. (B) Affected child from family 2 at age 13 years. Note the short trunk, facial asymmetry, dysplastic left ear, and normal hands and feet. (C) Affected child from family 5 at age 2 years and 11 months. Note the scoliosis, genu varum, and normal hands and feet. (D) Affected child from family 6 at age 8 years. Note the short trunk and scoliotic posture. (A) Individual from family 7; note the significant scoliosis. (B) Individual from family 3; note the absence of coxa vara, the presence of irregular metaphyses with corner fractures, and the presence of ovoid vertebral bodies. Additional radiographs from all families are available in Figure S1. All FN1 variants discovered in this study are absent from the ExAC Browser and affect highly conserved residues (Figure 4). The majority (4/6) affect cysteine residues, all of which form disulfide bonds that are critical for the three-dimensional structure of the type I fibronectin domains.15Baron M. Norman D. Willis A. Campbell I.D. Structure of the fibronectin type 1 module.Nature. 1990; 345: 642-646Crossref PubMed Scopus (92) Google Scholar Five of the mutations (c.260G>T [p.Cys87Phe], c.367T>C [p.Cys123Arg], c.675C>G [p.Cys225Trp], c.718T>G [p.Tyr240Asp], and c.778T>G [p.Cys260Gly]) are located in the N-terminal assembly domain (spanning fibronectin domains I-1 through I-5), which is important to initiating assembly on the cell surface.9Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (616) Google Scholar The remaining mutation (c.2425_2427del [p.Thr809del]) was found in the III-2 domain, which contains a fibronectin binding site and is involved in conformational changes promoting fibronectin assembly.9Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (616) Google Scholar The Tyr240 residue has previously been mutated into a Ser residue in vitro and shown to be critical for fibronectin binding to fibroblasts.16Sottile J. Schwarzbauer J. Selegue J. Mosher D.F. Five type I modules of fibronectin form a functional unit that binds to fibroblasts and Staphylococcus aureus.J. Biol. Chem. 1991; 266: 12840-12843Abstract Full Text PDF PubMed Google Scholar Heterozygous FN1 mutations have previously been reported in type 2 autosomal-dominant glomerulopathy with fibronectin deposits (MIM: 601894).17Ohtsubo H. Okada T. Nozu K. Takaoka Y. Shono A. Asanuma K. Zhang L. Nakanishi K. Taniguchi-Ikeda M. Kaito H. et al.Identification of mutations in FN1 leading to glomerulopathy with fibronectin deposits.Pediatr. Nephrol. 2016; 31: 1459-1467Crossref PubMed Scopus (22) Google Scholar Remarkably, all FN1 mutations previously implicated in this glomerulopathy cluster in more C-terminally-located regions important for heparin binding and integrin binding (Figure 4).17Ohtsubo H. Okada T. Nozu K. Takaoka Y. Shono A. Asanuma K. Zhang L. Nakanishi K. Taniguchi-Ikeda M. Kaito H. et al.Identification of mutations in FN1 leading to glomerulopathy with fibronectin deposits.Pediatr. Nephrol. 2016; 31: 1459-1467Crossref PubMed Scopus (22) Google Scholar, 18Castelletti F. Donadelli R. Banterla F. Hildebrandt F. Zipfel P.F. Bresin E. Otto E. Skerka C. Renieri A. Todeschini M. et al.Mutations in FN1 cause glomerulopathy with fibronectin deposits.Proc. Natl. Acad. Sci. USA. 2008; 105: 2538-2543Crossref PubMed Scopus (99) Google Scholar, 19Ertoy Baydar D. Kutlugun A.A. Bresin E. Piras R. A case of familial glomerulopathy with fibronectin deposits caused by the Y973C mutation in fibronectin.Am. J. Kidney Dis. 2013; 61: 514-518Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar In vitro, the mutations cause decreased heparin and integrin binding, reduced endothelial cell spreading, and cytoskeletal reorganization, which has been hypothesized to affect glomerular size selectivity and protein trafficking. Importantly, none of the SMD individuals in our cohort had any evidence of renal disease. As stated above, all cysteine residues affected by mutations in our cohort are involved in disulfide bonds in fibronectin type I domains (bridges form between Cys87 and Cys76, between Cys123 and Cys135, between Cys225 and Cys213, and between Cys260 and Cys231). We selected one of these (p.Cys87Phe) to model the consequence of the mutation on the three-dimensional structure of fibronectin domains (Figure 5A). The p.Cys87Phe substitution was predicted to destabilize the structure of the I-1 domain by breaking the disulfide bond with residue Cys76 and by displaying a hydrophobic residue at the surface. A similar disruptive impact was predicted for the other missense changes involving Cys123, Cys225, and Cys260. For the two other amino acid residues affected by mutations (Tyr240 and Thr809), 3D structures adequate for modeling the mutations were available only for p.Tyr240Asp (Thr809 was found only at the C-terminal end of a solution structure of III-2,20Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. Interdomain association in fibronectin: insight into cryptic sites and fibrillogenesis.EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar which limits the interpretation of its interactions). Modeling predicted that the p.Tyr240Asp variant would disrupt the pi-stacking (or π-π stacking) of the side chains of Tyr240 and Trp246 and thus destabilize the I-5 domain (Figure 5B). We next aimed to analyze the functional consequences of selected FN1 mutations on the fibronectin protein. Because cells from affected individuals were not available for this study, we generated a recombinant 70 kDa N-terminal fragment of fibronectin (rF70K), spanning the region where five of the six missense variants identified in this study are located, and a full-length recombinant fibronectin (rFN) (Figure 6A). Expression vectors for the human wild-type rF70K and rFN, as well as selected mutants, were generated by standard cloning procedures in the pcDNA3.1+ plasmid (Thermo Fisher Scientific, V79020) with the sequence for a C-terminal V5 epitope tag to facilitate detection and a hexa-histidine tag intended for chromatographic purification. At the 5′ ends, the vectors contained the coding sequence for either the BM40 signal peptide (rF70K) or the native fibronectin signal peptide (rFN) to ensure secretion of the recombinant protein through the secretory pathway. The rFN expression plasmid was constructed with the sequence for the alternatively spliced EDA and EDB domains. For this analysis, we selected two cysteine missense variants (p.Cys87Phe and p.Cys260Gly) because they represent the major group of the identified mutations, as well as a non-cysteine missense variant (p.Tyr240Asp). These point mutants were generated from the wild-type plasmids via the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, 200519). The plasmids were transfected into human HEK293 cells, which synthesize and secrete endogenous fibronectin and represent an efficient system for producing a range of correctly folded and posttranslationally modified ECM proteins (subfragments as well as mutants).21Kirschner R. Hubmacher D. Iyengar G. Kaur J. Fagotto-Kaufmann C. Brömme D. Bartels R. Reinhardt D.P. Classical and neonatal Marfan syndrome mutations in fibrillin-1 cause differential protease susceptibilities and protein function.J. Biol. Chem. 2011; 286: 32810-32823Crossref PubMed Scopus (43) Google Scholar, 22McKee K.K. Harrison D. Capizzi S. Yurchenco P.D. Role of laminin terminal globular domains in basement membrane assembly.J. Biol. Chem. 2007; 282: 21437-21447Crossref PubMed Scopus (133) Google Scholar, 23Fox J.W. Mayer U. Nischt R. Aumailley M. Reinhardt D. Wiedemann H. Mann K. Timpl R. Krieg T. Engel J. et al.Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.EMBO J. 1991; 10: 3137-3146Crossref PubMed Scopus (380) Google Scholar After the plasmids were stably transfected into human HEK293 cells as described previously,24Lin G. Tiedemann K. Vollbrandt T. Peters H. Batge B. Brinckmann J. Reinhardt D.P. Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils.J. Biol. Chem. 2002; 277: 50795-50804Crossref PubMed Scopus (120) Google Scholar the secreted wild-type recombinant proteins were readily identified in the cell-culture medium (conditioned for 2 days) with fibronectin-specific or anti-V5-specific antibodies (Figure 6B). Surprisingly, all three rF70K and rFN mutants were either undetectable in the culture medium or detectable at much lower levels than the wild-type protein. Analysis of recombinant mRNA with primers excluding the endogenous fibronectin mRNA revealed that the amount of mutant mRNA was similar to that of wild-type mRNA (Figure 6C). Immunofluorescence analysis demonstrated very low amounts of the wild-type proteins within the stably transfected cells because the proteins secreted into the culture medium (Figures 6D and 6E). In contrast to this finding, all three rF70K and rFN mutants consistently showed strong accumulation within the cells. These data clearly demonstrate that p.Cys87Phe, p.Tyr240Asp, and p.Cys260Gly exert a common molecular defect in fibronectin secretion. To assess whether the intracellular retention induced the unfolded protein response in HEK293 cells, we analyzed XBP1 splicing (IRE1 pathway) and the mRNA expression levels of CHOP and ATF4 (PERK pathway). However, no indication of increased XBP1 splicing or changes in the CHOP or ATF4 mRNA expression levels was detected (Figure S2). In addition, the mutant rFN cell clones did not undergo caspase-3-mediated apoptosis (Figure S3). Fibronectin is present throughout cartilage differentiation and persists in mature cartilaginous tissue.25Singh P. Schwarzbauer J.E. Fibronectin and stem cell differentiation - lessons from chondrogenesis.J. Cell Sci. 2012; 125: 3703-3712Crossref PubMed Scopus (144) Google Scholar However, little is known about the functional role of fibronectin in the development and homeostasis of cartilage. Given that fibronectin has been established as a key ECM protein that guides fibrillogenesis of several other ECM proteins, including proteins present in cartilage, such as fibrillin-1,26Keene D.R. Jordan C.D. Reinhardt D.P. Ridgway C.C. Ono R.N. Corson G.M. Fairhurst M. Sussman M.D. Memoli V.A. Sakai L.Y. Fibrillin-1 in human cartilage: developmental expression and formation of special banded fibers.J. Histochem. Cytochem. 1997; 45: 1069-1082Crossref PubMed Scopus (70) Google Scholar it is conceivable that a reduced amount of secreted fibronectin from chondrocytes could result in a deficient matrix network in cartilage. It is well known that mutations affecting another cartilage protein, cartilage oligomeric matrix protein (COMP [MIM: 600310]), lead to pseudoachondroplasia (MIM: 177170) and multiple epiphyseal dysplasia (MIM: 132400) through accumulation of mutant COMP in the endoplasmic reticulum of chondrocytes.27Maddox B.K. Keene D.R. Sakai L.Y. Charbonneau N.L. Morris N.P. Ridgway C.C. Boswell B.A. Sussman M.D. Horton W.A. Bächinger H.P. Hecht J.T. The fate of cartilage oligomeric matrix protein is determined by the cell type in the case of a novel mutation in pseudoachondroplasia.J. Biol. Chem. 1997; 272: 30993-30997Crossref PubMed Scopus (91) Google Scholar It is thought that this protein accumulation impairs secretion of other matrix proteins and leads to abnormal ECM and chondrocyte death.28Hecht J.T. Montufar-Solis D. Decker G. Lawler J. Daniels K. Duke P.J. Retention of cartilage oligomeric matrix protein (COMP) and cell death in redifferentiated pseudoachondroplasia chondrocytes.Matrix Biol. 1998; 17: 625-633Crossref PubMed Scopus (80) Google Scholar, 29Hashimoto Y. Tomiyama T. Yamano Y. Mori H. Mutation (D472Y) in the type 3 repeat domain of cartilage oligomeric matrix protein affects its early vesicle trafficking in endoplasmic reticulum and induces apoptosis.Am. J. Pathol. 2003; 163: 101-110Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar It is possible that a similar mechanism exists for retained mutant fibronectin and leads to the SMD phenotype observed in our cohort. Conditional double-knockout experiments of fibronectin in mouse cartilage and liver or knockin of some of the mutations are currently underway to further elucidate the molecular basis of fibronectin in the development and maintenance of cartilage. This project was supported in part by operating grants from the Canadian Institutes for Health Research (clinician-scientist award RN315908 to P.M.C.; MOP-137091 to D.P.R.), the Fonds de Recherche du Québec - Santé clinical research scholar award 30647 to P.M.C., the Quebec Network for Oral and Bone Health Research (RSBO Emerging Collaborating Project 2014-2015 to P.M.C. and D.P.R.), the NIH (UM1 HG006542 to the Baylor Hopkins Center for Mendelian Genomics), the Heart and Stroke Foundation of Canada (G-16-00014634 to D.P.R.), the Italian Ministry of Health (Ricerca Corrente 2016 to M.T. and M.N.), and Fondazione Bambino Gesù (Vite Coraggiose grant to M.T.). This work was also supported by NIH P01 HD070394, by HD024064 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development granted to the Baylor College of Medicine (BCM) Intellectual and Developmental Disabilities Research Center (for processing samples obtained and managing clinical protocols), by the BCM Advanced Technology Cores through funding from the NIH (AI036211, CA125123, and RR024574), by the Rolanette and Berdon Lawrence Bone Disease Program of Texas, and by the BCM Center for Skeletal Medicine and Biology (to B.H.L). Analysis of individual 5 was supported by the São Paulo Research Foundation (FAPESP 2015/21783-9; Centros de Pesquisa, Inovação, e Difusão [CEPID] 2013/08028-1) and National Council for Scientific and Technological Development (CNPq 302605/2013-4 and 304130/2016-8 to D. B). E.L. is supported by grants from the German Research Foundation (CRC 1140), the German Ministry for Education and Research (MaTrOC), and the European Union (SYBIL grant agreement no. 602300; RARENET). We thank Dr. Deane Mosher and Dr. Douglas Annis for providing the FN1 pAcGP67A plasmid. We thank Dr. Reggie Hamdy for referring individual 7 to Dr. Campeau and Dr. Amélie Damphousse for the interpretation of the radiographs. The accession numbers for the variants identified in this study are ClinVar: SCV000574553–SCV000574558. Download .pdf (4.65 MB) Help with pdf files Document S1. Figures S1–S3 and Table S1 and S2 Baylor-Hopkins Center for Mendelian Genetics (BH-CMG), http://bhcmg.org/ExAC Browser, http://exac.broadinstitute.org/GenBank, http://www.ncbi.nlm.nih.gov/genbank/GeneMatcher, https://www.genematcher.orgInternational Skeletal Dysplasia Society (ISDS), http://www.isds.ch/International Skeletal Dysplasia Registry (ISDR), http://ortho.ucla.edu/isdrOMIM, http://www.omim.org/RCSB Protein Data Bank, https://www.rcsb.org/pdb/home/home.doUniProt, http://www.uniprot.org/uniprot/

