Joseph D. Buxbaum

Eleven susceptibility loci for late-onset Alzheimer's disease (LOAD) were identified by previous studies; however, a large portion of the genetic risk for this disease remains unexplained. We conducted a large, two-stage meta-analysis of genome-wide association studies (GWAS) in individuals of European ancestry. In stage 1, we used genotyped and imputed data (7,055,881 SNPs) to perform meta-analysis on 4 previously published GWAS data sets consisting of 17,008 Alzheimer's disease cases and 37,154 controls. In stage 2, 11,632 SNPs were genotyped and tested for association in an independent set of 8,572 Alzheimer's disease cases and 11,312 controls. In addition to the APOE locus (encoding apolipoprotein E), 19 loci reached genome-wide significance (P < 5 × 10(-8)) in the combined stage 1 and stage 2 analysis, of which 11 are newly associated with Alzheimer's disease.

Naomi Wray and colleagues report an analysis of genome-wide association data sets from the Psychiatric Genomics Consortium for five psychiatric disorders. They find that common variation explains 17–29% of the variance in liability and provide further support for a shared genetic etiology for these related psychiatric disorders. Most psychiatric disorders are moderately to highly heritable. The degree to which genetic variation is unique to individual disorders or shared across disorders is unclear. To examine shared genetic etiology, we use genome-wide genotype data from the Psychiatric Genomics Consortium (PGC) for cases and controls in schizophrenia, bipolar disorder, major depressive disorder, autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD). We apply univariate and bivariate methods for the estimation of genetic variation within and covariation between disorders. SNPs explained 17–29% of the variance in liability. The genetic correlation calculated using common SNPs was high between schizophrenia and bipolar disorder (0.68 ± 0.04 s.e.), moderate between schizophrenia and major depressive disorder (0.43 ± 0.06 s.e.), bipolar disorder and major depressive disorder (0.47 ± 0.06 s.e.), and ADHD and major depressive disorder (0.32 ± 0.07 s.e.), low between schizophrenia and ASD (0.16 ± 0.06 s.e.) and non-significant for other pairs of disorders as well as between psychiatric disorders and the negative control of Crohn's disease. This empirical evidence of shared genetic etiology for psychiatric disorders can inform nosology and encourages the investigation of common pathophysiologies for related disorders.

The Alzheimer Disease Genetics Consortium (ADGC) performed a genome-wide association study of late-onset Alzheimer disease using a three-stage design consisting of a discovery stage (stage 1) and two replication stages (stages 2 and 3). Both joint analysis and meta-analysis approaches were used. We obtained genome-wide significant results at MS4A4A (rs4938933; stages 1 and 2, meta-analysis P (P(M)) = 1.7 × 10(-9), joint analysis P (P(J)) = 1.7 × 10(-9); stages 1, 2 and 3, P(M) = 8.2 × 10(-12)), CD2AP (rs9349407; stages 1, 2 and 3, P(M) = 8.6 × 10(-9)), EPHA1 (rs11767557; stages 1, 2 and 3, P(M) = 6.0 × 10(-10)) and CD33 (rs3865444; stages 1, 2 and 3, P(M) = 1.6 × 10(-9)). We also replicated previous associations at CR1 (rs6701713; P(M) = 4.6 × 10(-10), P(J) = 5.2 × 10(-11)), CLU (rs1532278; P(M) = 8.3 × 10(-8), P(J) = 1.9 × 10(-8)), BIN1 (rs7561528; P(M) = 4.0 × 10(-14), P(J) = 5.2 × 10(-14)) and PICALM (rs561655; P(M) = 7.0 × 10(-11), P(J) = 1.0 × 10(-10)), but not at EXOC3L2, to late-onset Alzheimer's disease susceptibility.

Autism spectrum disorder (ASD) is a highly heritable and heterogeneous group of neurodevelopmental phenotypes diagnosed in more than 1% of children. Common genetic variants contribute substantially to ASD susceptibility, but to date no individual variants have been robustly associated with ASD. With a marked sample-size increase from a unique Danish population resource, we report a genome-wide association meta-analysis of 18,381 individuals with ASD and 27,969 controls that identified five genome-wide-significant loci. Leveraging GWAS results from three phenotypes with significantly overlapping genetic architectures (schizophrenia, major depression, and educational attainment), we identified seven additional loci shared with other traits at equally strict significance levels. Dissecting the polygenic architecture, we found both quantitative and qualitative polygenic heterogeneity across ASD subtypes. These results highlight biological insights, particularly relating to neuronal function and corticogenesis, and establish that GWAS performed at scale will be much more productive in the near term in ASD.

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.

Autism spectrum disorders (ASDs) are common, heritable neurodevelopmental conditions. The genetic architecture of ASDs is complex, requiring large samples to overcome heterogeneity. Here we broaden coverage and sample size relative to other studies of ASDs by using Affymetrix 10K SNP arrays and 1,181 [corrected] families with at least two affected individuals, performing the largest linkage scan to date while also analyzing copy number variation in these families. Linkage and copy number variation analyses implicate chromosome 11p12-p13 and neurexins, respectively, among other candidate loci. Neurexins team with previously implicated neuroligins for glutamatergic synaptogenesis, highlighting glutamate-related genes as promising candidates for contributing to ASDs.

Autism spectrum disorders (ASDs) are childhood neurodevelopmental disorders with complex genetic origins. Previous studies focusing on candidate genes or genomic regions have identified several copy number variations (CNVs) that are associated with an increased risk of ASDs. Here we present the results from a whole-genome CNV study on a cohort of 859 ASD cases and 1,409 healthy children of European ancestry who were genotyped with approximately 550,000 single nucleotide polymorphism markers, in an attempt to comprehensively identify CNVs conferring susceptibility to ASDs. Positive findings were evaluated in an independent cohort of 1,336 ASD cases and 1,110 controls of European ancestry. Besides previously reported ASD candidate genes, such as NRXN1 (ref. 10) and CNTN4 (refs 11, 12), several new susceptibility genes encoding neuronal cell-adhesion molecules, including NLGN1 and ASTN2, were enriched with CNVs in ASD cases compared to controls (P = 9.5 x 10(-3)). Furthermore, CNVs within or surrounding genes involved in the ubiquitin pathways, including UBE3A, PARK2, RFWD2 and FBXO40, were affected by CNVs not observed in controls (P = 3.3 x 10(-3)). We also identified duplications 55 kilobases upstream of complementary DNA AK123120 (P = 3.6 x 10(-6)). Although these variants may be individually rare, they target genes involved in neuronal cell-adhesion or ubiquitin degradation, indicating that these two important gene networks expressed within the central nervous system may contribute to the genetic susceptibility of ASD.

Analysis of de novo CNVs (dnCNVs) from the full Simons Simplex Collection (SSC) (N = 2,591 families) replicates prior findings of strong association with autism spectrum disorders (ASDs) and confirms six risk loci (1q21.1, 3q29, 7q11.23, 16p11.2, 15q11.2-13, and 22q11.2). The addition of published CNV data from the Autism Genome Project (AGP) and exome sequencing data from the SSC and the Autism Sequencing Consortium (ASC) shows that genes within small de novo deletions, but not within large dnCNVs, significantly overlap the high-effect risk genes identified by sequencing. Alternatively, large dnCNVs are found likely to contain multiple modest-effect risk genes. Overall, we find strong evidence that de novo mutations are associated with ASD apart from the risk for intellectual disability. Extending the transmission and de novo association test (TADA) to include small de novo deletions reveals 71 ASD risk loci, including 6 CNV regions (noted above) and 65 risk genes (FDR ≤ 0.1).

Alzheimer disease (AD) is characterized neuropathologically by the presence of amyloid beta-peptide (Abeta)-containing plaques and neurofibrillary tangles composed of abnormal tau protein. Considerable controversy exists as to whether the extent of accumulation of Abeta correlates with dementia and whether Abeta alterations precede or follow changes in tau.To determine whether accumulation of Abeta correlates with the earliest signs of cognitive deterioration and to define the relationship between Abeta accumulation and early tau changes.Postmortem cross-sectional study of 79 nursing home residents with Clinical Dementia Rating (CDR) scale scores of 0.0 to 5.0 who died between 1986 and 1997, comparing the levels of Abeta variants in the cortices of the subjects with no (CDR score, 0.0 [n = 16]), questionable (CDR score, 0.5 [n = 11]), mild (CDR score, 1.0 [n = 22]), moderate (CDR score, 2.0 [n = 15]), or severe (CDR score, 4.0 or 5.0 [n = 15]) dementia.Levels of total Abeta peptides with intact or truncated amino termini and ending in either amino acid 40 (A(beta)x-40) or 42 (A(beta)x-42) in 5 neocortical brain regions as well as levels of tau protein undergoing early conformational changes in frontal cortex, as a function of CDR score.The levels of both A(beta)x-40 and A(beta)x-42 were elevated even in cases classified as having questionable dementia (CDR score = 0.5), and increases of both peptides correlated with progression of dementia. Levels of the more fibril-prone A(beta)x-42 peptide were higher than those of A(beta)x-40 in nondemented cases and remained higher throughout progression of disease in all regions examined. Finally, increases in A(beta)x-40 and A(beta)x-42 precede significant tau pathology at least in the frontal cortex, an area chosen for examination because of the absence of neuritic changes in the absence of disease.In this study, levels of total A(beta)x-40 and A(beta)x-42 were elevated early in dementia and levels of both peptides were strongly correlated with cognitive decline. Of particular interest, in the frontal cortex, Abeta was elevated before the occurrence of significant tau pathology. These results support an important role for Abeta in mediating initial pathogenic events in AD dementia and suggest that treatment strategies targeting the formation, accumulation, or cytotoxic effects of Abeta should be pursued.

