Yavuz Oktay

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O(2) at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F(1)F(0) ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.

<h2>Summary</h2> RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, we localized polynucleotide phosphorylase (PNPASE), a 3′ → 5′ exoribonuclease and poly-A polymerase, in the mitochondrial intermembrane space, a location lacking resident RNAs. Here, we show a new role for PNPASE in regulating the import of nuclear-encoded RNAs into the mitochondrial matrix. PNPASE reduction impaired mitochondrial RNA processing and polycistronic transcripts accumulated. Augmented import of <i>RNase P</i>, <i>5S rRNA</i>, and <i>MRP</i> RNAs depended on PNPASE expression and PNPASE–imported RNA interactions were identified. PNPASE RNA processing and import activities were separable and a mitochondrial RNA targeting signal was isolated that enabled RNA import in a PNPASE-dependent manner. Combined, these data strongly support an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria.

Defined mutations in the mitochondrial ADP/ATP carrier (AAC) are associated with certain types of progressive external ophthalmoplegia. AAC is required for oxidative phosphorylation (OXPHOS), and dysregulation of AAC has been implicated in apoptosis. Little is known about the AAC interactome, aside from a known requirement for the phospholipid cardiolipin (CL) and that it is thought to function as a homodimer. Using a newly developed dual affinity tag, we demonstrate that yeast AAC2 physically participates in several protein complexes of distinct size and composition. The respiratory supercomplex and several smaller AAC2-containing complexes, including other members of the mitochondrial carrier family, are identified here. In the absence of CL, most of the defined interactions are destabilized or undetectable. The absence of CL and/or AAC2 results in distinct yet additive alterations in respiratory supercomplex structure and respiratory function. Thus, a single lipid can significantly alter the functional interactome of an individual protein.

Abstract Erythropoiesis in the adult mammal depends critically on erythropoietin, an inducible cytokine with pluripotent effects. Erythropoietin gene expression increases under conditions associated with lowered oxygen content such as anemia and hypoxia. HIF-1α, the founding member of the hypoxia-inducible factor (HIF) alpha class, was identified by its ability to bind and activate the hypoxia-responsive enhancer in the erythropoietin regulatory region in vitro. The existence of multiple HIF alpha members raises the question of which HIF alpha member or members regulates erythropoietin expression in vivo. We previously reported that mice lacking wild-type HIF-2α, encoded by the EPAS1 gene, exhibit pancytopenia. In this study, we have characterized the etiology of this hematopoietic phenotype. Molecular studies of EPAS1-null kidneys reveal dramatically decreased erythropoietin gene expression. EPAS1-null as well as heterozygous mice have impaired renal erythropoietin induction in response to hypoxia. Treatment of EPAS1-null mice with exogenous erythropoietin reverses the hematopoietic and other defects. We propose that HIF-2α is an essential regulator of murine erythropoietin production. Impairments in HIF signaling, involving either HIF-1α or HIF-2α, may play a prominent role in conditions involving altered hematopoietic or erythropoietin homeostasis.

Abstract Hypoxic stress plays a role in pathophysiologic states such as myocardial infarction and cerebral vascular events as well as in normal physiologic conditions including development and hematopoiesis. Members of the hypoxia inducible factor (HIF) family function as transcriptional regulators of genes involved in the hypoxic response. After generating adult mice that globally lack endothelial PAS domain protein 1 (EPAS1, also known as HIF-2α/HRF/HLF/MOP3), the second member of the HIF family, characterization of the hematopoietic cell population indicated that the loss of EPAS1/HIF-2α resulted in pancytopenia. Using bone marrow reconstitution experiments of lethally irradiated hosts, we have defined the extent and site of hematopoietic impairment in the EPAS1/HIF-2α null mice. These data suggest a critical role for EPAS1/HIF-2α in maintaining a functional microenvironment in the bone marrow for effective hematopoiesis.

Breast cancer cells are suggested to be organized in a hierarchical manner with a subpopulation of stem cells, termed as breast cancer stem cells (BCSCs), which contribute significantly to tumorigenesis, cancer recurrence and metastasis. BCSCs have been demonstrated to exhibit significant resistance to conventional chemo- and radiotherapy. Recent evidence suggests that treatment of breast cancers with radiation or chemotherapy agents induces stem cell-like properties in non-stem cells. Herein, we provide an overview of the key determinants of resistance to chemotherapy and radiation in BCSCs. To this end and by the use of bioinformatics, the molecular pathways, the defining markers, as well as the microenvironmental and genetic factors, which are implicated in the maintenance of stemness, chemo- and radioresistance in BCSCs, are identified and presented. Our findings could provide the foundation for the design of targeted chemo- or radiotherapeutic regimens in order to eliminate or sensitize BCSCs to cytotoxic therapies and prevent tumor relapse and metastasis.

COVID-19, caused by the novel SARS-CoV-2 virus, started in China in late 2019, and soon became a global pandemic. With the help of thousands of viral genome sequences that have been accumulating, it has become possible to track the evolution of the viral genome over time as it spread across the world. An important question that still needs to be answered is whether any of the common mutations affect the viral properties, and therefore the disease characteristics. Therefore, we sought to understand the effects of mutations in RNA-dependent RNA polymerase (RdRp), particularly the common 14408C>T mutation, on mutation rate and viral spread. By focusing on mutations in the slowly evolving M or E genes, we aimed to minimize the effects of selective pressure. Our results indicate that 14408C>T mutation increases the mutation rate, while the third-most common RdRp mutation, 15324C>T, has the opposite effect. It is possible that 14408C>T mutation may have contributed to the dominance of its co-mutations in Europe and elsewhere.

SARS-CoV-2 is a betacoronavirus responsible for COVID-19, a pandemic with global impact that first emerged in late 2019. Since then, the viral genome has shown considerable variance as the disease spread across the world, in part due to the zoonotic origins of the virus and the human host adaptation process. As a virus with an RNA genome that codes for its own genomic replication proteins, mutations in these proteins can significantly impact the variance rate of the genome, affecting both the survival and infection rate of the virus, and attempts at combating the disease. In this study, we analyzed the mutation densities of viral isolates carrying frequently observed mutations for four proteins in the RNA synthesis complex over time in comparison to wildtype isolates. Our observations suggest mutations in nsp14, an error-correcting exonuclease protein, have the strongest association with increased mutation load without selective pressure and across the genome, compared to nsp7, nsp8 and nsp12, which form the core polymerase complex. We propose nsp14 as a priority research target for understanding genomic variance rate in SARS-CoV-2 isolates and nsp14 mutations as potential predictors for high mutability strains.

Mice lacking <i>Epas1,</i> encoding the transcription factor Hypoxia-inducible Factor 2α (HIF-2α), exhibit an apparent mitochondrial disease state. Similarities between knock-outs of <i>Epas1</i> and of <i>Sod2,</i> encoding the mitochondrial antioxidant enzyme manganese superoxide dismutase, led to the identification of <i>Sod2</i> as a HIF-2α target gene. However, <i>Sod2</i> levels in <i>Epas1</i>^–<i>^/</i>^– liver are intermediate between that of <i>Sod</i>⁺<i>^/</i>^– and <i>Sod2</i>^–<i>^/</i>^– mice, which have subtle or severe phenotypes, respectively. This suggests that additional HIF-2α target genes besides <i>Sod2</i> contribute to the <i>Epas1</i>^–<i>^/</i>^– mitochondrial disease state. To define the nature of the mitochondrial defect in <i>Epas1</i>^–<i>^/</i>^– liver, we performed biophysical, biochemical, and molecular studies. In the setting of decreased <i>Sod2</i> levels and increased oxidative stress, we found reduced respiration, sensitized mitochondrial permeability transition pore opening, intact electron transport chain activities, and impaired mitochondrial aconitase activity. Mitochondrial aconitase protein levels were preserved, whereas mRNA and protein levels for frataxin, the oxidative stress-regulated mitochondrial aconitase chaperone protein, were markedly reduced in <i>Epas1</i>^–<i>^/</i>^– livers. The mouse <i>Fxn</i> promoter was preferentially activated by HIF-2α through a consensus HIF-responsive enhancer element. In summary, the studies reveal that <i>Fxn</i>, like <i>Sod2</i>, is a nuclear-encoded, mitochondrial-localized HIF-2α target gene required for optimal mitochondrial homeostasis. These findings expand upon the previously defined role of HIF-2α in the cellular response to oxidative stress and identify a novel link of HIF-2α with mitochondrial homeostasis.