Proteins anchored to the cell surface via glycosylphosphatidylinositol (GPI) play various key roles in the human body, particularly in development and neurogenesis. As such, many developmental disorders are caused by mutations in genes involved in the GPI biosynthesis and remodeling pathway. We describe ten unrelated families with bi-allelic mutations in PIGB, a gene that encodes phosphatidylinositol glycan class B, which transfers the third mannose to the GPI. Ten different PIGB variants were found in these individuals. Flow cytometric analysis of blood cells and fibroblasts from the affected individuals showed decreased cell surface presence of GPI-anchored proteins. Most of the affected individuals have global developmental and/or intellectual delay, all had seizures, two had polymicrogyria, and four had a peripheral neuropathy. Eight children passed away before four years old. Two of them had a clinical diagnosis of DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures), a condition that includes sensorineural deafness, shortened terminal phalanges with small finger and toenails, intellectual disability, and seizures; this condition overlaps with the severe phenotypes associated with inherited GPI deficiency. Most individuals tested showed elevated alkaline phosphatase, which is a characteristic of the inherited GPI deficiency but not DOORS syndrome. It is notable that two severely affected individuals showed 2-oxoglutaric aciduria, which can be seen in DOORS syndrome, suggesting that severe cases of inherited GPI deficiency and DOORS syndrome might share some molecular pathway disruptions. Proteins anchored to the cell surface via glycosylphosphatidylinositol (GPI) play various key roles in the human body, particularly in development and neurogenesis. As such, many developmental disorders are caused by mutations in genes involved in the GPI biosynthesis and remodeling pathway. We describe ten unrelated families with bi-allelic mutations in PIGB, a gene that encodes phosphatidylinositol glycan class B, which transfers the third mannose to the GPI. Ten different PIGB variants were found in these individuals. Flow cytometric analysis of blood cells and fibroblasts from the affected individuals showed decreased cell surface presence of GPI-anchored proteins. Most of the affected individuals have global developmental and/or intellectual delay, all had seizures, two had polymicrogyria, and four had a peripheral neuropathy. Eight children passed away before four years old. Two of them had a clinical diagnosis of DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures), a condition that includes sensorineural deafness, shortened terminal phalanges with small finger and toenails, intellectual disability, and seizures; this condition overlaps with the severe phenotypes associated with inherited GPI deficiency. Most individuals tested showed elevated alkaline phosphatase, which is a characteristic of the inherited GPI deficiency but not DOORS syndrome. It is notable that two severely affected individuals showed 2-oxoglutaric aciduria, which can be seen in DOORS syndrome, suggesting that severe cases of inherited GPI deficiency and DOORS syndrome might share some molecular pathway disruptions.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD. Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