Neuropathological and brain imaging studies suggest that schizophrenia may result from neurodevelopmental defects. Cytoarchitectural studies indicate cellular abnormalities suggestive of a disruption in neuronal connectivity in schizophrenia, particularly in the dorsolateral prefrontal cortex. Yet, the molecular mechanisms underlying these findings remain unclear. To identify molecular substrates associated with schizophrenia, DNA microarray analysis was used to assay gene expression levels in postmortem dorsolateral prefrontal cortex of schizophrenic and control patients. Genes determined to have altered expression levels in schizophrenics relative to controls are involved in a number of biological processes, including synaptic plasticity, neuronal development, neurotransmission, and signal transduction. Most notable was the differential expression of myelination-related genes suggesting a disruption in oligodendrocyte function in schizophrenia.

A key component of genetic architecture is the allelic spectrum influencing trait variability. For autism spectrum disorder (herein termed autism), the nature of the allelic spectrum is uncertain. Individual risk-associated genes have been identified from rare variation, especially de novo mutations. From this evidence, one might conclude that rare variation dominates the allelic spectrum in autism, yet recent studies show that common variation, individually of small effect, has substantial impact en masse. At issue is how much of an impact relative to rare variation this common variation has. Using a unique epidemiological sample from Sweden, new methods that distinguish total narrow-sense heritability from that due to common variation and synthesis of results from other studies, we reach several conclusions about autism's genetic architecture: its narrow-sense heritability is ∼52.4%, with most due to common variation, and rare de novo mutations contribute substantially to individual liability, yet their contribution to variance in liability, 2.6%, is modest compared to that for heritable variation.

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyperactivity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.

Over 100 genetic loci harbor schizophrenia-associated variants, yet how these variants confer liability is uncertain. The CommonMind Consortium sequenced RNA from dorsolateral prefrontal cortex of people with schizophrenia (N = 258) and control subjects (N = 279), creating a resource of gene expression and its genetic regulation. Using this resource, ∼20% of schizophrenia loci have variants that could contribute to altered gene expression and liability. In five loci, only a single gene was involved: FURIN, TSNARE1, CNTN4, CLCN3 or SNAP91. Altering expression of FURIN, TSNARE1 or CNTN4 changed neurodevelopment in zebrafish; knockdown of FURIN in human neural progenitor cells yielded abnormal migration. Of 693 genes showing significant case-versus-control differential expression, their fold changes were ≤ 1.33, and an independent cohort yielded similar results. Gene co-expression implicates a network relevant for schizophrenia. Our findings show that schizophrenia is polygenic and highlight the utility of this resource for mechanistic interpretations of genetic liability for brain diseases.

Spontaneously arising (de novo) mutations have an important role in medical genetics. For diseases with extensive locus heterogeneity, such as autism spectrum disorders (ASDs), the signal from de novo mutations is distributed across many genes, making it difficult to distinguish disease-relevant mutations from background variation. Here we provide a statistical framework for the analysis of excesses in de novo mutation per gene and gene set by calibrating a model of de novo mutation. We applied this framework to de novo mutations collected from 1,078 ASD family trios, and, whereas we affirmed a significant role for loss-of-function mutations, we found no excess of de novo loss-of-function mutations in cases with IQ above 100, suggesting that the role of de novo mutations in ASDs might reside in fundamental neurodevelopmental processes. We also used our model to identify ∼1,000 genes that are significantly lacking in functional coding variation in non-ASD samples and are enriched for de novo loss-of-function mutations identified in ASD cases.