The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies."

Abstract Neuromuscular diseases (NMDs) affect ∼15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However, most affected families are in low-to-middle income countries (LMICs) with limited access to DNA-based diagnosis. Most (86%) published genetic data is derived from European ancestry. This marked genetic data inequality hampers understanding of genetic diversity and hinders accurate genetic diagnosis in all income settings. We developed a cloud-based transcontinental partnership to build diverse, deeply-phenotyped and genetically characterized cohorts to improve genetic architecture knowledge, and potentially advance diagnosis and clinical management. We connected 18 centres in Brazil, India, South Africa, Turkey, Zambia, Netherlands and the UK. We co-developed a cloud-based data solution and trained 17 international neurology fellows in clinical genomic data interpretation. Single gene and whole exome data were analysed via a bespoke bioinformatics pipeline and reviewed alongside clinical and phenotypic data in global webinars to inform genetic outcome decisions. We recruited 6001 participants in the first 43 months. Initial genetic analyses ‘solved’ or ‘possibly solved’ ∼56% probands overall. In-depth genetic data review of the four commonest clinical categories (limb girdle muscular dystrophy, inherited peripheral neuropathies, congenital myopathy/muscular dystrophies and Duchenne/Becker muscular dystrophy) delivered a ∼59% ‘solved’ and ∼13% ‘possibly solved’ outcome. Almost 29% of disease causing variants were novel, increasing diverse pathogenic variant knowledge. Unsolved participants represent a new discovery cohort. The dataset provides a large resource from under-represented populations for genetic and translational research. In conclusion, we established a remote transcontinental partnership to assess genetic architecture of NMDs across diverse populations. It supported DNA-based diagnosis, potentially enabling genetic counselling, care pathways and eligibility for gene-specific trials. Similar virtual partnerships could be adopted by other areas of global genomic neurological practice to reduce genetic data inequality and benefit patients globally.

The single nucleotide polymorphism rs55705857, located in a non-coding but evolutionarily conserved region at 8q24.21, is strongly associated with IDH-mutant glioma development and was suggested to be a causal variant. However, the molecular mechanism underlying this association has remained unknown. With a case control study in 285 gliomas, 316 healthy controls, 380 systemic cancers, 31 other CNS-tumors, and 120 IDH-mutant cartilaginous tumors, we identified that the association was specific to IDH-mutant gliomas. Odds-ratios were 9.25 (5.17-16.52; 95% CI) for IDH-mutated gliomas and 12.85 (5.94-27.83; 95% CI) for IDH-mutated, 1p/19q co-deleted gliomas. Decreasing strength with increasing anaplasia implied a modulatory effect. No somatic mutations were noted at this locus in 114 blood-tumor pairs, nor was there a copy number difference between risk-allele and only-ancestral allele carriers. CCDC26 RNA-expression was rare and not different between the two groups. There were only minor subtype-specific differences in common glioma driver genes. RNA sequencing and LC-MS/MS comparisons pointed to significantly altered MYC-signaling. Baseline enhancer activity of the conserved region specifically on the MYC promoter and its further positive modulation by the SNP risk-allele was shown in vitro. Our findings implicate MYC deregulation as the underlying cause of the observed association.

OBJECTIVE Recent studies have established that hemispheric diffuse gliomas may be grouped into subsets on the basis of molecular markers; these subsets are loosely correlated with the histopathological diagnosis but are strong predictors of clinical tumor behavior. Based on an analysis of molecular and clinical parameters, the authors hypothesized that mutations of the telomerase promoter (TERTp-mut) mark separate oncogenic programs among isocitrate dehydrogenase 1 and/or 2 (IDH) mutant (IDH-mut) and IDH wild-type (IDH-wt) diffuse gliomas independent of histopathology or WHO grade. METHODS Four molecular subsets of the combined statuses of IDH and TERT-promoter mutations (double mutant, IDH only, TERT only, and double negative) were defined. Differences in age, anatomical location, molecular genetics, and survival rates in a surgical cohort of 299 patients with a total of 356 hemispheric diffuse gliomas (WHO Grade II, III, or IV) were analyzed. RESULTS TERTp-mut were present in 38.8% of IDH-mut and 70.2% of IDH-wt gliomas. The mutational status was stable in each patient at 57 recurrence events over a 2645-month cumulative follow-up period. Among patients with IDH-mut gliomas, those in the double-mutant subset had better survival and a lower incidence of malignant degeneration than those in the IDH-only subset. Of patients in the double-mutant subset, 96.3% were also positive for 1p/19q codeletions. All patients with 1p/19q codeletions had TERTp-mut. In patients with IDH-mut glioma, epidermal growth factor receptor or phosphatase and tensin homolog mutations were not observed, and copy-number variations were uncommon. Among IDH-wt gliomas, the TERT-only subset was associated with significantly higher age, higher Ki-67 labeling index, primary glioblastoma-specific oncogenic changes, and poor survival. The double-negative subset was genetically and biologically heterogeneous. Survival analyses (Kaplan-Meier, multivariate, and regression-tree analyses) confirmed that patients in the 4 molecular subsets had distinct prognoses. CONCLUSIONS Molecular subsets result in different tumor biology and clinical behaviors in hemispheric diffuse gliomas.

Since its emergence in Wuhan, China in late 2019, the origin and evolution of SARS-CoV-2 have been among the most debated issues related to COVID-19. Throughout its spread around the world, the viral genome continued acquiring new mutations and some of them became widespread. Among them, 14408 C>T and 23403 A>G mutations in RdRp and S, respectively, became dominant in Europe and the US, which led to debates regarding their effects on the mutability and transmissibility of the virus. In this study, we aimed to investigate possible differences between time-dependent variation of mutation densities (MDe) of viral strains that carry these two mutations and those that do not. Our analyses at the genome and gene level led to two important findings: First, time-dependent changes in the average MDe of circulating SARS-CoV-2 genomes showed different characteristics before and after the beginning of April, when daily new case numbers started levelling off. Second, this pattern was much delayed or even non-existent for the "mutant" (MT) strain that harbored both 14408 C>T and 23403 A>G mutations. Although these differences were not limited to a few hotspots, it is intriguing that the MDe increase is most evident in two critical genes, S and Orf1ab, which are also the genes that harbor the defining mutations of the MT genotype. The nature of these unexpected relationships warrants further research.