IFAP syndrome is a rare genetic disorder characterized by ichthyosis follicularis, atrichia, and photophobia. Previous research found that mutations in <i>MBTPS2</i>, encoding site-2-protease (S2P), underlie X-linked IFAP syndrome. The present report describes the identification via whole-exome sequencing of three heterozygous mutations in <i>SREBF1</i> in 11 unrelated, ethnically diverse individuals with autosomal-dominant IFAP syndrome. <i>SREBF1</i> encodes sterol regulatory element-binding protein 1 (SREBP1), which promotes the transcription of lipogenes involved in the biosynthesis of fatty acids and cholesterols. This process requires cleavage of SREBP1 by site-1-protease (S1P) and S2P and subsequent translocation into the nucleus where it binds to sterol regulatory elements (SRE). The three detected <i>SREBF1</i> mutations caused substitution or deletion of residues 527, 528, and 530, which are crucial for S1P cleavage. <i>In vitro</i> investigation of SREBP1 variants demonstrated impaired S1P cleavage, which prohibited nuclear translocation of the transcriptionally active form of SREBP1. As a result, SREBP1 variants exhibited significantly lower transcriptional activity compared to the wild-type, as demonstrated via luciferase reporter assay. RNA sequencing of the scalp skin from IFAP-affected individuals revealed a dramatic reduction in transcript levels of low-density lipoprotein receptor (<i>LDLR</i>) and of keratin genes known to be expressed in the outer root sheath of hair follicles. An increased rate of <i>in situ</i> keratinocyte apoptosis, which might contribute to skin hyperkeratosis and hypotrichosis, was also detected in scalp samples from affected individuals. Together with previous research, the present findings suggest that SREBP signaling plays an essential role in epidermal differentiation, skin barrier formation, hair growth, and eye function.