Alzheimer's disease (AD) is characterized by the deposition of senile plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are composed of aggregated β-amyloid (Aβ) 40/42(43) peptides. Evidence implicates a central role for Aβ in the pathophysiology of AD. Mutations in βAPP and presenilin 1 (PS1) lead to elevated secretion of Aβ, especially the more amyloidogenic Aβ42. Immunohistochemical studies have also emphasized the importance of Aβ42 in initiating plaque pathology. Cell biological studies have demonstrated that Aβ is generated intracellularly. Recently, endogenous Aβ42 staining was demonstrated within cultured neurons by confocal immunofluorescence microscopy and within neurons of PS1 mutant transgenic mice. A central question about the role of Aβ in disease concerns whether extracellular Aβ deposition or intracellular Aβ accumulation initiates the disease process. Here we report that human neurons in AD-vulnerable brain regions specifically accumulate γ-cleaved Aβ42 and suggest that this intraneuronal Aβ42 immunoreactivity appears to precede both NFT and Aβ plaque deposition. This study suggests that intracellular Aβ42 accumulation is an early event in neuronal dysfunction and that preventing intraneuronal Aβ42 aggregation may be an important therapeutic direction for the treatment of AD. Alzheimer's disease (AD) is characterized by the deposition of senile plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are composed of aggregated β-amyloid (Aβ) 40/42(43) peptides. Evidence implicates a central role for Aβ in the pathophysiology of AD. Mutations in βAPP and presenilin 1 (PS1) lead to elevated secretion of Aβ, especially the more amyloidogenic Aβ42. Immunohistochemical studies have also emphasized the importance of Aβ42 in initiating plaque pathology. Cell biological studies have demonstrated that Aβ is generated intracellularly. Recently, endogenous Aβ42 staining was demonstrated within cultured neurons by confocal immunofluorescence microscopy and within neurons of PS1 mutant transgenic mice. A central question about the role of Aβ in disease concerns whether extracellular Aβ deposition or intracellular Aβ accumulation initiates the disease process. Here we report that human neurons in AD-vulnerable brain regions specifically accumulate γ-cleaved Aβ42 and suggest that this intraneuronal Aβ42 immunoreactivity appears to precede both NFT and Aβ plaque deposition. This study suggests that intracellular Aβ42 accumulation is an early event in neuronal dysfunction and that preventing intraneuronal Aβ42 aggregation may be an important therapeutic direction for the treatment of AD. Alzheimer's disease (AD) neuropathology is classically characterized by the accumulation of senile plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are composed of parenchymal and cerebrovascular aggregates of β-amyloid (Aβ) 40/42(43) peptides. Increasing evidence indicates that Aβ plays a central role in the pathophysiology of AD. Individuals with Down's syndrome (DS) have an extra copy of chromosome 21, where the gene encoding the β-amyloid precursor protein (βAPP) is localized, and invariably develop AD pathology at an early age. Mutations in βAPP segregate with some forms of autosomal dominant familial AD (FAD). Transgenic mice bearing FAD βAPP mutations develop striking AD-like senile plaque pathology.1Selkoe DJ The cell biology of β-amyloid precursor protein and presenilin in Alzheimer's disease.Trends Cell Biol. 1998; 8: 447-453Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar FAD mutations in βAPP and presenilin 1 (PS1) lead to elevated secretion of Aβ, especially the more amyloidogenic Aβ42. In addition, immunohistochemical studies have underscored the importance of Aβ42 as the initiator of plaque pathology in AD and DS.2Iwatsubo T Odaka A Suzuki N Mizusawa H Nukina N Ihara Y Visualization of A β 42(43) and A β 40 in senile plaques with end-specific A β monoclonals: evidence that an initially deposited species is A β 42(43).Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1582) Google Scholar, 3Lemere CA Blusztajn JK Yamaguchi H Wisniewski T Saido TC Selkoe DJ Sequence of deposition of heterogeneous amyloid β-peptides and APO E in Down syndrome: implications for initial events in amyloid plaque formation.Neurobiol Dis. 1996; 3: 16-32Crossref PubMed Scopus (469) Google Scholar Over the past few years cell biological studies support the view that Aβ is generated intracellularly1Selkoe DJ The cell biology of β-amyloid precursor protein and presenilin in Alzheimer's disease.Trends Cell Biol. 1998; 8: 447-453Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar, 4Xu H Sweeney D Wang R Thinakaran G Lo AC Sisodia SS Greengard P Gandy S Generation of Alzheimer β-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation.Proc Natl Acad Sci USA. 1997; 94: 3748-3752Crossref PubMed Scopus (255) Google Scholar, 5Wild-Bode C Yamazaki T Capell A Leimer U Steiner H Ihara Y Haass C Intracellular generation and accumulation of amyloid β-peptide terminating at amino acid 42.J Biol Chem. 1997; 272: 16085-16088Crossref PubMed Scopus (290) Google Scholar, 6Skovronsky DM Doms RW Lee VM Detection of a novel intraneuronal pool of insoluble amyloid β protein that accumulates with time in culture.J Cell Biol. 1998; 141: 1031-1039Crossref PubMed Scopus (270) Google Scholar, 7Hartmann T Bieger SC Bruhl B Tienari PJ Ida N Allsop D Roberts GW Masters CL Dotti CG Unsicker K Beyreuther K Distinct sites of intracellular production for Alzheimer's disease A β40/42 amyloid peptides.Nat Med. 1997; 3: 1016-1020Crossref PubMed Scopus (656) Google Scholar, 8Cook DG Forman MS Sung JC Leight S Kolson DL Iwatsubo T Lee VM Doms RW Alzheimer's A β(1–42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells.Nat Med. 1997; 3: 1021-1023Crossref PubMed Scopus (437) Google Scholar, 9Greenfield JP Tsai J Gouras GK Hai B Thinakaran G Checler F Sisodia SS Greengard P Xu H Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer β-amyloid peptides.Proc Natl Acad Sci USA. 1999; 96: 742-747Crossref PubMed Scopus (336) Google Scholar, 10Perez RG Soriano S Hayes JD Ostaszewski B Xia W Selkoe DJ Chen X Stokin GB Koo EH Mutagenesis identifies new signals for β-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42.J Biol Chem. 1999; 274: 18851-18856Crossref PubMed Scopus (346) Google Scholar from the endoplasmic reticulum (ER)1Selkoe DJ The cell biology of β-amyloid precursor protein and presenilin in Alzheimer's disease.Trends Cell Biol. 1998; 8: 447-453Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar, 7Hartmann T Bieger SC Bruhl B Tienari PJ Ida N Allsop D Roberts GW Masters CL Dotti CG Unsicker K Beyreuther K Distinct sites of intracellular production for Alzheimer's disease A β40/42 amyloid peptides.Nat Med. 1997; 3: 1016-1020Crossref PubMed Scopus (656) Google Scholar, 8Cook DG Forman MS Sung JC Leight S Kolson DL Iwatsubo T Lee VM Doms RW Alzheimer's A β(1–42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells.Nat Med. 1997; 3: 1021-1023Crossref PubMed Scopus (437) Google Scholar to the trans-Golgi network (TGN),4Xu H Sweeney D Wang R Thinakaran G Lo AC Sisodia SS Greengard P Gandy S Generation of Alzheimer β-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation.Proc Natl Acad Sci USA. 1997; 94: 3748-3752Crossref PubMed Scopus (255) Google Scholar and the endosomal-lysosomal system.10Perez RG Soriano S Hayes JD Ostaszewski B Xia W Selkoe DJ Chen X Stokin GB Koo EH Mutagenesis identifies new signals for β-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42.J Biol Chem. 1999; 274: 18851-18856Crossref PubMed Scopus (346) Google Scholar Recently, endogenous Aβ42 staining was demonstrated within cultured primary neurons by confocal immunofluorescence microscopy9Greenfield JP Tsai J Gouras GK Hai B Thinakaran G Checler F Sisodia SS Greengard P Xu H Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer β-amyloid peptides.Proc Natl Acad Sci USA. 1999; 96: 742-747Crossref PubMed Scopus (336) Google Scholar and within neurons of human PS1 mutant transgenic mice by immunocytochemical light microscopy.11Chui DH Tanahashi H Ozawa K Ikeda S Checler F Ueda O Suzuki H Araki W Inoue H Shirotani K Takahashi K Gallyas F Tabira T Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.Nat Med. 1999; 5: 560-564Crossref PubMed Scopus (323) Google Scholar A central question on the role of Aβ in AD is whether extracellular Aβ deposition or intracellular Aβ accumulation is initiating the disease process. Several groups had postulated the presence of intraneuronal Aβ immunostaining. However, the Aβ immunoreactivity observed in these studies was compromised by that of full-length βAPP, because these Aβ antibodies also recognize full-length βAPP.12Stern RA Otvos Jr, L Trojanowski JQ Lee VM Monoclonal antibodies to a synthetic peptide homologous with the first 28 amino acids of Alzheimer's disease β-protein recognize amyloid and diverse glial and neuronal cell types in the central nervous system.Am J Pathol. 1989; 134: 973-978PubMed Google Scholar, 13Mak K Yang F Vinters HV Frautschy SA Cole GM Polyclonals to β-amyloid(1–42) identify most plaque and vascular deposits in Alzheimer cortex, but not striatum.Brain Res. 1994; 667: 138-142Crossref PubMed Scopus (66) Google Scholar, 14LaFerla FM Troncoso JC Strickland DK Kawas CH Jay G Neuronal cell death in Alzheimer's disease correlates with apoE uptake and intracellular Abeta stabilization.J Clin Invest. 1997; 100: 310-320Crossref PubMed Scopus (151) Google Scholar In addition, NFTs had previously been reported to be immunoreactive to Aβ.15Masters CL Multhaup G Simms G Pottgiesser J Martins RN Beyreuther K Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels.EMBO J. 1985; 4: 2757-2763Crossref PubMed Scopus (809) Google Scholar, 16Hyman BT Van Hoesen GW Beyreuther K Masters CL A4 amyloid protein immunoreactivity is present in Alzheimer's disease neurofibrillary tangles.Neurosci Lett. 1989; 101: 352-355Crossref PubMed Scopus (57) Google Scholar This association of Aβ with NFTs was subsequently believed to be the result of artifactual “shared” epitopes.17Allsop D Haga S Bruton C Ishii T Roberts GW Neurofibrillary tangles in some cases of dementia pugilistica share antigens with amyloid β-protein of Alzheimer's disease.Am J Pathol. 1990; 136: 255-260PubMed Google Scholar We now report that human neurons in AD-vulnerable brain regions specifically accumulate γ-cleaved Aβ42 but not the more abundantly secreted Aβ40. We also demonstrate intraneuronal Aβ42 staining in neurons in both the absence and presence of NFTs. Our observations in adjacent sections of intraneuronal Aβ42 staining and hyperphosphorylated tau staining suggest that neuronal Aβ42 staining is more abundant and therefore may precede NFTs, which would exclude the possibility of cross-reactivity of shared epitopes. Furthermore, we observe the earliest Aβ42 immunoreactive SPs developing along the projections and at terminals of early Aβ42 accumulating neurons, suggesting a mechanism for the previously hypothesized regional specificity of AD disease progression within the brain.18Hyman BT Van Hoesen GW Kromer LJ Damasio AR Perforant pathway changes and the memory impairment of Alzheimer's disease.Ann Neurol. 1986; 20: 472-481Crossref PubMed Scopus (574) Google Scholar Polyclonal rabbit Aβ40 (RU226) and Aβ42 (RU228) C-terminal specific antibodies were generated at Rockefeller University (RU). Polyclonal rabbit Aβ40 and Aβ42 C-terminal antibodies were also obtained commercially (QCB). The results obtained with these two sets of antibodies were similar and were confirmed using well-characterized polyclonal rabbit Aβ40 (FCA3340) and Aβ42 (FCA3542. antibodies19Barelli H Lebeau A Vizzavona J Delaere P Chevallier N Drouot C Marambaud P Ancolio K Buxbaum JD Khorkova O Heroux J Sahasrabudhe S Martinez J Warter JM Mohr M Checler F Characterization of new polyclonal antibodies specific for 40 and 42 amino acid-long amyloid β peptides: their use to examine the cell biology of presenilins and the immunohistochemistry of sporadic Alzheimer's disease and cerebral amyloid angiopathy cases.Mol Med. 1997; 3: 695-707Crossref PubMed Google Scholar (kindly provided by F. Checler). Antibody 4G8 recognizes amino acids 17–24 of Aβ (Senetek). Hyperphosphorylated tau was recognized by antibody AT8 (Polymedco). ApoE was visualized with a mouse monoclonal anti-ApoE antibody (Boehringer-Mannheim). Postmortem brain tissue was examined from representative neurologically normal controls (ages 3 months and 3, 30, 44, 58, and 79 years); elderly nursing home residents without dementia (Clinical Dementia Rating (CDR) 0; ages 64, 69, 71, 72, 82, and 91 years) or with mild cognitive dysfunction (CDR 0.5; ages 67, 81, 87, 93, and 94 years. or mild (CDR 1; ages 79, 83, 84, 87, and 90 years), moderate (CDR 2. ages 83, 85, 90, 93, 94, and 94 years), or severe dementia (ages 64, 72, 79 years); and subjects with DS of varying ages (3 months and 3, 12, 13, and 24 years). The normal control and DS tissue were from New York Hospital, and the CDR tissue was from Mt. Sinai Medical Center. Postmortem intervals ranged from 6 to 18 hours. Ten percent formalin-fixed, paraffin-embedded brain sections (8 μm) were deparaffinized, washed in phosphate-buffered saline (PBS), incubated for 30 minutes at room temperature in 90% formic acid, washed again in PBS, incubated in 0.4% Triton X-100 (Tx) for 30 minutes, quenched for endogenous peroxidase with 3% hydrogen peroxide for 5 minutes, and preincubated in 3% serum from the species of the secondary antibody in 0.1% Tx/PBS for 1 hour to prevent nonspecific staining. Thereafter, slides were incubated with the appropriate antibody in 3% serum from the species of the secondary antibody/0.1%Tx/PBS overnight: anti-ApoE antibody (1:500), AT8 antibody (1:500), anti-Aβ40, or 42 C-terminal specific antibodies (typically 1:500 for RU and 1:100 for QCB antibodies). Slides were washed with PBS and incubated with secondary antibody (anti-primary antibody species antibody) (Vectastain ABC kit. Vector) in 1.5% serum from the species of the secondary antibody/0.1%Tx/PBS at room temperature for 1 hour. Slides were incubated with avidin-biotin and developed with diaminobenzidine (DAB) (ABC kit) for 2 minutes. Except for some representative sections counterstained with hematoxylin and eosin (H&E), most sections were not counterstained, so as not to obscure the immunohistochemical staining. Primary neuronal cultures were derived from the cerebral cortices of embryonic day 15 (E15) CD1 mice (Charles River) as previously described.20Gouras GK Xu H Jovanovic JN Buxbaum JD Wang R Greengard P Relkin NR Gandy S Generation and regulation of amyloid-β peptide variants in neurons.J Neurochem. 1998; 71: 1920-1925Crossref PubMed Scopus (107) Google Scholar Brains were removed, cortices were isolated, and the meninges were removed. Cortices were triturated in glass pipettes until cells were dissociated. Cells were counted in a hemocytometer and plated in serum-free Neurobasal media with N2 supplement (Gibco) and 0.5 mmol/L l-glutamine on poly-d-lysine-treated (0.1 mg/ml; Sigma) 100-mm dishes. Cortical cultures plated 3–4 days previously or murine N2a neuroblastoma cells doubly transfected with human βAPP695 and the Δ10e FAD mutant human PS121Borchelt DR Thinakaran G Eckman CB Lee MK Davenport F Ratovitsky T Prada CM Kim G Seekins S Yager D Slunt HH Wang R Seeger M Levey AI Gandy SE Copeland NG Jenkins NA Price DL Younkin SG Sisodia SS Familial Alzheimer's disease-linked presenilin 1 variants elevate Abeta1–42/1–40 ratio in vitro and in vivo.Neuron. 