Corrigendum18 April 2016free access UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells Jin Zhang Search for more papers by this author Ivan Khvorostov Search for more papers by this author Jason S Hong Search for more papers by this author Yavuz Oktay Search for more papers by this author Laurent Vergnes Search for more papers by this author Esther Nuebel Search for more papers by this author Paulin N Wahjudi Search for more papers by this author Kiyoko Setoguchi Search for more papers by this author Geng Wang Search for more papers by this author Anna Do Search for more papers by this author Hea-Jin Jung Search for more papers by this author J Michael McCaffery Search for more papers by this author Irwin J Kurland Search for more papers by this author Karen Reue Search for more papers by this author Wai-Nang P Lee Search for more papers by this author Carla M Koehler Search for more papers by this author Michael A Teitell Search for more papers by this author Jin Zhang Search for more papers by this author Ivan Khvorostov Search for more papers by this author Jason S Hong Search for more papers by this author Yavuz Oktay Search for more papers by this author Laurent Vergnes Search for more papers by this author Esther Nuebel Search for more papers by this author Paulin N Wahjudi Search for more papers by this author Kiyoko Setoguchi Search for more papers by this author Geng Wang Search for more papers by this author Anna Do Search for more papers by this author Hea-Jin Jung Search for more papers by this author J Michael McCaffery Search for more papers by this author Irwin J Kurland Search for more papers by this author Karen Reue Search for more papers by this author Wai-Nang P Lee Search for more papers by this author Carla M Koehler Search for more papers by this author Michael A Teitell Search for more papers by this author Author Information Jin Zhang, Ivan Khvorostov, Jason S Hong, Yavuz Oktay, Laurent Vergnes, Esther Nuebel, Paulin N Wahjudi, Kiyoko Setoguchi, Geng Wang, Anna Do, Hea-Jin Jung, J Michael McCaffery, Irwin J Kurland, Karen Reue, Wai-Nang P Lee, Carla M Koehler and Michael A Teitell EMBO J (2016)35:899-899https://doi.org/10.15252/embj.201694054 This article corrects the following: UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells15 November 2011 ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Authors’ statement We wish to clarify that Fig 4A and B intentionally displayed duplicate controls and that the first panel in Figs 1A and S1A was intentionally duplicated. The low (control) and high (FCCP) traces were purposefully duplicated in panels from Fig 4A and B to allow for direct, experimentally unbiased comparisons between the effects of antimycin and sodium oxamate on pluripotent stem and differentiated cells. Panels shown in Fig 4A and B are representative of one single experiment for N = 2 equivalently performed experiments. The panels were separated into parts 4A (antimycin) and 4B (sodium oxamate) for clarity of presentation since the traces and the colors of these traces have overlaps, which makes clear visualization difficult. We apologize for not explicitly stating that these traces were derived from a single, representative experiment. In addition, the first panel in Fig 1A and the first panel of Supplementary Fig S1A were purposefully reproduced to highlight different points. Figure 1A was provided to show how the steady-state pluripotent mitochondrial network appears in relation to the networks shown from three additional pluripotent stem cell lines and one differentiated cell type in the other panels of Fig 1A. Fig S1A was provided to clearly show the progressive effect of a differentiation time course induced by removal of basic fibroblast growth factor over 5 days on the mitochondrial network using a side-by-side comparison with the network in the pluripotent state. We apologize for not explicitly stating that these images were duplicated to highlight these specific, different features. All authors concur with this statement, and we regret not being more explicit in the purposeful use of these duplicated materials in the original publication. Next ArticlePrevious Article Read MoreAbout the coverClose modalView large imageVolume 35,Issue 8,15 April 2016Cover: Cilium scales brain size in microcephaly. From Elke Gabriel, Jay Gopalakrishnan and colleagues: CPAP promotes timely cilium disassembly to maintain neural progenitor pool. For details, see the Article on p 803. Scientific images by Elke Gabriel and Jay Gopalakrishnan (Center for Molecular Medicine and Institute for Biochemistry I of the University of Cologne, Robert-Koch-Str. 21 50931 Cologne, Germany) and Marco Gottardo, Giuliano Callaini (Department of Life Sciences, University of Siena, 53100 Siena, Italy) Volume 35Issue 815 April 2016In this issue RelatedDetailsLoading ...

Abstract Consanguineous marriages have a prevalence rate of 24% in Turkey. These carry an increased risk of autosomal recessive genetic conditions, leading to severe disability or premature death, with a significant health and economic burden. A definitive molecular diagnosis could not be achieved in these children previously, as infrastructures and access to sophisticated diagnostic options were limited. We studied the cause of neurogenetic disease in 246 children from 190 consanguineous families recruited in three Turkish hospitals between 2016 and 2020. All patients underwent deep phenotyping and trio whole exome sequencing, and data were integrated in advanced international bioinformatics platforms. We detected causative variants in 119 known disease genes in 72% of families. Due to overlapping phenotypes 52% of the confirmed genetic diagnoses would have been missed on targeted diagnostic gene panels. Likely pathogenic variants in 27 novel genes in 14% of the families increased the diagnostic yield to 86%. Eighty-two per cent of causative variants (141/172) were homozygous, 11 of which were detected in genes previously only associated with autosomal dominant inheritance. Eight families carried two pathogenic variants in different disease genes. De novo (9.3%), X-linked recessive (5.2%) and compound heterozygous (3.5%) variants were less frequent compared to non-consanguineous populations. This cohort provided a unique opportunity to better understand the genetic characteristics of neurogenetic diseases in a consanguineous population. Contrary to what may be expected, causative variants were often not on the longest run of homozygosity and the diagnostic yield was lower in families with the highest degree of consanguinity, due to the high number of homozygous variants in these patients. Pathway analysis highlighted that protein synthesis/degradation defects and metabolic diseases are the most common pathways underlying paediatric neurogenetic disease. In our cohort 164 families (86%) received a diagnosis, enabling prevention of transmission and targeted treatments in 24 patients (10%). We generated an important body of genomic data with lasting impacts on the health and wellbeing of consanguineous families and economic benefit for the healthcare system in Turkey and elsewhere. We demonstrate that an untargeted next generation sequencing approach is far superior to a more targeted gene panel approach, and can be performed without specialized bioinformatics knowledge by clinicians using established pipelines in populations with high rates of consanguinity.

Autozygosity is associated with an increased risk of genetic rare disease, thus being a relevant factor for clinical genetic studies. More than 2400 exome sequencing data sets were analyzed and screened for autozygosity on the basis of detection of >1 Mbp runs of homozygosity (ROHs). A model was built to predict if an individual is likely to be a consanguineous offspring (accuracy, 98%), and probability of consanguinity ranges were established according to the total ROH size. Application of the model resulted in the reclassification of the consanguinity status of 12% of the patients. The analysis of a subset of 79 consanguineous cases with the Rare Disease (RD)-Connect Genome-Phenome Analysis Platform, combining variant filtering and homozygosity mapping, enabled a 50% reduction in the number of candidate variants and the identification of homozygous pathogenic variants in 41 patients, with an overall diagnostic yield of 52%. The newly defined consanguinity ranges provide, for the first time, specific ROH thresholds to estimate inbreeding within a pedigree on disparate exome sequencing data, enabling confirmation or (re)classification of consanguineous status, hence increasing the efficiency of molecular diagnosis and reporting on secondary consanguinity findings, as recommended by American College of Medical Genetics and Genomics guidelines.

Abstract COVID-19, caused by the novel SARS-CoV-2 virus, started in China in late 2019, and soon became a global pandemic. With the help of thousands of viral genome sequences that have been accumulating, it has become possible to track the evolution of viral genome over time as it spread across the world. An important question that still needs to be answered is whether any of the common mutations affect the viral properties, and therefore the disease characteristics. Therefore, we sought to understand the effects of mutations in RNA-dependent RNA polymerase (RdRp), particularly the common 14408C&gt;T mutation, on mutation rate and viral spread. By focusing on mutations in the slowly evolving M or E genes, we aimed to minimize the effects of selective pressure. Our results indicate that 14408C&gt;T mutation increases the mutation rate, while the third-most common RdRp mutation, 15324C&gt;T, has the opposite effect. It is possible that 14408C&gt;T mutation may have contributed to the dominance of its co-mutations in Europe and elsewhere.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.

SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic that has affected millions of people worldwide. Pharmaceutical research against COVID-19 and the most frequently used tests for SARS-CoV-2 both depend on the genomic and peptide sequences of the virus for their robustness. Therefore, understanding the mutation rates and content of the virus is critical. Two key proteins for SARS-CoV-2 infection and replication are the S protein, responsible for viral entry into the cells, and RdRp, the RNA polymerase responsible for replicating the viral genome. Due to their roles in the viral cycle, these proteins are crucial for the fitness and infectiousness of the virus. Our previous findings had shown that the two most frequently observed mutations in the SARS-CoV-2 genome, 14408C>T in the RdRp coding region, and 23403A>G in the S gene, are correlated with higher mutation density over time. In this study, we further detail the selection dynamics and the mutation rates of SARS-CoV-2 genes, comparing them between isolates carrying both mutations, and isolates carrying neither. We find that the S gene and the RdRp coding region show the highest variance between the genotypes, and their selection dynamics contrast each other over time. The S gene displays higher tolerance for positive selection in mutant isolates early during the appearance of the double mutant genotype, and undergoes increasing negative selection over time, whereas the RdRp region in the mutant isolates shows strong negative selection throughout the pandemic.

Abstract Pontocerebellar hypoplasia (PCH) is a heterogeneous group of neurodegenerative disorders characterized by hypoplasia and degeneration of the cerebellum and pons. We aimed to identify the clinical, laboratory, and imaging findings of the patients with diagnosed PCH with confirmed genetic analysis. We collected available clinical data, laboratory, and imaging findings in our retrospective multicenter national study of 64 patients with PCH in Turkey. The genetic analysis included the whole-exome sequencing (WES), targeted next-generation sequencing (NGS), or single gene analysis. Sixty-four patients with PCH were 28 female (43.8%) and 36 (56.3%) male. The patients revealed homozygous mutation in 89.1%, consanguinity in 79.7%, pregnancy at term in 85.2%, microcephaly in 91.3%, psychomotor retardation in 98.4%, abnormal neurological findings in 100%, seizure in 63.8%, normal biochemistry and metabolic investigations in 92.2%, and dysmorphic findings in 51.2%. The missense mutation was found to be the most common variant type in all patients with PCH. It was detected as CLP1 ( n = 17) was the most common PCH related gene. The homozygous missense variant c.419G &gt; A (p.Arg140His) was identified in all patients with CLP1. Moreover, all patients showed the same homozygous missense variant c.919G &gt; T (p.A307S) in TSEN54 group ( n = 6). In Turkey, CLP1 was identified as the most common causative gene with the identical variant c.419G &gt; A; p.Arg140His. The current study supports that genotype data on PCH leads to phenotypic variability over a wide phenotypic spectrum.

OBJECTIVE Processes that cause or contribute to cancer, such as aging, exposure to carcinogens, or DNA damage repair deficiency (DDRd), create predictable and traceable nucleotide alterations in one’s genetic code (termed “mutational signatures”). Large studies have previously identified various such mutational signatures across cancers that can be attributed to the specific causative processes. To gain further insight into the processes in glioma development, the authors analyzed mutational signatures in adult diffuse gliomas (DGs). METHODS Twenty-five DGs and paired blood samples were whole exome sequenced. Somatic mutational signatures were identified using 2 different methods. Associations of the signatures with age at diagnosis, molecular subset, and mutational load were investigated. As DDRd-related signatures were frequently observed, germline and somatic DDR gene mutations as well as microsatellite instability (MSI) status were determined for all samples. For validation of signature prevalence, publicly available data from The Cancer Genome Atlas (TCGA) were used. RESULTS Each tumor had a unique combination of signatures. The most common signatures were signature 1 (88%, aging related), signature 3 (52%, homologous recombination related), and signature 15 (56%, mismatch repair related). Eighty-four percent of the tumors contained at least 1 DDRd signature. The findings were validated using public TCGA data. The weight of signature 1 positively correlated with age (r = 0.43) while cumulative weight of DDRd signatures negatively correlated with age (r = −0.16). Each subject had at least 1 germline/somatic alteration in a DDR gene, the most common being the risk single nucleotide polymorphism rs1800734 in MLH1. The rs1800734-AA genotype had a higher cumulative DDRd weight as well as higher mutational load; TP53 was the most common somatically altered DDR gene. MSI was observed in 24% of the tumors. No significant associations of MSI status with mutational load, rs1800734, or the cumulative weight of DDRd signatures were identified. CONCLUSIONS Current findings suggest that DDRd may act as a fundamental mechanism in gliomagenesis rather than being a random, secondary event.

Dihydropyridine receptor congenital myopathy is a recently described congenital myopathy caused by dominant or recessive mutations in the CACNA1S gene. To date, only 11 cases from 7 families were described in a single report. Here, we describe a consanguineous family with three affected children, p resenting congenital hypotonia, contractures, ophthalmoplegia and respiratory insufficiency, with a novel homozygous mutation in the CACNA1S gene. They also showed cognitive delay, pes equinovarus deformity and neurogenic changes that have not been associated with this myopathy in the previous reports. This report expands the phenotypic spectrum of dihydropyridine receptor congenital myopathy and underscores the importance of whole exome sequencing in early onset neuromuscular disorders.

As the underlying pathogen for the COVID-19 pandemic that has affected tens of millions of lives worldwide, SARS-CoV-2 and its mutations are among the most urgent research topics worldwide. Mutations in the virus genome can complicate attempts at accurate testing or developing a working treatment for the disease. Furthermore, because the virus uses its own proteins to replicate its genome, rather than host proteins, mutations in the replication proteins can have cascading effects on the mutation load of the virus genome. Due to the global, rapidly developing nature of the COVID-19 pandemic, local demographics of the virus can be difficult to accurately analyze and track, disproportionate to the importance of such information. Here, we analyzed available, high-quality genome data of SARS-CoV-2 isolates from Turkey and identified their mutations, in comparison to the reference genome, to understand how the local mutatome compares to the global genomes. Our results indicate that viral genomes in Turkey has one of the highest mutation loads and certain mutations are remarkably frequent compared to global genomes. We also made the data on Turkey isolates available on an online database to facilitate further research on SARS-CoV-2 mutations in Turkey.

<h3>Objective</h3> This study presents the neurologic phenotypes of 2 brothers with a novel homozygous <i>COL4A1</i> mutation that was identified in a large Turkish consanguineous cohort of neurogenetic diseases. <h3>Methods</h3> Whole-exome sequencing and bioinformatic analysis of consanguineous families with children affected by early-onset, neurogenetic disorders was performed using the RD-Connect Genome-Phenome Analysis Platform. We also performed clinical, EEG, and neuroimaging analyses in unaffected siblings and parents. <h3>Results</h3> We have identified a homozygous missense mutation in <i>COL4A1</i> (p.Gly1278Ser, NM_001845.5:c.3832G&gt;T) in 2 siblings affected by small vessel brain disease with periventricular leukoencephalopathy and ocular defects. Presenting symptoms included mild weakness, hemiparetic gait, pyramidal findings, and seizures, whereas their intellectual and behavioral functions were normal. Both parents and 5 of the siblings (3 boys and 2 girls) were heterozygous for the variant. They did not show any clinical or laboratory signs of small vessel disease. <h3>Conclusions</h3> <i>COL4A1</i> has previously been associated with dominant small vessel disease of the brain and other organs, manifesting with high penetrance in heterozygous mutation carriers. Our findings provide evidence that <i>COL4A1</i>-related encephalopathy can be inherited in an autosomal recessive manner, which is important for counseling, prognosis, and treatment. Genotype-phenotype correlations remain to be established.

Congenital disorders of glycosylation (CDG) is a heterogeneous group of congenital metabolic diseases with multisystem clinical involvement. ALG3-CDG is a very rare subtype with only 24 cases reported so far.Here, we report two siblings with dysmorphic features, growth retardation, microcephaly, intractable epilepsy, and hemangioma in the frontal, occipital and lumbosacral regions.We studied two siblings by whole exome sequencing. A pathogenic variant in ALG3 (NM_005787.6: c.165C > T; p.Gly55=) that had been previously associated with congenital glycolysis defect type 1d was identified. Their intractable seizures were controlled by ketogenic diet.Although prominent findings of growth retardation and microcephaly seen in our patients have been extensively reported before, presence of hemangioma is a novel finding that may be used as an indication for ALG3-CDG diagnosis. Our patients are the first reported cases whose intractable seizures were controlled with ketogenic diet. This report adds ketogenic diet as an option for treatment of intractable epilepsy in ALG3-CDG.