Given the genomic uniqueness, a local data set is most desired for Indians, who are underrepresented in existing public databases. We hypothesize patients with rare monogenic disorders and their family members can provide a reliable source of common variants in the population. Exome sequencing (ES) data from families with rare Mendelian disorders was aggregated from five centers in India. The dataset was refined by excluding related individuals and removing the disease-causing variants (refined cohort). The efficiency of these data sets was assessed in a new set of 50 exomes against gnomAD and GenomeAsia. Our original cohort comprised 1455 individuals from 1203 families. The refined cohort had 836 unrelated individuals that retained 1,251,064 variants with 181,125 population-specific and 489,618 common variants. The allele frequencies from our cohort helped to define 97,609 rare variants in gnomAD and 44,520 rare variants in GenomeAsia as common variants in our population. Our variant dataset provided an additional 1.7% and 0.1% efficiency for prioritizing heterozygous and homozygous variants respectively for rare monogenic disorders. We observed additional 19 genes/human knockouts. We list carrier frequency for 142 recessive disorders. This is a large and useful resource of exonic variants for Indians. Despite limitations, datasets from patients are efficient tools for variant prioritization in a resource-limited setting.

<h2>Abstract</h2><h3>Purpose</h3> This study aimed to undertake a multidisciplinary characterization of the phenotype associated with <i>SOX11</i> variants. <h3>Methods</h3> Individuals with protein altering variants in <i>SOX11</i> were identified through exome and genome sequencing and international data sharing. Deep clinical phenotyping was undertaken by referring clinicians. Blood DNA methylation was assessed using Infinium MethylationEPIC array. The expression pattern of <i>SOX11</i> in developing human brain was defined using RNAscope. <h3>Results</h3> We reported 38 new patients with <i>SOX11</i> variants. Idiopathic hypogonadotropic hypogonadism was confirmed as a feature of <i>SOX11</i> syndrome. A distinctive pattern of blood DNA methylation was identified in <i>SOX11</i> syndrome, separating <i>SOX11</i> syndrome from other BAFopathies. <h3>Conclusion</h3> <i>SOX11</i> syndrome is a distinct clinical entity with characteristic clinical features and episignature differentiating it from BAFopathies.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

Aberrant forms of endoplasmic reticulum (ER)-resident chaperones are implicated in loss of protein quality control in rare diseases. Here we report a novel mutation (p.Asp233Asn) in the ER retention signal of MESD by whole exome sequencing of an individual diagnosed with osteogenesis imperfecta (OI) type XX. While MESDD233N has similar stability and chaperone activity as wild-type MESD, its mislocalization to cytoplasm leads to imbalance of ER proteostasis, resulting in improper folding and aggregation of proteins, including LRP5 and type I collagen. Aggregated LRP5 loses its plasma membrane localization to disrupt the expression of WNT-responsive genes, such as BMP2, BMP4, in proband fibroblasts. We show that MESD is a direct chaperone of pro-α1(I) [COL1A1], and absence of MESDD233N in ER results in cytosolic type I collagen aggregates that remain mostly not secreted. While cytosolic type I collagen aggregates block the intercellular nanotubes, decreased extracellular type I collagen also results in loss of interaction of ITGB1 with type I collagen and weaker attachment of fibroblasts to matrix. Although proband fibroblasts show increased autophagy to degrade the aggregated type I collagen, an overall cellular stress overwhelms the proband fibroblasts. In summary, we present an essential chaperone function of MESD for LRP5 and type I collagen and demonstrating how the D233N mutation in MESD correlates with impaired WNT signaling and proteostasis in OI.