1996; 17: 1005-1013Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar were washed with PBS and incubated at 37°C for 20–30 minutes in methionine-free/glutamine-free Dulbecco's minimum essential medium (Gibco). Cells were labeled with 750 μCi/ml [35S]methionine (NEN/Dupont) (1 Ci = 37 GBq) in methionine-free medium supplemented with N2 and l-glutamine for 4 hours. Cells were scraped into ice-cold PBS with a rubber policeman. The supernatant was aspirated after brief centrifugation, and lysis buffer (100 μl) (0.5% deoxycholate, 0.5% NP-40, Trasylol (5 μg/ml), leupeptin (5 μg/ml), and phenylmethylsulfonyl fluoride (0.25 mmol/L)) was added. The lysate was subjected to agitation, repeat centrifugation, and collection of supernatant. Samples were treated with 0.5% sodium dodecyl sulfate, and the solutions were heated for 2 minutes at 75°C. Samples were adjusted to 190 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.3), 6 mmol/L EDTA, and 2.5% Triton X-100. Samples were incubated overnight with either antibody 4G8 or Aβ40/42 antibodies, followed by secondary rabbit anti-mouse antibody (Cappell) for 1 hour and protein A-Sepharose (Pharmacia) beads for 2 hours (all at 4°C). Proteins were analyzed with 10–20% tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography on Kodak X-OMAT AR5 film. Sucrose gradients used to prepare ER- and Golgi-enriched fractions were prepared as previously described.9Greenfield JP Tsai J Gouras GK Hai B Thinakaran G Checler F Sisodia SS Greengard P Xu H Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer β-amyloid peptides.Proc Natl Acad Sci USA. 1999; 96: 742-747Crossref PubMed Scopus (336) Google Scholar Metabolically labeled cells were homogenized in 0.25 mol/L sucrose, 10 mmol/L Tris-HCl (pH 7.4), 1 mmol/L MgAc2, and a protease inhibitor cocktail (Boehringer-Mannheim). The homogenate was loaded on a step gradient of 2 mol/L, 1.3 mol/L, 1.16 mol/L, and 0.8 mol/L sucrose. Gradients were centrifuged for 2.5 hours at 100,000 × g. Fractions were collected from the top of each gradient, immunoprecipitated with Aβ40/42 antibodies, and visualized as described above. Brain tissue from a 64-year-old representative subject with mild cognitive impairment (Clinical Dementia Rating Scale 0.5 (CDR 0.5); n = 5), stained with antibodies specific to the C-terminus of Aβ42, revealed significant amounts of region-specific intraneuronal immunoreactivity (Figure 1a, left), compared with relatively little Aβ40 immunoreactivity (Figure 1a, right). This intraneuronal Aβ42 staining was especially evident within pyramidal neurons of areas such as the hippocampus/entorhinal cortex, which are prone to developing early AD neuropathology. Aβ42 staining was less evident in sections from brain regions less affected by AD, such as primary sensory and motor cortices. The non-β-pleated nature of this intracellular Aβ42 is supported by a lack of Bielschowsky silver staining, the absence of Congo red birefringence under polarized light, the lack of thioflavin S staining, and the presence of Aβ42 immunostaining without formic acid pretreatment. The Aβ42 immunoreactivity was seen equivalently by three different Aβ42 antibodies, was abolished by synthetic Aβ1–42 peptide competition (Figure 1b), and was not detected with the use of preimmune serum or in the absence of the primary antibody (data not shown). These Aβ42 antibodies have negligible cross-reactivity to full-length βAPP. Intraneuronal Aβ42 immunoreactivity in a representative normal 3-month-old brain (Figure 1c, left) was of markedly less intensity than that in the brain of a 3-year-old with DS (Figure 1c, center) or the brain of a nondemented 76-year-old (Figure 1c, right). Thus neurons from neurologically normal controls (n = 6, ages 3 months to 79 years) showed intraneuronal Aβ42 staining that appeared to increase in relation to the subject's age at death. Analogous to the variability of SP deposition that exists among anatomical regions in an individual and between the same anatomical regions of different subjects, there was variability in the degree of intraneuronal Aβ42 immunoreactivity between anatomical regions (ie, CA1 compared with CA4) within and between individuals. Because intraneuronal Aβ42 accumulation occurs with early AD pathology, it is possible that extracellular Aβ plaques may develop from this intraneuronally accumulating pool of Aβ42. Consistent with this possibility, we observed instances where Aβ42 appears to aggregate within the cytoplasm of neurons (Figure 1d, left) and where Aβ plaque staining was neuronal in shape (Figure 1d, center). As has been described by others, we also observed diffuse plaque-like Aβ42 immunoreactivity that appears to be located directly outside neurons (Figure 1d, right). Early Aβ42 immunoreactivity was observed along the axonal projections (perforant path) of early Aβ42 accumulating neurons of the entorhinal cortex and at their terminal fields, the outer molecular layer of the dentate gyrus. It is of particular interest that with increasing cognitive dysfunction and Aβ plaque deposition (CDR 2 subjects, n = 6, and severe AD, n = 3), we observed that intraneuronal Aβ42 immunoreactivity tended to become less apparent. For example, in layer 2 neurons (islands of Calleja) of the entorhinal cortex from a CDR 1 patient, marked intraneuronal Aβ42 immunoreactivity was observed (Figure 2a), whereas in the patient with more advanced CDR 2 this staining was lost, presumably resulting from death or severe dysfunction of these neurons. In contrast, the emergence of Aβ40 immunoreactive plaques can be seen in the patient with more advanced CDR 2 compared to the CDR1 patient, which is known to occur with disease progression. In an attempt to elucidate whether Aβ42 immunoreactivity may precede NFT formation, we stained representative sections, taken from several subjects with marked intraneuronal Aβ42 immunoreactivity, with antibody AT8 for hyperphosphorylated tau, the principal component of NFTs. Neurons with Aβ42 immmunoreactivity were more numerous than those with hyperphosphorylated tau staining (Figure 2b), suggesting that Aβ42 accumulation may occur in the absence of appreciable tau pathology. In agreement with previous reports describing the presence of intraneuronal apoE,14LaFerla FM Troncoso JC Strickland DK Kawas CH Jay G Neuronal cell death in Alzheimer's disease correlates with apoE uptake and intracellular Abeta stabilization.J Clin Invest. 1997; 100: 310-320Crossref PubMed Scopus (151) Google Scholar we also observed that neurons with marked intracellular Aβ42 immunoreactivity also seemed to stain positively for apoE (Figure 2c), suggesting a possible involvement of apoE in these intracellular events. To corroborate our light microscopic observations of intraneuronal Aβ42 immunoreactivity, we used metabolic labeling-immunoprecipitation to demonstrate endogenous Aβ42 in primary rodent neuronal cultures. Pulse-labeling of these neuronal cultures, followed by immunoprecipitation of conditioned media by the use of Aβ40 and Aβ42 C-terminus-specific antibodies, revealed the expected predominance of secreted Aβ40 over secreted Aβ42 species (Figure 3a, top). In agreement with observations made using Aβ40/Aβ42 enzyme-linked immu-nosorbent assay in NT2 cells,6Skovronsky DM Doms RW Lee VM Detection of a novel intraneuronal pool of insoluble amyloid β protein that accumulates with time in culture.J Cell Biol. 1998; 141: 1031-1039Crossref PubMed Scopus (270) Google Scholar we observed relatively greater ratios of intracellular Aβ1–42/Aβ1–40 and of Aβx-42/Aβx-40 in neuronal lysates than in conditioned media. In fact, almost equal amounts of Aβx-40 and Aβx-42 species were detected with the use of a standard detergent lysis buffer (Figure 3a, bottom). To more readily detect intracellular Aβ42, we used a murine neuroblastoma N2a cell line harboring the human Δe10 FAD PS1 mutation, which is known to produce elevated levels of Aβ42.21Borchelt DR Thinakaran G Eckman CB Lee MK Davenport F Ratovitsky T Prada CM Kim G Seekins S Yager D Slunt HH Wang R Seeger M Levey AI Gandy SE Copeland NG Jenkins NA Price DL Younkin SG Sisodia SS Familial Alzheimer's disease-linked presenilin 1 variants elevate Abeta1–42/1–40 ratio in vitro and in vivo.Neuron. 1996; 17: 1005-1013Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar Aβ42 was readily detected in the ER- and Golgi-enriched fractions, with most of the secreted Aβ1–42 in the Golgi-enriched fraction and most of the Aβx-42 in the ER-enriched fraction (Figure 3b). Aβ40 species were detected mainly in the Golgi-enriched fraction (Figure 3b).9Greenfield JP Tsai J Gouras GK Hai B Thinakaran G Checler F Sisodia SS Greengard P Xu H Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer β-amyloid peptides.Proc Natl Acad Sci USA. 1999; 96: 742-747Crossref PubMed Scopus (336) Google Scholar Our immunohistochemical results support the concept that Aβ42 accumulation within neurons is an early pathological step in the cascade of events underlying AD neuropathology. Our immunohistochemical data cannot define the N-termini of the Aβ42 peptides, because our antibodies differentiate only the C-termini of Aβ. In addition to traditional Aβ1–40/42, various NH2-terminal truncated Aβ species have been described and suggested to be pathologically important.9Greenfield JP Tsai J Gouras GK Hai B Thinakaran G Checler F Sisodia SS Greengard P Xu H Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer β-amyloid peptides.Proc Natl Acad Sci USA. 1999; 96: 742-747Crossref PubMed Scopus (336) Google Scholar, 20Gouras GK Xu H Jovanovic JN Buxbaum JD Wang R Greengard P Relkin NR Gandy S Generation and regulation of amyloid-β peptide variants in neurons.J Neurochem. 1998; 71: 1920-1925Crossref PubMed Scopus (107) Google Scholar, 22Saido TC Iwatsubo T Mann DM Shimada H Ihara Y Kawashima S Dominant and differential deposition of distinct β-amyloid peptide species, A β N3(pE), in senile plaques.Neuron. 1995; 14: 457-466Abstract Full Text PDF PubMed Scopus (517) Google Scholar, 23Xu H Gouras GK Greenfield JP Vincent B Naslund J Mazzarelli L Fried G Jovanovic JN Seeger M Relkin NR Liao F Checler F Buxbaum JD Chait BT Thinakaran G Sisodia SS Wang R Greengard P Gandy S Estrogen reduces neuronal generation of Alzheimer β-amyloid peptides.Nat Med. 1998; 4: 447-451Crossref PubMed Scopus (519) Google Scholar Similar to the earliest Aβ42 deposited in SPs, intraneuronally accumulating Aβ42 also appears to be N-terminally truncated, as evidenced by the relative paucity of Aβasp1 and 6E10 (directed at Aβ1–10 epitope) as compared with Aβ42 and 4G8 (directed at Aβ17–24 epitope) antibody immunoreactivities (G. K. Gouras, personal observations). The possibility of this Aβ42 staining being due to artifactually shared epitope(s) appears unlikely, because intraneuronal Aβ42 immunoreactivity was replicated by three sets of antibodies and was not found to be present either with the use of preimmune serum or after Aβ1–42 peptide competition. Because intraneuronal Aβ42 immunoreactivity becomes less noticeable with disease progression, it seems that Aβ42-containing neurons may be lost and/or replaced by “ghost” tangles and/or plaques. The abundance of Aβ within senile plaques may also compete for antibody with the less abundant intracellular Aβ. The apparent disappearance of this staining, early on in the process of dementia, may provide an explanation for why intraneuronal Aβ immunoreactivity has not been appreciated by earlier investigators. The subcellular compartment(s) within which Aβ42 peptides accumulate remains to be identified. One interesting study reported disruption of the Golgi apparatus as an early event in AD neuropathology and postulated that this may even proceed NFT development.24Stieber A Mourelatos Z Gonatas NK In Alzheimer's disease the Golgi apparatus of a population of neurons without neurofibrillary tangles is fragmented and atrophic.Am J Pathol. 1996; 148: 415-426PubMed Google Scholar Given the growing body of evidence that both Aβ40 and Aβ42 formation occurs in the Golgi,4Xu H Sweeney D Wang R Thinakaran G Lo AC Sisodia SS Greengard P Gandy S Generation of Alzheimer β-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation.Proc Natl Acad Sci USA. 1997; 94: 3748-3752Crossref PubMed Scopus (255) Google Scholar, 9Greenfield JP Tsai J Gouras GK Hai B Thinakaran G Checler F Sisodia SS Greengard P Xu H Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer β-amyloid peptides.Proc Natl Acad Sci USA. 1999; 96: 742-747Crossref PubMed Scopus (336) Google Scholar it is conceivable that Aβ42 may begin accumulating abnormally within this organelle. However, more recent evidence indicates that Aβ42 cleavage can also occur earlier in the secretory pathway in the ER, with retention of the peptide within this compartment.7Hartmann T Bieger SC Bruhl B Tienari PJ Ida N Allsop D Roberts GW Masters CL Dotti CG Unsicker K Beyreuther K Distinct sites of intracellular production for Alzheimer's disease A β40/42 amyloid peptides.Nat Med. 1997; 3: 1016-1020Crossref PubMed Scopus (656) Google Scholar, 8Cook DG Forman MS Sung JC Leight S Kolson DL Iwatsubo T Lee VM Doms RW Alzheimer's A β(1–42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells.Nat Med. 1997; 3: 1021-1023Crossref PubMed Scopus (437) Google Scholar, 9Greenfield JP Tsai J Gouras GK Hai B Thinakaran G Checler F Sisodia SS Greengard P Xu H Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer β-amyloid peptides.Proc Natl Acad Sci USA. 1999; 96: 742-747Crossref PubMed Scopus (336) Google Scholar Accumulating Aβ42 may cause disruption of the cytoskeleton and initiate the formation of aggregated intracellular tau. Our proposal that intracellular accumulation of Aβ42 disrupts the normal functioning of neurons is supported by increasing reports of cellular dysfunction within AD-susceptible neurons, such as the presence of markers of apoptosis14LaFerla FM Troncoso JC Strickland DK Kawas CH Jay G Neuronal cell death in Alzheimer's disease correlates with apoE uptake and intracellular Abeta stabilization.J Clin Invest. 1997; 100: 310-320Crossref PubMed Scopus (151) Google Scholar and oxidative injury,25Guo Q Fu W Xie J Luo H Sells SF Geddes JW Bondada V Rangnekar VM Mattson MP Par-4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer disease.Nat Med. 1998; 4: 957-962Crossref PubMed Scopus (256) Google Scholar even before senile plaque and NFT formation. This proposal is further supported by the recent report of intraneuronal Aβ42 accumulation and neural degeneration in FAD PS1 mutant transgenic mice in the absence of Aβ plaque deposition.11Chui DH Tanahashi H Ozawa K Ikeda S Checler F Ueda O Suzuki H Araki W Inoue H Shirotani K Takahashi K Gallyas F Tabira T Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.Nat Med. 1999; 5: 560-564Crossref PubMed Scopus (323) Google Scholar Neuronal dysfunction arising from aggregating intraneuronal Aβ42 may also explain recent studies reporting plaque-independent functional and structural disruption of neural circuits in βAPP transgenic mice.26Hsia AY Masliah E McConlogue L Yu GQ Tatsuno G Hu K Kholodenko D Malenka RC Nicoll RA Mucke L Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models.Proc Natl Acad Sci USA. 1999; 96: 3228-3233Crossref PubMed Scopus (1001) Google Scholar, 27Moechars D Dewachter I Lorent K Reverse D Baekelandt V Naidu A Tesseur I Spittaels K Haute CV Checler F Godaux E Cordell B Van Leuven F Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain.J Biol Chem. 1999; 274: 6483-6492Crossref PubMed Scopus (618) Google Scholar The role of apoE in AD remains incompletely understood. The decrease in plaque load of βAPP transgenic mice crossed to apoE knockouts suggests an important relationship between apoE and aggregated Aβ.28Bales KR Verina T Dodel RC Du Y Altstiel L Bender M Hyslop P Johnstone EM Little SP Cummins DJ Piccardo P Ghetti B Paul SM Lack of apolipoprotein E dramatically reduces amyloid β-peptide deposition.Nat Genet. 1997; 17: 263-264Crossref PubMed Scopus (713) Google Scholar With Aβ accumulation and neuronal dysfunction, neuronal or astrocyte-generated apoE may potentially bind to Aβ intraneuronally and/or extracellularly with subsequent neuronal internalization, explaining the observation of apparent increased apoE immunoreactivity in Aβ42 immunoreactive neurons. Our observations of early intraneuronal accumulation of Aβ42 within those brain areas that are affected earliest by AD suggest a mechanism that may explain AD disease progression within the brain. Intraneuronal Aβ42 may act as a nidus for Aβ deposition, intraneuronally and extracellularly, at the soma and along processes and terminals of affected neurons. The resultant accumulation of Aβ in the parenchyma may hasten the pathological process, providing a potential mechanism for the “spread” of Aβ-related pathology.