Background In 2009, we identified TACO1 as a novel mitochondrial disease gene in a single family, however no second family has been described to confirm the role of TACO1 in mitochondrial disease. Objective In this report, we describe two independent consanguineous families carrying pathogenic variants in TACO1, confirming the phenotype. Methods Detailed clinical investigations and whole exome sequencing with haplotype analysis have been performed in several members of the two reported families. Results Clinical phenotype of the patients confirms the originally reported phenotype of a childhood-onset progressive cerebellar and pyramidal syndrome with optic atrophy and learning difficulties. Brain MRI showed periventricular white matter lesions with multiple cystic defects, suggesting leukoencephalopathy in both patients. One patient carried the previously described homozygous TACO1 variant (p.His158ProfsTer8) and haplotype analysis suggested that this variant is a rare founder mutation. The second patient from another family carried a homozygous novel frame shift variant (p.Cys85PhefsTer15). Conclusions The identification of two Turkish families with similar characteristic clinical presentation and an additional homozygous nonsense mutation confirms that TACO1 is a human mitochondrial disease gene. Although most patients with this clinical presentation undergo next generation sequencing analysis, screening for selected founder mutations in the Turkish population based on the precise clinical presentation may reduce time and cost of finding the genetic diagnosis even in the era of massively parallel sequencing.

Low-grade gliomas (LGG) are central nervous system Grade I tumors, and as they progress they are becoming one of the deadliest brain tumors. There is still great need for timely and accurate diagnosis and prognosis of LGG. Herein, we aimed to identify diagnostic and prognostic biomarkers associated with LGG, by employing diverse computational approaches. For this purpose, differential gene expression analysis on high-throughput transcriptomics data of LGG versus corresponding healthy brain tissue, derived from TCGA and GTEx, respectively, was performed. Weighted gene co-expression network analysis of the detected differentially expressed genes was carried out in order to identify modules of co-expressed genes significantly correlated with LGG clinical traits. The genes comprising these modules were further used to construct gene co-expression and protein-protein interaction networks. Based on the network analyses, we derived a consensus of eighteen hub genes, namely, CD74, CD86, CDC25A, CYBB, HLA-DMA, ITGB2, KIF11, KIFC1, LAPTM5, LMNB1, MKI67, NCKAP1L, NUSAP1, SLC7A7, TBXAS1, TOP2A, TYROBP, and WDFY4. All detected hub genes were up-regulated in LGG, and were also associated with unfavorable prognosis in LGG patients. The findings of this study could be applicable in the clinical setting for diagnosing and monitoring LGG.

Leiomyosarcomas of the breast are rare tumors. Less than 16 such cases have been reported in the literature so far. We present a case of a 44 year female patient who was found to have primary leiomyosarcoma of the breast.

Abstract A distinct neurodevelopmental phenotype characterised mainly by mild motor and language delay and facial dysmorphism, caused by heterozygous de novo or dominant variants in the TLK2 gene has recently been described. All cases reported carried either truncating variants located throughout the gene, or missense changes principally located at the C-terminal end of the protein mostly resulting in haploinsufficiency of TLK2 . Through whole exome sequencing, we identified a homozygous missense variant in TLK2 in a patient showing more severe symptoms than those previously described, including cerebellar vermis hypoplasia and West syndrome. Both parents are heterozygous for the variant and clinically unaffected highlighting that recessive variants in TLK2 can also be disease causing and may act through a different pathomechanism.

<h2>Summary</h2> Microtubules help building the cytoskeleton of neurons and other cells. Several components of the gamma-tubulin (γ-tubulin) complex have been previously reported in human neurodevelopmental diseases. We describe two siblings from a consanguineous Turkish family with dysmorphic features, developmental delay, brain malformation, and epilepsy carrying a homozygous mutation (p.Glu311Lys) in <i>TUBGCP2</i> encoding the γ-tubulin complex 2 (GCP2) protein. This variant is predicted to disrupt the electrostatic interaction of GCP2 with GCP3. In primary fibroblasts carrying the variant, we observed a faint delocalization of γ-tubulin during the cell cycle but normal GCP2 protein levels. Through mass spectrometry, we observed dysregulation of multiple proteins involved in the assembly and organization of the cytoskeleton and the extracellular matrix, controlling cellular adhesion and of proteins crucial for neuronal homeostasis including axon guidance. In summary, our functional and proteomic studies link TUBGCP2 and the γ-tubulin complex to the development of the central nervous system in humans.

Goltz syndrome (GS) is a X-linked disorder defined by defects of mesodermal- and ectodermal-derived structures and caused by PORCN mutations. Features include striated skin-pigmentation, ocular and skeletal malformations and supernumerary or hypoplastic nipples. Generally, GS is associated with in utero lethality in males and most of the reported male patients show mosaicism (only three non-mosaic surviving males have been described so far). Also, precise descriptions of neurological deficits in GS are rare and less severe phenotypes might not only be caused by mosaicism but also by less pathogenic mutations suggesting the need of a molecular genetics and functional work-up of these rare variants.We report two cases: one girl suffering from typical skin and skeletal abnormalities, developmental delay, microcephaly, thin corpus callosum, periventricular gliosis and drug-resistant epilepsy caused by a PORCN nonsense-mutation (c.283C > T, p.Arg95Ter). Presence of these combined neurological features indicates that CNS-vulnerability might be a guiding symptom in the diagnosis of GS patients. The other patient is a boy with a supernumerary nipple and skeletal anomalies but also, developmental delay, microcephaly, cerebral atrophy with delayed myelination and drug-resistant epilepsy as predominant features. Skin abnormalities were not observed. Genotyping revealed a novel PORCN missense-mutation (c.847G > C, p.Asp283His) absent in the Genome Aggregation Database (gnomAD) but also identified in his asymptomatic mother. Given that non-random X-chromosome inactivation was excluded in the mother, fibroblasts of the index had been analyzed for PORCN protein-abundance and -distribution, vulnerability against additional ER-stress burden as well as for protein secretion revealing changes.Our combined findings may suggest incomplete penetrance for the p.Asp283His variant and provide novel insights into the molecular etiology of GS by adding impaired ER-function and altered protein secretion to the list of pathophysiological processes resulting in the clinical manifestation of GS.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with high heritability, however, understanding the complexity of the underlying genetic basis has proven to be a challenging task. We hypothesized that dissecting the aberrations in alternative splicing (AS) and their effects on expression networks might provide insight. Therefore, we performed AS and co-expression analyses of total RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) of two pairs of dizygotic (DZ) twins with non-syndromic autism and their parents. We identified 183 differential AS events in 146 genes, seven of them being Simons Foundation Autism Research Initiative (SFARI) Category 1-3 genes, three of which had previously been reported to be alternatively spliced in ASD post-mortem brains. Gene co-expression analysis identified 7 modules with 513 genes, 5 of which were SFARI Category 1 or Category 2 genes. Among differentially AS genes within the modules, ZNF322 and NR4A1 could be potentially interesting targets for further investigations.