Despite large sequencing and data sharing efforts, previously characterized pathogenic variants only account for a fraction of Mendelian disease patients, which highlights the need for accurate identification and interpretation of novel variants. In a large Mendelian cohort of 4577 molecularly characterized families, numerous scenarios in which variant identification and interpretation can be challenging are encountered. We describe categories of challenges that cover the phenotype (e.g. novel allelic disorders), pedigree structure (e.g. imprinting disorders masquerading as autosomal recessive phenotypes), positional mapping (e.g. double recombination events abrogating candidate autozygous intervals), gene (e.g. novel gene-disease assertion) and variant (e.g. complex compound inheritance). Overall, we estimate a probability of 34.3% for encountering at least one of these challenges. Importantly, our data show that by only addressing non-sequencing-based challenges, around 71% increase in the diagnostic yield can be expected. Indeed, by applying these lessons to a cohort of 314 cases with negative clinical exome or genome reports, we could identify the likely causal variant in 54.5%. Our work highlights the need to have a thorough approach to undiagnosed diseases by considering a wide range of challenges rather than a narrow focus on sequencing technologies. It is hoped that by sharing this experience, the yield of undiagnosed disease programs globally can be improved.

The ex vivo gene therapy approach for Duchenne muscular dystrophy is promising since myoblast transplantation in primates is now very efficient. One obstacle to this treatment is the low transfection efficiency of large DNA constructs in human primary myoblasts. Small plasmids can be easily transfected with the new phosphonolipid described in this study. However, a dramatic drop in transfection efficiency is observed with plasmids of 12 kb or more containing EGFP minidystrophin and EGFP dystrophin fusion genes. The transfection of human primary myoblasts with such large plasmids could only be achieved when the DNA was linked to an adenovirus with the use of polyethylenimine (PEI), with efficiencies ranging between 3 and 5% of transitory transfection. Branched 2 kDa PEI was less toxic in PEI adenofection than branched 25 kDa PEI or linear 22 kDa PEI. The adenovirus was an absolute necessity for an efficient transfection. An integrin-binding peptide, a nuclear localization signal peptide, chloroquine, glycerol or cell cycle synchronization using aphidicolin did not enhance PEI adenofection. Following PEI adenofection, the adenoviral proteins were detected using a polyclonal antibody. The detected antigens fell below the detectable level after 12 days in culture. We thus provide in this study an efficient and reproducible method to permit efficient delivery of large plasmids to human primary myoblasts for the ex vivo gene therapy of Duchenne muscular dystrophy.

Our objective was to study demographic features, clinical features, and karyotype analyses of patients with Down syndrome (DS). Our study design was a retrospective analysis, while the study was conducted in the Genetic Clinic of a tertiary-care teaching hospital.Retrospective analysis of cases referred to the Genetic Clinic was performed. Case proformas of the patients presenting with phenotypic features of DS were analyzed. The following information was recorded from the proformas: age at presentation; sex; maternal age; craniofacial and other physical features; presence and type of congenital heart disease; gastrointestinal abnormalities, and results of cytogenetic evaluation. Clinical features in neonates with DS were separately analyzed.Analysis included cases of DS presenting over a period of 7.5 years; a total of 524 patients were studied (303 males and 221 females; M:F ratio 1.37:1). Average age at presentation was 19.4 months (range: 1 day-26 years). Average maternal age at birth of the affected child was 26.8 years (range: 16-45 years). Craniofacial features noted in >50% of the cases included mongoloid slant (83.9%), ear abnormalities (66.9%), epicanthic folds (56.9%), and flat facial profile (50.9%). A total of 76.3% cases had hypotonia. Characteristic limb and dermatoglyphic anomalies were seen in less than one half of cases. These included sandle sign (46.2%), unilateral or bilateral simian crease (33.2%), clinodactyly (36.1%), and brachydactyly (11.1%). Ophthalmologic abnormalities included hypertelorism (33.9%), nystagmus (3.2%), Brushfield spots (3.2%), squint (2.7%), and cataracts (1.9%). Congenital heart disease was clinically diagnosed in 96 cases (18.3%). The nature of the cardiac defect was ascertained by color Doppler examination and/or 2D-echocardiography in 58 cases. The most common cardiac anomalies were ventricular septal defect (25.8%), tetralogy of Fallot (15.5%), and atrial septal defect (12.1%). Gastrointestinal anomalies were noted in seven cases and included three cases with imperforate anus, two with Hirschsprung disease, and one each with duodenal atresia and Morgagni hernia. Results of cytogenetic abnormalities were available in 42.2%. Free trisomy (non-dysjunction) was present in 95%, 3.2% had translocation, and 1.8% were mosaics. In neonates, common features noted were mongoloid slant, ear abnormalities, flat facial profile, hypotonia, sandle sign, and clinodactyly+/-brachydactyly.All characteristic craniofacial and physical features of DS need not be present in every case. Major features noted in the present study were mongoloid slant, ear abnormalities, epicanthic folds, flat facies, and hypotonia. Congenital heart disease was present in 18.3% of cases, with ventricular septal defect being the most common type of defect. Non-dysjunction was the most common cause of the chromosomal anomaly.