Autism spectrum disorders (ASDs) represent a group of childhood neurodevelopmental and neuropsychiatric disorders characterized by deficits in verbal communication, impairment of social interaction, and restricted and repetitive patterns of interests and behaviour. To identify common genetic risk factors underlying ASDs, here we present the results of genome-wide association studies on a cohort of 780 families (3,101 subjects) with affected children, and a second cohort of 1,204 affected subjects and 6,491 control subjects, all of whom were of European ancestry. Six single nucleotide polymorphisms between cadherin 10 (CDH10) and cadherin 9 (CDH9)-two genes encoding neuronal cell-adhesion molecules-revealed strong association signals, with the most significant SNP being rs4307059 (P = 3.4 x 10(-8), odds ratio = 1.19). These signals were replicated in two independent cohorts, with combined P values ranging from 7.4 x 10(-8) to 2.1 x 10(-10). Our results implicate neuronal cell-adhesion molecules in the pathogenesis of ASDs, and represent, to our knowledge, the first demonstration of genome-wide significant association of common variants with susceptibility to ASDs.

The amyloid protein, A␤, which accumulates in the brains of Alzheimer patients, is derived by proteolysis of the amyloid protein precursor (APP).APP can undergo endoproteolytic processing at three sites, one at the amino terminus of the A␤ domain (␤-cleavage), one within the A␤ domain (␣-cleavage), and one at the carboxyl terminus of the A␤ domain (␥-cleavage).The enzymes responsible for these activities have not been unambiguously identified.By the use of gene disruption (knockout), we now demonstrate that TACE (tumor necrosis factor ␣ converting enzyme), a member of the ADAM family (a disintegrin and metalloprotease-family) of proteases, plays a central role in regulated ␣-cleavage of APP.Our data suggest that TACE may be the ␣-secretase responsible for the majority of regulated ␣-cleavage in cultured cells.Furthermore, we show that inhibiting this enzyme affects both APP secretion and A␤ formation in cultured cells.The amyloid protein, A␤, which accumulates in the brains of Alzheimer patients, is derived by proteolysis of the amyloid protein precursor (APP) 1 (1-3).APP can undergo endoproteolytic processing at three sites, one at the amino terminus of the A␤ domain (␤-cleavage), one within the A␤ domain (␣-cleavage), and one at the carboxyl terminus of the A␤ domain (␥-