Little is known about the mutational processes that shape the genetic landscape of gliomas. Numerous mutational processes leave marks on the genome in the form of mutations, copy number alterations, rearrangements or their combinations. To explore gliomagenesis, we hypothesized that gliomas with different underlying oncogenic mechanisms would have differences in the burden of various forms of these genomic alterations. This was an analysis on adult diffuse gliomas, but IDH-mutant gliomas as well as diffuse midline gliomas H3-K27M were excluded to search for the possible presence of new entities among the very heterogenous group of IDH-WT glioblastomas. The cohort was divided into two molecular subsets: (1) Molecularly-defined GBM (mGBM) as those that carried molecular features of glioblastomas (including TERT promoter mutations, 7/10 pattern, or EGFR-amplification), and (2) those who did not (others). Whole exome sequencing was performed for 37 primary tumors and matched blood samples as well as 8 recurrences. Single nucleotide variations (SNV), short insertion or deletions (indels) and copy number alterations (CNA) were quantified using 5 quantitative metrics (SNV burden, indel burden, copy number alteration frequency-wGII, chromosomal arm event ratio-CAER, copy number amplitude) as well as 4 parameters that explored underlying oncogenic mechanisms (chromothripsis, double minutes, microsatellite instability and mutational signatures). Findings were validated in the TCGA pan-glioma cohort. mGBM and “Others” differed significantly in their SNV (only in the TCGA cohort) and CNA metrics but not indel burden. SNV burden increased with increasing age at diagnosis and at recurrences and was driven by mismatch repair deficiency. On the contrary, indel and CNA metrics remained stable over increasing age at diagnosis and with recurrences. Copy number alteration frequency (wGII) correlated significantly with chromothripsis while CAER and CN amplitude correlated significantly with the presence of double minutes, suggesting separate underlying mechanisms for different forms of CNA.

Although the underlying genetic causes of intellectual disability (ID) continue to be rapidly identified, the biological pathways and processes that could be targets for a potential molecular therapy are not yet known. This study aimed to identify ID-related shared pathways and processes utilizing enrichment analyses.In this multicenter study, causative genes of patients with ID were used as input for Disease Ontology (DO), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes enrichment analysis.Genetic test results of 720 patients from 27 centers were obtained. Patients with chromosomal deletion/duplication, non-ID genes, novel genes, and results with changes in more than one gene were excluded. A total of 558 patients with 341 different causative genes were included in the study. Pathway-based enrichment analysis of the ID-related genes via ClusterProfiler revealed 18 shared pathways, with lysine degradation and nicotine addiction being the most common. The most common of the 25 overrepresented DO terms was ID. The most frequently overrepresented GO biological process, cellular component, and molecular function terms were regulation of membrane potential, ion channel complex, and voltage-gated ion channel activity/voltage-gated channel activity, respectively.Lysine degradation, nicotine addiction, and thyroid hormone signaling pathways are well-suited to be research areas for the discovery of new targeted therapies in ID patients.

Congenital glycosylation disorders are multisystem diseases with heterogeneous clinical manifestations caused by defects in the synthesis of the glycan moiety of glycoproteins or glycolipids or the binding of glycans to proteins and lipids. DPAGT1 (UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphotransferase) is an initiating protein in the biosynthetic pathway of dolichol-linked oligosaccharides required for protein N-glycosylation. Pathogenic variants in DPAGT1 (UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphotransferase) gene cause a rare type of congenital glycosylation disorder called DPAGT1-CDG (formerly CDG-Ij) (OMIM #608093). It is a rare autosomal recessive disease or a milder version with congenital myasthenic syndrome known as DPAGT1-CMS. A severe disease course with hypotonia, cataracts, skeletal deformities, resistant epilepsy, intellectual disability, global developmental delay, premature death has been described in most patients with DPAGT1-CDG.We describe two patients with variants in the DPAGT1 gene: an 8-month-old boy with a homozygous, missense DPAGT1:c.339T>G (p.Phe113Leu) novel variant and a 13-year-old female patient with compound heterozygous variants, DPAGT1:c.466C>T (p.Arg156Cys, R156C) and DPAGT1:c.161+5G>A. While the 8-month-old patient was diagnosed with congenital cataract at the age of 1 month, had dysmorphic findings, and epilepsy, clinical symptoms in the other patient appeared later but with more prominent muscle weakness, behavioral disorder, dysmorphic findings, and no epilepsy.Cholinesterase inhibitor therapy was found to be effective in patients against muscle weakness, supporting DPAGT1 deficiency as the underlying etiology. We started pyridostigmine treatment in our patient with more pronounced muscle weakness, and we saw its benefit. We aimed to present our patients diagnosed with DPAGT1-CDG due to different variants in the same gene and different clinical presentations, treatment and to compare them with other patients in the literature.

Small molecules that enter chemical reactions as a substrate inside or outside the cell, or that emerge as a result of the reactions are known as metabolites. So far, only a fraction of the existing metabolites, numbering in the millions, have yet been identified, and even a smaller portion have been measured. Two main technologies are commonly used for metabolite analysis: analytes, which are usually separated by liquid or gas chromatography, are examined with the aid of either mass spectrometry or nuclear magnetic resonance. With the understanding that metabolites play roles in gene regulation by influencing epigenetic mechanisms, the interest in this field has grown and the field of ‘metabolomics’ emerged, akin to the ‘omics’ approaches applied to the examination of DNA, RNA, and proteins. In addition to the ‘shotgun’ approach, in which a large number of metabolites are measured simultaneously, targeted approaches that monitor the changes of a small number of metabolites under various conditions are also frequently used. In this section, the issues that should be considered in every metabolomics study, regardless of the methodological approach adopted, are discussed; examples of the use of metabolomics approaches, especially in the field of cancer metabolism, are presented; and the importance of big data and biobanks are emphasized.

ÖzObjective: Isocitrate dehydrogenase 1/2 (IDH 1/2) mutations define a group of low-grade gliomas (LGGs) that display more favorable prognosis compared with LGGs without them.Although IDH wild-type (IDHwt) LGGs are classified as low-grade, they almost invariably progress to higher grades and rarely respond to aggressive treatment regimes.Here, we employed a comparative transcriptomic approach to identify key genes that could significantly contribute to the aggressive progression of IDHwt LGGs.Methods: Using The Cancer Genome Atlas LGG cohort data and weighted gene coexpression network analysis methodology, we identified modules that correlated significantly with clinical features.We focused on modules that correlated with telomerase reverse transcriptase (TERT) promoter mutation status, as TERT promoter mutations are shared between glioblastomas and oligodendrogliomas, however, with two opposite prognostic outcomes.We selected module pathways shared between IDH mutant (IDHmt) and IDHwt LGGs and identified genes that were differentially expressed between the two groups.Results: Several synaptic proteins are down-regulated in IDHwt compared with IDHmt, while GNG12 and VIPR2 are up-regulated.Finally, we identified known drugs that could target many of those genes and therefore could be tested against IDHwt LGGs. Conclusion:Targeting of multiple candidate genes identified in this study could provide novel approaches toward the treatment of IDHwt LGGs.

Background/aim: Glioblastoma is the most heterogeneous and the most difficult-to-treat type of brain tumor and one of the deadliest among all cancers. The high plasticity of glioma cancer stem cells and the resistance they develop against multiple modalities of therapy, along with their high heterogeneity, are the main challenges faced during treatment of glioblastoma. Therefore, a better understanding of the stemness characteristics of glioblastoma cells is needed. With the development of various single-cell technologies and increasing applications of machine learning, indices based on transcriptomic and/or epigenomic data have been developed to quantitatively measure cellular states and stemness. In this study, we aimed to develop a glioma-specific stemness score model using scATAC-seq data for the first time. Materials and methods: We first applied three powerful machine-learning algorithms, i.e. random forest, gradient boosting, and extreme gradient boosting, to glioblastoma scRNA-seq data to discover the most important genes associated with cellular states. We then identified promoter and enhancer regions associated with these genes. After downloading the scATAC-seq peaks and their read counts for each patient, we identified the overlapping regions between the single-cell peaks and the peaks of genes obtained through machine-learning algorithms. Then we calculated read counts that were mapped to these overlapping regions. We finally developed a model capable of estimating the stemness score for each glioma cell using overlapping regions and the importance of genes predictive of glioblastoma cellular states. We also created an R package, accessible to all researchers regardless of their coding proficiency. Results: Our results showed that mesenchymal-like stem cells display higher stemness scores compared to neural-progenitor-, oligodendrocyte-progenitor-, and astrocyte-like cells. Conclusion: scATAC-seq can be used to assess heterogeneity in glioblastoma and identify cells with high stemness characteristics. The package is publicly available at https://github.com/Necla/StemnesScoRe and includes documentation with implementation of a real-data experiment.