Patients with neonatal urea cycle defects (UCDs) typically have high mortality and poor neurological outcome unless they receive liver transplantation. Neurologic outcome may be better with liver transplantation before age one year. We report on a follow up on an initial prospective study performed to assess developmental outcome after early liver transplant using the Griffiths Scales. Developmental testing up to 7years after transplantation showed average developmental quotients (DQs) of 69 for four children who underwent transplantation before one year of age (latest DQs were 47, 63, 95 and 96), and 80 for a patient who underwent transplantation at 3years of age (latest DQ was 88). We conclude that a combination of early liver transplantation, aggressive metabolic management and early childhood intervention improve the neurologic outcome of children with UCDs.

Acrodysostosis is characterized by nasal hypoplasia, peripheral dysostosis, variable short stature, and intellectual impairment. Recently, mutations in PRKAR1A were reported in patients with acrodysostosis and hormone resistance. Subsequently, mutations in a phosphodiesterase gene (PDE4D) were identified in seven sporadic cases. We sequenced PDE4D in seven acrodysostosis patients from five families. Missense mutations were identified in all cases. Families showed de novo inheritance except one family with three affected children whose father was subsequently found to have subtle features of acrodysostosis. There were no recurrent mutations. Short stature and endocrine resistance are rare in this series; however, cognitive involvement and obesity were frequent. This last finding is relevant given PDE4D is insulin responsive and potentially involved in lipolysis. PDE4D encodes a cyclic AMP regulator and places PDE4D-related acrodysostosis within the same family of diseases as pseudohypoparathyroidism, pseudopseudohypoparathyroidism, PRKAR1A-related acrodysostosis and brachydactyly-mental retardation syndrome; all characterized by cognitive impairment and short distal extremities.

Coarctation of the aorta (CoA) and hypoplastic left heart syndrome (HLHS) have been reported in rare individuals with large terminal deletions of chromosome 15q26. However, no single gene important for left ventricular outflow tract (LVOT) development has been identified in this region. Using array-comparative genomic hybridization, we identified two half-siblings with CoA with a 2.2 Mb deletion on 15q26.2, inherited from their mother, who was mosaic for this deletion. This interval contains an evolutionary conserved, protein-coding gene, MCTP2 (multiple C2-domains with two transmembrane regions 2). Using gene-specific array screening in 146 individuals with non-syndromic LVOT obstructive defects, another individual with HLHS and CoA was found to have a de novo 41 kb intragenic duplication within MCTP2, predicted to result in premature truncation, p.F697X. Alteration of Mctp2 gene expression in Xenopus laevis embryos by morpholino knockdown and mRNA overexpression resulted in the failure of proper OT development, confirming the functional importance of this dosage-sensitive gene for cardiogenesis. Our results identify MCTP2 as a novel genetic cause of CoA and related cardiac malformations.

Undernutrition contributes to half of all childhood deaths under the age of 5 y, and confers upon survivors a life-long predisposition to obesity, type 2 diabetes, and cardiovascular disease. Mechanisms underlying the link between early nutrient deprivation and noncommunicable diseases are unknown. Using outbred CD1 neonatal mice, we measured metabolic profile differences between conventionally reared mice given unrestricted access to nursing, the control group, and undernourished mice subjected to protein-calorie deprivation through timed separation from lactating mothers. After 11 d of undernutrition, urine, plasma, liver, ileal fluid, cecal fluid, and stool were harvested from 8 pools of 4 neonatal mice per group. The metabolome was identified using a multiplatform mass spectrometry-based approach, and random forest metrics were used to identify the most important metabolites that distinguished the undernourished from the control group. Our data reveal striking metabolic changes in undernourished mice consistent with the known mammalian response to starvation, including evidence of muscle and fat catabolism and increased reliance on the tricarboxylic acid cycle for energy. However, we also revealed evidence of liver and biliary injury, anomalies in bile acid metabolism, oxidative stress and inflammation, accelerated heme breakdown, and altered regulation of DNA methylation. Among the metabolites that most strongly distinguished the 2 groups were 2-hydroxyisobutyrate, increased 3-fold in plasma of undernourished mice (P = 2.19 × 10(-11)); urobilinogen, increased 11-fold in urine of undernourished mice (P = 4.22 × 10(-7)); deoxycholate, decreased 94% in stool of undernourished mice (P = 3.0 × 10(-4)); and 12 different products of the enzyme γ-glutamyltransferase, increased in all 6 compartments of undernourished mice. This model of the undernourished neonatal metabolome illustrates the wide range of pathways disrupted by undernutrition in early development, and suggests mechanistic links between early starvation and persistent metabolic diseases.