The predisposition to neuropsychiatric disease involves a complex, polygenic, and pleiotropic genetic architecture. However, little is known about how genetic variants impart brain dysfunction or pathology. We used transcriptomic profiling as a quantitative readout of molecular brain-based phenotypes across five major psychiatric disorders-autism, schizophrenia, bipolar disorder, depression, and alcoholism-compared with matched controls. We identified patterns of shared and distinct gene-expression perturbations across these conditions. The degree of sharing of transcriptional dysregulation is related to polygenic (single-nucleotide polymorphism-based) overlap across disorders, suggesting a substantial causal genetic component. This comprehensive systems-level view of the neurobiological architecture of major neuropsychiatric illness demonstrates pathways of molecular convergence and specificity.

Numerous lines of inquiry implicate connectivity as a central abnormality in schizophrenia. Myelination and factors that affect myelination, such as the function of oligodendroglia, are critical processes that could profoundly affect neuronal connectivity, especially given the diffuse distribution of oligodendrocytes and the widespread distribution of brain regions that have been implicated in schizophrenia. Multiple lines of evidence now converge to implicate oligodendroglia and myelin in schizophrenia. Imaging and neurocytochemical evidence, similarities with demyelinating diseases, age-related changes in white matter, myelin-related gene abnormalities, and morphologic abnormalities in the oligodendroglia demonstrated in schizophrenic brains are all examined in light of the hypothesis that oligodendroglial dysfunction and even death, with subsequent abnormalities in myelin maintenance and repair, contribute to the schizophrenic syndrome.

Schizophrenia and bipolar disorder are two distinct diagnoses that share symptomology. Understanding the genetic factors contributing to the shared and disorder-specific symptoms will be crucial for improving diagnosis and treatment. In genetic data consisting of 53,555 cases (20,129 bipolar disorder [BD], 33,426 schizophrenia [SCZ]) and 54,065 controls, we identified 114 genome-wide significant loci implicating synaptic and neuronal pathways shared between disorders. Comparing SCZ to BD (23,585 SCZ, 15,270 BD) identified four genomic regions including one with disorder-independent causal variants and potassium ion response genes as contributing to differences in biology between the disorders. Polygenic risk score (PRS) analyses identified several significant correlations within case-only phenotypes including SCZ PRS with psychotic features and age of onset in BD. For the first time, we discover specific loci that distinguish between BD and SCZ and identify polygenic components underlying multiple symptom dimensions. These results point to the utility of genetics to inform symptomology and potential treatment.

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

Although autism is a highly heritable neurodevelopmental disorder, attempts to identify specific susceptibility genes have thus far met with limited success. Genome-wide association studies using half a million or more markers, particularly those with very large sample sizes achieved through meta-analysis, have shown great success in mapping genes for other complex genetic traits. Consequently, we initiated a linkage and association mapping study using half a million genome-wide single nucleotide polymorphisms (SNPs) in a common set of 1,031 multiplex autism families (1,553 affected offspring). We identified regions of suggestive and significant linkage on chromosomes 6q27 and 20p13, respectively. Initial analysis did not yield genome-wide significant associations; however, genotyping of top hits in additional families revealed an SNP on chromosome 5p15 (between SEMA5A and TAS2R1) that was significantly associated with autism (P = 2 x 10(-7)). We also demonstrated that expression of SEMA5A is reduced in brains from autistic patients, further implicating SEMA5A as an autism susceptibility gene. The linkage regions reported here provide targets for rare variation screening whereas the discovery of a single novel association demonstrates the action of common variants.

Activation of protein kinase C by phorbol esters is known to accelerate the processing and secretion of the beta/A4 amyloid protein precursor. We have now examined various first messengers that increase protein kinase C activity of target cells for their ability to affect beta/A4 amyloid protein precursor metabolism. Acetylcholine and interleukin 1, which are altered in Alzheimer disease, were shown to increase processing of the beta/A4 amyloid protein precursor via the secretory cleavage pathway. Cholinergic agonists stimulated secretion in human glioma and neuroblastoma cells as well as in PC12 cells transfected with the M1 receptor, while interleukin 1 stimulated secretion in human endothelial and glioma cells.

Over the past few years, substantial effort has been put into the functional annotation of variation in human genome sequences. Such annotations can have a critical role in identifying putatively causal variants for a disease or trait among the abundant natural variation that occurs at a locus of interest. The main challenges in using these various annotations include their large numbers and their diversity. Here we develop an unsupervised approach to integrate these different annotations into one measure of functional importance (Eigen) that, unlike most existing methods, is not based on any labeled training data. We show that the resulting meta-score has better discriminatory ability using disease-associated and putatively benign variants from published studies (in both coding and noncoding regions) than the recently proposed CADD score. Across varied scenarios, the Eigen score performs generally better than any single individual annotation, representing a powerful single functional score that can be incorporated in fine-mapping studies.