Even though the effect of inflammation on pathogenesis of obsessive compulsive disorder (OCD) is known, information regarding the underlying mechanisms are yet to be revealed. The NLRP3 inflammasome complex is an important component of the innate immune system that initiates and mediates inflammatory response to a variety of stimuli. This study aims to inquire into a possible association between NLRP3 inflammasome complex and OCD.This case-control study included 103 participants (51 cases with OCD and 52 healthy controls). All participants were evaluated with the Yale Brown Obsessive Compulsive Scale, Hamilton Depression Scale, and Hewitt Multidimensional Perfectionism Scale. RNA and proteins were extracted from peripheral blood mononuclear cells. Expression of NLRP3 inflammasome components were determined using quantitative real-time polymerase chain reaction (PCR) and Western blotting. Levels of Serum IL-1beta and IL-18 cytokine were determined by ELISA.NEK7 and CASP1 mRNA levels were significantly higher in OCD patients, compared to controls. Pro-caspase-1 protein levels were elevated, as well. Regression analysis showed that NEK7 mRNA and pro-caspase-1 protein levels can differentiate OCD and healthy control groups.Our results provide insight into the molecular alterations that could explain the inflammation-OCD association.

ABSTRACT Background Inflammation has a well-known role in the pathogenesis of a range of neuropsychiatric disorders such as major depressive disorder and schizophrenia. Previous studies provided evidence regarding its possible involvement in the etiology of obsessive-compulsive disorder (OCD). However, mechanisms explaining the association of inflammation with OCD are lacking. The NLRP3 inflammasome complex initiates and mediates inflammatory response, which explain one of the most important key mechanisms behind inflammatory response. In this study, we aimed to determine a possible association between NLRP3 inflammasome and OCD, and to evaluate its relationship with clinical features. Methods This case-control study included 103 participants (51 OCD and 52 healthy controls). OCD patients were diagnosed using DSM-IV criteria. All participants were evaluated by psychiatric inventories, i.e. Yale Brown Obsessive Compulsive Scale, Hamilton Depression Scale, and Hewitt Multidimensional Perfectionism Scale. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples. RNA and protein were extracted from PBMCs and expression of NLRP3 inflammasome components were evaluated by quantitative real-time PCR (qPCR) and Western blotting. Serum IL-1beta and IL-18 cytokine levels were determined by ELISA. Results NEK7 and CASP1 mRNA levels were significantly higher in OCD patients, compared to controls. Pro-Caspase-1 protein levels were elevated, as well. Regression analysis showed that NEK7 mRNA and Caspase-1 protein levels can differentiate OCD and healthy control groups. Discussion pro-Caspase-1 mRNA and protein levels in PBMCs, as well as NEK7 mRNA levels are potential biomarker candidates in OCD. Our results may prompt research into possible role of NLRP3 inflammasome in OCD etiology.

Abstract SARS-CoV-2 is a betacoronavirus responsible for human cases of COVID-19, a pandemic with global impact that first emerged in late 2019. Since then, the viral genome has shown considerable variance as the disease spread across the world, in part due to the zoonotic origins of the virus and the human host adaptation process. As a virus with an RNA genome that codes for its own genomic replication proteins, mutations in these proteins can significantly impact the variance rate of the genome, affecting both the survival and infection rate of the virus, and attempts at combating the disease. In this study, we analyzed the mutation densities of viral isolates carrying frequently observed mutations for four proteins in the RNA synthesis complex over time in comparison to wildtype isolates. Our observations suggest mutations in nsp14, an error-correcting exonuclease protein, have the strongest association with increased mutation load in both regions without selective pressure and across the genome, compared to nsp7, 8, and 12, which form the core polymerase complex. We propose nsp14 as a priority research target for understanding genomic variance rate in SARS-CoV-2 isolates, and nsp14 mutations as potential predictors for high mutability strains.

Abstract SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic that has affected millions of people worldwide, with no dedicated treatment or vaccine currently available. As pharmaceutical research against and the most frequently used tests for SARS-CoV-2 infection both depend on the genomic and peptide sequences of the virus for their efficacy, understanding the mutation rates and content of the virus is critical. Two key proteins for SARS-CoV-2 infection and replication are the S protein, responsible for viral entry into the cells, and RdRp, the RNA polymerase responsible for replicating the viral genome. Due to their roles in the viral cycle, these proteins are crucial for the fitness and infectiousness of the virus. Our previous findings had shown that the two most frequently observed mutations in the SARS-CoV-2 genome, 14408C&gt;T in the RdRp coding region, and 23403A&gt;G in the S gene, are correlated with higher mutation density over time. In this study, we further detail the selection dynamics and the mutation rates of SARS-CoV-2 genes, comparing them between isolates carrying both mutations, and isolates carrying neither. We find that the S gene and the RdRp coding region show the highest variance between the genotypes, and their selection dynamics contrast each other over time. The S gene displays higher positive selection in mutant isolates early on, and undergoes increasing negative selection over time, whereas the RdRp region in the mutant isolates shows strong negative selection throughout the pandemic.

Abstract It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbor a fused, branching mitochondrial network, with oxidative phosphorylation (OXPHOS) as the main energy source. A role for mitochondria in hPSC bioenergetics therefore remains uncertain. Here we show that hPSCs have functional respiratory complexes that consume O2 at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis. To help maintain hPSC mitochondrial membrane potential, cell proliferation, and viability, glycolytic ATP is hydrolyzed by the mitochondrial F1F0ATP synthase. Uncoupling protein 2 (UCP2) regulates this unique pattern of hPSC energy metabolism while suppressing reactive oxygen species. Upon differentiation, UCP2 is repressed and cells shift from glycolysis and fatty acid oxidation to glucose oxidation dependency. Ectopic UCP2 expression during differentiation blocks this metabolic transition and blocks proper embryoid body formation. Combined, our data show that hPSC have distinct bioenergetics regulated by UCP2 and repression of UCP2 is required for metabolic reprogramming along differentiation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4360. doi:10.1158/1538-7445.AM2011-4360

Abstract Since its emergence in Wuhan, China in late 2019, the origin and evolution of SARS-CoV-2 have been among the most debated issues related to COVID-19. Throughout its spread around the world, the viral genome continued acquiring new mutations and some of them became widespread. Among them, 14408 C&gt;T and 23403 A&gt;G mutations in RdRp and S, respectively, became dominant in Europe and the US, which led to debates regarding their effects on the mutability and transmissibility of the virus. In this study, we aimed to investigate possible differences between time-dependent variation of mutation densities (MDe) of viral strains that carry these two mutations and those that do not. Our analyses at the genome and gene level led to two important findings: First, time-dependent changes in the average MDe of circulating SARS-CoV-2 genomes showed different characteristics before and after the beginning of April, when daily new case numbers started levelling off. Second, this pattern was much delayed or even non-existent for the “mutant” (MT) strain that harbored both 14408 C&gt;T and 23403 A&gt;G mutations. Although these differences were not limited to a few hotspots, it is intriguing that the MDe increase is most evident in two critical genes, S and Orf1ab, which are also the genes that harbor the defining mutations of the MT genotype. The nature of these unexpected relationships warrant further research.