DOORS syndrome (Deafness, Onychodystrophy, Osteodystrophy, mental Retardation, Seizures) is characterized mainly by sensorineural deafness, shortened terminal phalanges with small nails of hands and feet, intellectual deficiency, and seizures. Half of the patients with all clinical features have mutations in TBC1D24. We review here the manifestations of patients clinically diagnosed with DOORS syndrome. In this cohort of 32 families (36 patients) we detected 13 individuals from 10 families with TBC1D24 mutations. Subsequent whole exome sequencing in the cohort showed the same de novoSMARCB1 mutation (c.1130G>A), known to cause Coffin-Siris syndrome, in two patients. Distinguishing features include retinal anomalies, Dandy-Walker malformation, scoliosis, rocker bottom feet, respiratory difficulties and absence of seizures, and 2-oxoglutaric aciduria in the patients with the SMARCB1 mutation. We briefly discuss the heterogeneity of the DOORS syndrome phenotype and the differential diagnosis of this condition.

There are over 150 known human proteins which are tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. These proteins play a variety of important roles in development, and particularly in neurogenesis. Not surprisingly, mutations in the GPI anchor biosynthesis and remodeling pathway cause a number of developmental disorders. This group of conditions has been termed inherited GPI deficiencies (IGDs), a subgroup of congenital disorders of glycosylation; they present with variable phenotypes, often including seizures, hypotonia and intellectual disability. Here, we report two siblings with compound heterozygous variants in the gene phosphatidylinositol glycan anchor biosynthesis, class P (PIGP) (NM_153681.2: c.74T > C;p.Met25Thr and c.456delA;p.Glu153AsnFs*34). PIGP encodes a subunit of the enzyme that catalyzes the first step of GPI anchor biosynthesis. Both children presented with early-onset refractory seizures, hypotonia, and profound global developmental delay, reminiscent of other IGD phenotypes. Functional studies with patient cells showed reduced PIGP mRNA levels, and an associated reduction of GPI-anchored cell surface proteins, which was rescued by exogenous expression of wild-type PIGP. This work associates mutations in the PIGP gene with a novel autosomal recessive IGD, and expands our knowledge of the role of PIG genes in human development.

Myhre syndrome is characterized by short stature, brachydactyly, facial features, pseudomuscular hypertrophy, joint limitation and hearing loss. We identified SMAD4 mutations as the cause of Myhre syndrome. SMAD4 mutations have also been identified in laryngotracheal stenosis, arthropathy, prognathism and short stature syndrome (LAPS). This study aimed to review the features of Myhre and LAPS patients to define the clinical spectrum of SMAD4 mutations. We included 17 females and 15 males ranging in age from 8 to 48 years. Thirty were diagnosed with Myhre syndrome and two with LAPS. SMAD4 coding sequence was analyzed by Sanger sequencing. Clinical and radiological features were collected from a questionnaire completed by the referring physicians. All patients displayed a typical facial gestalt, thickened skin, joint limitation and muscular pseudohypertrophy. Growth retardation was common (68.7%) and was variable in severity (from -5.5 to -2 SD), as was mild-to-moderate intellectual deficiency (87.5%) with additional behavioral problems in 56.2% of the patients. Significant health concerns like obesity, arterial hypertension, bronchopulmonary insufficiency, laryngotracheal stenosis, pericarditis and early death occurred in four. Twenty-nine patients had a de novo heterozygous SMAD4 mutation, including both patients with LAPS. In 27 cases mutation affected Ile500 and in two cases Arg496. The three patients without SMAD4 mutations had typical findings of Myhre syndrome. Myhre-LAPS syndrome is a clinically homogenous condition with life threatening complications in the course of the disease. Our identification of SMAD4 mutations in 29/32 cases confirms that SMAD4 is the major gene responsible for Myhre syndrome.