Abstract Background SHANK3 is a protein in the core of the postsynaptic density (PSD) and has a critical role in recruiting many key functional elements to the PSD and to the synapse, including components of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA), metabotropic glutamate (mGlu) and N -methyl-D-aspartic acid (NMDA) glutamate receptors, as well as cytoskeletal elements. Loss of a functional copy of the SHANK3 gene leads to the neurobehavioral manifestations of 22q13 deletion syndrome and/or to autism spectrum disorders. The goal of this study was to examine the effects of haploinsufficiency of full-length Shank3 in mice, focusing on synaptic development, transmission and plasticity, as well as on social behaviors, as a model for understanding SHANK3 haploinsufficiency in humans. Methods We used mice with a targeted disruption of Shank3 in which exons coding for the ankyrin repeat domain were deleted and expression of full-length Shank3 was disrupted. We studied synaptic transmission and plasticity by multiple methods, including patch-clamp whole cell recording, two-photon time-lapse imaging and extracellular recordings of field excitatory postsynaptic potentials. We also studied the density of GluR1-immunoreactive puncta in the CA1 stratum radiatum and carried out assessments of social behaviors. Results In Shank3 heterozygous mice, there was reduced amplitude of miniature excitatory postsynaptic currents from hippocampal CA1 pyramidal neurons and the input-output (I/O) relationship at Schaffer collateral-CA1 synapses in acute hippocampal slices was significantly depressed; both of these findings indicate a reduction in basal neurotransmission. Studies with specific inhibitors demonstrated that the decrease in basal transmission reflected reduced AMPA receptor-mediated transmission. This was further supported by the observation of reduced numbers of GluR1-immunoreactive puncta in the stratum radiatum. Long-term potentiation (LTP), induced either with θ-burst pairing (TBP) or high-frequency stimulation, was impaired in Shank3 heterozygous mice, with no significant change in long-term depression (LTD). In concordance with the LTP results, persistent expansion of spines was observed in control mice after TBP-induced LTP; however, only transient spine expansion was observed in Shank3 heterozygous mice. Male Shank3 heterozygotes displayed less social sniffing and emitted fewer ultrasonic vocalizations during interactions with estrus female mice, as compared to wild-type littermate controls. Conclusions We documented specific deficits in synaptic function and plasticity, along with reduced reciprocal social interactions in Shank3 heterozygous mice. Our results are consistent with altered synaptic development and function in Shank3 haploinsufficiency, highlighting the importance of Shank3 in synaptic function and supporting a link between deficits in synapse function and neurodevelopmental disorders. The reduced glutamatergic transmission that we observed in the Shank3 heterozygous mice represents an interesting therapeutic target in Shank3 -haploinsufficiency syndromes.

The turnover and processing of the Alzheimer beta/A4 amyloid precursor protein (beta APP) has been studied in PC12 cells after treatment with agents that regulate protein phosphorylation. Phorbol 12,13-dibutyrate, an agent that stimulates protein kinase C, decreased the levels of mature beta APP and increased the levels of 15- and 19-kDa peptides. These peptides appeared to be COOH-terminal fragments of beta APP, which arose when phorbol 12,13-dibutyrate increased the rate of proteolytic processing of mature forms of beta APP. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, also led to decreased levels of mature beta APP and increased levels of the 15- and 19-kDa peptides. H-7, an inhibitor of protein kinase C and of several other protein kinases, apparently decreased the rate of proteolytic processing of mature beta APP. The sizes of the putative COOH-terminal fragments observed after treatment with either phorbol 12,13-dibutyrate or okadaic acid suggest that one or both may contain the entire beta/A4 region of beta APP and thus be amyloidogenic. Our results support the hypothesis that abnormal protein phosphorylation may play a role in the development of the cerebral amyloidosis that accompanies Alzheimer disease.

Recent developments in sequencing technologies have made it possible to uncover both rare and common genetic variants. Genome-wide association studies (GWASs) can test for the effect of common variants, whereas sequence-based association studies can evaluate the cumulative effect of both rare and common variants on disease risk. Many groupwise association tests, including burden tests and variance-component tests, have been proposed for this purpose. Although such tests do not exclude common variants from their evaluation, they focus mostly on testing the effect of rare variants by upweighting rare-variant effects and downweighting common-variant effects and can therefore lose substantial power when both rare and common genetic variants in a region influence trait susceptibility. There is increasing evidence that the allelic spectrum of risk variants at a given locus might include novel, rare, low-frequency, and common genetic variants. Here, we introduce several sequence kernel association tests to evaluate the cumulative effect of rare and common variants. The proposed tests are computationally efficient and are applicable to both binary and continuous traits. Furthermore, they can readily combine GWAS and whole-exome-sequencing data on the same individuals, when available, and are also applicable to deep-resequencing data of GWAS loci. We evaluate these tests on data simulated under comprehensive scenarios and show that compared with the most commonly used tests, including the burden and variance-component tests, they can achieve substantial increases in power. We next show applications to sequencing studies for Crohn disease and autism spectrum disorders. The proposed tests have been incorporated into the software package SKAT.

The genetics underlying the autism spectrum disorders (ASDs) is complex and remains poorly understood. Previous work has demonstrated an important role for structural variation in a subset of cases, but has lacked the resolution necessary to move beyond detection of large regions of potential interest to identification of individual genes. To pinpoint genes likely to contribute to ASD etiology, we performed high density genotyping in 912 multiplex families from the Autism Genetics Resource Exchange (AGRE) collection and contrasted results to those obtained for 1,488 healthy controls. Through prioritization of exonic deletions (eDels), exonic duplications (eDups), and whole gene duplication events (gDups), we identified more than 150 loci harboring rare variants in multiple unrelated probands, but no controls. Importantly, 27 of these were confirmed on examination of an independent replication cohort comprised of 859 cases and an additional 1,051 controls. Rare variants at known loci, including exonic deletions at NRXN1 and whole gene duplications encompassing UBE3A and several other genes in the 15q11–q13 region, were observed in the course of these analyses. Strong support was likewise observed for previously unreported genes such as BZRAP1, an adaptor molecule known to regulate synaptic transmission, with eDels or eDups observed in twelve unrelated cases but no controls (p = 2.3×10−5). Less is known about MDGA2, likewise observed to be case-specific (p = 1.3×10−4). But, it is notable that the encoded protein shows an unexpectedly high similarity to Contactin 4 (BLAST E-value = 3×10−39), which has also been linked to disease. That hundreds of distinct rare variants were each seen only once further highlights complexity in the ASDs and points to the continued need for larger cohorts.

To determine whether genotypes at CLU, PICALM, and CR1 confer risk for Alzheimer disease (AD) and whether risk for AD associated with these genes is influenced by apolipoprotein E (APOE) genotypes.Association study of AD and CLU, PICALM, CR1, and APOE genotypes.Academic research institutions in the United States, Canada, and Israel.Seven thousand seventy cases with AD, 3055 with autopsies, and 8169 elderly cognitively normal controls, 1092 with autopsies, from 12 different studies, including white, African American, Israeli-Arab, and Caribbean Hispanic individuals.Unadjusted, CLU (odds ratio [OR], 0.91; 95% confidence interval [CI], 0.85-0.96 for single-nucleotide polymorphism [SNP] rs11136000), CR1 (OR, 1.14; 95% CI, 1.07-1.22; SNP rs3818361), and PICALM (OR, 0.89; 95% CI, 0.84-0.94, SNP rs3851179) were associated with AD in white individuals. None were significantly associated with AD in the other ethnic groups. APOE ε4 was significantly associated with AD (ORs, 1.80-9.05) in all but 1 small white cohort and in the Arab cohort. Adjusting for age, sex, and the presence of at least 1 APOE ε4 allele greatly reduced evidence for association with PICALM but not CR1 or CLU. Models with the main SNP effect, presence or absence of APOE ε4, and an interaction term showed significant interaction between presence or absence of APOE ε4 and PICALM.We confirm in a completely independent data set that CR1, CLU, and PICALM are AD susceptibility loci in European ancestry populations. Genotypes at PICALM confer risk predominantly in APOE ε4-positive subjects. Thus, APOE and PICALM synergistically interact.

Neurobiology of speech and language has previously been studied in the KE family, in which half of the members have severe impairment in both speech and language. The gene responsible for the phenotype was mapped to chromosome 7q31 and identified as the FOXP2 gene, coding for a transcription factor containing a polyglutamine tract and a forkhead DNA-binding domain. Because of linkage studies implicating 7q31 in autism, where language impairment is a component of the disorder, and in specific language impairment, FOXP2 has also been considered as a potential susceptibility locus for the language deficits in autism and/or specific language impairment. In this study, we characterized mice with a disruption in the murine Foxp2 gene. Disruption of both copies of the Foxp2 gene caused severe motor impairment, premature death, and an absence of ultrasonic vocalizations that are elicited when pups are removed from their mothers. Disruption of a single copy of the gene led to modest developmental delay but a significant alteration in ultrasonic vocalization in response to such separation. Learning and memory appear normal in the heterozygous animals. Cerebellar abnormalities were observed in mice with disruptions in Foxp2, with Purkinje cells particularly affected. Our findings support a role for Foxp2 in cerebellar development and in a developmental process that subsumes social communication functions in diverse organisms.