ABSTRACT Background Association between inflammation and depression has been known for a long time. Activation of pro-inflammatory molecular complexes such as inflammasomes in depression was suggested as the most relevant hypothesis among many others. Psychological stress is considered to cause sterile inflammation through inflammasomes, and the NLRP3 inflammasome was proposed as a crucial molecule for the pro-inflammatory response in depression. Objective In the current study, we aimed to explore the relationship of NLRP3 inflammasome and its regulatory protein NEK7 with major depressive disorder in a drug naïve study sample. Methods In total 58 patients with major depressive disorder and 58 age and gender-matched healthy persons were included. The mRNA expressions of NLRP3, ASC, caspase-1 and NEK7 coding proteins were evaluated with quantitative PCR, plasma IL-1β levels were detected by ELISA. Results Patients with major depressive disorder had higher gene expressions of NLRP3 (p= 0.03) and ASC (p= 0.002) compared to healthy persons. Higher gene expressions of NLRP3 (OR= 1.17, 95% CI= 1.01, 1.37, p= 0.04), ASC (OR= 1.45, 95% CI= 1.15, 1.82, p= 0.002) and NEK7 (OR= 1.33, 95% CI= 1.08, 1.63, p= 0.007) were related to the increased likelihood of having major depressive disorder. Conclusion The results of this study support the role of NLRP3 inflammasome in the increased risk for major depressive disorder.

Objective:The proteome is all the proteins produced or modified by an organism or system.Proteomics, which is an important component of functional genomics, describes the detection and characterization of a complete set of proteins present in a cell, organ, or organism at a given time.The katanin-like 2 (KATNAL2) gene encodes a protein with a microtubule-severing function, based on studies in cell cultures and lower organisms.However, there are no functional studies investigating the function of this gene in humans, yet.In this study, we aimed to perform proteomic analysis to investigate the effects of the variant in the KATNAL2 gene on the binding of various proteins to microtubules in the fibroblasts of a patient with epilepsy, autistic symptoms and intellectual disability with a homozygous pathogenic variant in the KATNAL2 gene.Methods: Fibroblasts obtained from the patient by skin biopsy were grown in cell culture and protein isolation was performed from them.Following pelleting of microtubules and associated proteins, proteomic analysis was performed using liquid chromatography followed by tandem mass spectrometry.Results: Quantitative data analysis was performed using the SAINTexpress tool on 5 different fractions cut from the gel, identifying 1246 proteins, 38 of which differentially bound to microtubules between patient and control cells.The data obtained from the proteomic and the bioinformatic analyzes indicate that because of the mutation in the KATNAL2 gene in the patient, the extracellular matrix proteins such as collagens that interact with the cytoskeleton are organized differently from those in normal cells. Conclusion:Here, we leveraged the power of proteomics to identify the altered interactome of the microtubules in skin fibroblasts with a missense mutation in KATNAL2.Our results point to altered extracellular matrix-cell interactions in KATNAL2-mutant fibroblasts.

Cardiovascular risk in depression has been investigated in small clinical samples and population-based studies revealing inconclusive results. However, cardiovascular risk in drug-naive depressed patients has not been tested extensively.Body mass index-based Framingham Cardiovascular Risk Scores and soluble intercellular adhesion molecule-1 (sICAM-1) levels were used to assess the risk of cardiovascular disease in drug-naive depressed patients and healthy volunteers.There were no significant differences in Framingham Cardiovascular Risk Scores and individually assessed risk variables between patients and healthy controls (HC). Both groups were comparable in terms of sICAM-1.The widely recognized association between cardiovascular risk and major depression might be more prominent in older depressed patients and patients with recurring episodes.

Additional file 1. Summary of clinical findings described in male PORCN patients.

Alzheimer's disease (AD) is a neurodegenerative disorder that is the most common cause of dementia in the elderly, and intracellular neurofibrillary tangles (NFTs) are one of the pathological features of AD. Recent studies have suggested long noncoding RNAs (lncRNAs) play important roles in AD. Competing endogenous RNAs (ceRNAs) is a mechanism that has recently been proposed, in which lncRNAs compete for common miRNA-binding sites with mRNAs. However, the roles of lncRNAs and ceRNA in AD NFTs is limited. In this study, we constructed a global triple network based on ceRNA theory, then an AD NFT lncRNA–mRNA network (NFTLMN) was generated. By analyzing the NFTLMN, three lncRNAs (AP000265.1, KB-1460A1.5 and RP11-145M9.4), which are highly related with AD NFTs were identified. To further explore the cross-talk between mRNAs and lncRNAs, a clustering module analysis was performed on the NFTLMN and two AD NFT related modules were identified. Our study provides a better understanding of the molecular basis of AD NFTs and may offer novel treatment strategies for AD.

Mitochondrial diseases are a clinically heterogeneous group of disorders caused by dysfunction of the mitochondrial respiratory chain. Some mitochondrial disorders affect a single organ, while many involve multiple systems such as skeletal muscle, brain, heart and liver, leading to diagnostic difficulties. Here we present three patients who were originally suspected to have a primary disease of skeletal muscle, leukodystrophy and brain malformation. Patients were recruited from three paediatric neurology clinics in Turkey: Izmir, Malatya and Diyarbakir. Whole exome sequencing (WES) was performed using Illumina exome capture (38 Mb target). Data analysis was carried out on the RD-Connect Genome-Phenome Analysis Platform (https://platform.rd-connect.eu/). Standard filtering criteria with MAF<1% and high/moderate VEP were used, as well as a list consisting of >5,000 medically interpretable genes. We identified a homozygous frameshift variant (p.Glu41GlyfsTer10) in NDUFA12 and a homozygous missense variant (p.Gln85His) in NDUFS3, both associated with Leigh syndrome due to mitochondrial complex I deficiency (OMIM# 256000). We also identifid a homozygous nonsense variant (p.His158ProfsTer8) in TACO1 associated with mitochondrial complex IV deficiency (OMIM# 220110), the second patient to be described worldwide so far. All the variants were highly pathogenic and were absent in the control population, suggesting they were disease-causing. Critical clinical review and metabolic analysis confirmed the mitochondrial deficiency. Next generation sequencing has the advantage of allowing an unbiased genetic diagnosis. We described three cases that had been initially diagnosed as myopathy, brain malformation and leukodystrophy, and WES resulted in the diagnoses of mitochondrial disorders. Importantly, this will allow for appropriate clinical management of these patients.

The role of hematogenous (hMΦ) and microglial (mMΦ) macrophages following spinal cord injury (SCI) remains unclear as they are not distinguished easily from each other in the lesion area. We have recently described the temporal and spatial response to SCI of each MΦ population using the lys-EGFP-ki mouse that enables EGFP+ hMΦ to be distinguished from EGFP− mMΦ at the lesion site. In the present study, we characterized the response of monocyte and hMΦ subsets and mMΦ to SCI. We describe, for the first time, the responses of circulating classical (pro-inflammatory) and non-classical monocyte subsets to SCI. Additionally, we show the presence of classical and non-classical hMΦ at the SCI lesion. Importantly, we demonstrate that the ‘classical pro-inflammatory’ hMΦ respond in the acute (1 d, 3 d) stages of SCI while the ‘non-classical’ hMΦ respond in the sub-acute (7 d, 14 d) phase of SCI. At later time points (6 weeks post injury) classical hMΦ return to the injury site. Our study offers new insight into the cellular inflammatory response that occurs after SCI and suggests that the timing and targets of anti-inflammatory therapies may be crucial to maximize neuroprotection at the acute and more chronic stages of SCI.

COVID-19, caused by the novel SARS-CoV-2 virus, started in China in late 2019, and soon became a global pandemic.With the help of thousands of viral genome sequences that have been accumulating, it has become possible to track the evolution of viral genome over time as it spread across the world.An important question that still needs to be answered is whether any of the common mutations affect the viral properties, and therefore the disease characteristics.Therefore, we sought to understand the effects of mutations in RNA-dependent RNA polymerase (RdRp), particularly the common 14408C>T mutation, on mutation rate and viral spread.By focusing on mutations in the slowly evolving M or E genes, we aimed to minimize the effects of selective pressure.Our results indicate that 14408C>T mutation increases the mutation rate, while the third-most common RdRp mutation, 15324C>T, has the opposite effect.It is possible that 14408C>T mutation may have contributed to the dominance of its co-mutations in Europe and elsewhere.