Genetic variants associated with susceptibility to late-onset Alzheimer disease are known for individuals of European ancestry, but whether the same or different variants account for the genetic risk of Alzheimer disease in African American individuals is unknown. Identification of disease-associated variants helps identify targets for genetic testing, prevention, and treatment.To identify genetic loci associated with late-onset Alzheimer disease in African Americans.The Alzheimer Disease Genetics Consortium (ADGC) assembled multiple data sets representing a total of 5896 African Americans (1968 case participants, 3928 control participants) 60 years or older that were collected between 1989 and 2011 at multiple sites. The association of Alzheimer disease with genotyped and imputed single-nucleotide polymorphisms (SNPs) was assessed in case-control and in family-based data sets. Results from individual data sets were combined to perform an inverse variance-weighted meta-analysis, first with genome-wide analyses and subsequently with gene-based tests for previously reported loci.Presence of Alzheimer disease according to standardized criteria.Genome-wide significance in fully adjusted models (sex, age, APOE genotype, population stratification) was observed for a SNP in ABCA7 (rs115550680, allele = G; frequency, 0.09 cases and 0.06 controls; odds ratio [OR], 1.79 [95% CI, 1.47-2.12]; P = 2.2 × 10(-9)), which is in linkage disequilibrium with SNPs previously associated with Alzheimer disease in Europeans (0.8 < D' < 0.9). The effect size for the SNP in ABCA7 was comparable with that of the APOE ϵ4-determining SNP rs429358 (allele = C; frequency, 0.30 cases and 0.18 controls; OR, 2.31 [95% CI, 2.19-2.42]; P = 5.5 × 10(-47)). Several loci previously associated with Alzheimer disease but not reaching significance in genome-wide analyses were replicated in gene-based analyses accounting for linkage disequilibrium between markers and correcting for number of tests performed per gene (CR1, BIN1, EPHA1, CD33; 0.0005 < empirical P < .001).In this meta-analysis of data from African American participants, Alzheimer disease was significantly associated with variants in ABCA7 and with other genes that have been associated with Alzheimer disease in individuals of European ancestry. Replication and functional validation of this finding is needed before this information is used in clinical settings.

Hakon Hakonarson and colleagues report a genome-wide copy number variation study in 3,506 cases of attention-deficit hyperactivity disorder. The authors identify a statistically significant enrichment of CNVs impacting metabotropic glutamate receptor genes. Attention deficit hyperactivity disorder (ADHD) is a common, heritable neuropsychiatric disorder of unknown etiology. We performed a whole-genome copy number variation (CNV) study on 1,013 cases with ADHD and 4,105 healthy children of European ancestry using 550,000 SNPs. We evaluated statistically significant findings in multiple independent cohorts, with a total of 2,493 cases with ADHD and 9,222 controls of European ancestry, using matched platforms. CNVs affecting metabotropic glutamate receptor genes were enriched across all cohorts (P = 2.1 × 10−9). We saw GRM5 (encoding glutamate receptor, metabotropic 5) deletions in ten cases and one control (P = 1.36 × 10−6). We saw GRM7 deletions in six cases, and we saw GRM8 deletions in eight cases and no controls. GRM1 was duplicated in eight cases. We experimentally validated the observed variants using quantitative RT-PCR. A gene network analysis showed that genes interacting with the genes in the GRM family are enriched for CNVs in ∼10% of the cases (P = 4.38 × 10−10) after correction for occurrence in the controls. We identified rare recurrent CNVs affecting glutamatergic neurotransmission genes that were overrepresented in multiple ADHD cohorts.

Autism spectrum disorder (ASD) has a major impact on the development and social integration of affected individuals and is the most heritable of psychiatric disorders. An increase in the incidence of ASD cases has prompted a surge in research efforts on the underlying neuropathologic processes. We present an overview of current findings in neuropathology studies of ASD using two investigational approaches, postmortem human brains and ASD animal models, and discuss the overlap, limitations, and significance of each. Postmortem examination of ASD brains has revealed global changes including disorganized gray and white matter, increased number of neurons, decreased volume of neuronal soma, and increased neuropil, the last reflecting changes in densities of dendritic spines, cerebral vasculature and glia. Both cortical and non-cortical areas show region-specific abnormalities in neuronal morphology and cytoarchitectural organization, with consistent findings reported from the prefrontal cortex, fusiform gyrus, frontoinsular cortex, cingulate cortex, hippocampus, amygdala, cerebellum and brainstem. The paucity of postmortem human studies linking neuropathology to the underlying etiology has been partly addressed using animal models to explore the impact of genetic and non-genetic factors clinically relevant for the ASD phenotype. Genetically modified models include those based on well-studied monogenic ASD genes (NLGN3, NLGN4, NRXN1, CNTNAP2, SHANK3, MECP2, FMR1, TSC1/2), emerging risk genes (CHD8, SCN2A, SYNGAP1, ARID1B, GRIN2B, DSCAM, TBR1), and copy number variants (15q11-q13 deletion, 15q13.3 microdeletion, 15q11-13 duplication, 16p11.2 deletion and duplication, 22q11.2 deletion). Models of idiopathic ASD include inbred rodent strains that mimic ASD behaviors as well as models developed by environmental interventions such as prenatal exposure to sodium valproate, maternal autoantibodies, and maternal immune activation. In addition to replicating some of the neuropathologic features seen in postmortem studies, a common finding in several animal models of ASD is altered density of dendritic spines, with the direction of the change depending on the specific genetic modification, age and brain region. Overall, postmortem neuropathologic studies with larger sample sizes representative of the various ASD risk genes and diverse clinical phenotypes are warranted to clarify putative etiopathogenic pathways further and to promote the emergence of clinically relevant diagnostic and therapeutic tools. In addition, as genetic alterations may render certain individuals more vulnerable to developing the pathological changes at the synapse underlying the behavioral manifestations of ASD, neuropathologic investigation using genetically modified animal models will help to improve our understanding of the disease mechanisms and enhance the development of targeted treatments.

<h3>Importance</h3> The origins and development of autism spectrum disorder (ASD) remain unresolved. No individual-level study has provided estimates of additive genetic, maternal, and environmental effects in ASD across several countries. <h3>Objective</h3> To estimate the additive genetic, maternal, and environmental effects in ASD. <h3>Design, Setting, and Participants</h3> Population-based, multinational cohort study including full birth cohorts of children from Denmark, Finland, Sweden, Israel, and Western Australia born between January 1, 1998, and December 31, 2011, and followed up to age 16 years. Data were analyzed from September 23, 2016 through February 4, 2018. <h3>Main Outcomes and Measures</h3> Across 5 countries, models were fitted to estimate variance components describing the total variance in risk for ASD occurrence owing to additive genetics, maternal, and shared and nonshared environmental effects. <h3>Results</h3> The analytic sample included 2 001 631 individuals, of whom 1 027 546 (51.3%) were male. Among the entire sample, 22 156 were diagnosed with ASD. The median (95% CI) ASD heritability was 80.8% (73.2%-85.5%) for country-specific point estimates, ranging from 50.9% (25.1%-75.6%) (Finland) to 86.8% (69.8%-100.0%) (Israel). For the Nordic countries combined, heritability estimates ranged from 81.2% (73.9%-85.3%) to 82.7% (79.1%-86.0%). Maternal effect was estimated to range from 0.4% to 1.6%. Estimates of genetic, maternal, and environmental effects for autistic disorder were similar with ASD. <h3>Conclusions and Relevance</h3> Based on population data from 5 countries, the heritability of ASD was estimated to be approximately 80%, indicating that the variation in ASD occurrence in the population is mostly owing to inherited genetic influences, with no support for contribution from maternal effects. The results suggest possible modest differences in the sources of ASD risk between countries.

Early endosomes are a major site of amyloid precursor protein (APP) processing and a convergence point for molecules of pathologic relevance to Alzheimer's disease (AD). Neuronal endosome enlargement, reflecting altered endocytic function, is a disease-specific response that develops years before the earliest stage of AD and Down syndrome (DS). We examined how endocytic dysfunction is related to Abeta accumulation and distribution in early stage AD and DS. We found by ELISA and immunocytochemistry that the appearance of enlarged endosomes coincided with an initial rise in soluble Abeta40 and Abeta42 peptides, which preceded amyloid deposition. Double-immunofluorescence using numerous Abeta antibodies showed that intracellular Abeta localized principally to rab5-positive endosomes in neurons from AD brains and was prominent in enlarged endosomes. Abeta was not detectable in neurons from normal controls and was diminished after amyloid deposition in neuropathologically confirmed AD. These studies support growing evidence that endosomal pathology contributes significantly to Abeta overproduction and accumulation in sporadic AD and in AD associated with DS and may signify earlier disease-relevant disturbances of the signaling functions of endosomes.

To explore mechanisms through which altered peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) expression may influence Alzheimer disease (AD) amyloid neuropathology and to test the hypothesis that promotion of PGC-1alpha expression in neurons might be developed as a novel therapeutic strategy in AD.Case-control. Patients Human postmortem brain (hippocampal formation) samples from AD cases and age-matched non-AD cases.Using genome-wide complementary DNA microarray analysis, we found that PGC-1alpha messenger RNA expression was significantly decreased as a function of progression of clinical dementia in the AD brain. Following confirmatory real-time polymerase chain reaction assay, we continued to explore the role of PGC-1alpha in clinical dementia and found that PGC-1alpha protein content was negatively associated with both AD-type neuritic plaque pathology and beta-amyloid (Abeta)(X-42) contents. Moreover, we found that the predicted elevation of amyloidogenic Abeta(1-42) and Abeta(1-40) peptide accumulation in embryonic cortico-hippocampal neurons derived from Tg2576 AD mice under hyperglycemic conditions (glucose level, 182-273 mg/dL) coincided with a dose-dependent attenuation in PGC-1alpha expression. Most importantly, we found that the reconstitution of exogenous PGC-1alpha expression in Tg2576 neurons attenuated the hyperglycemic-mediated beta-amyloidogenesis through mechanisms involving the promotion of the "nonamyloidogenic" alpha-secretase processing of amyloid precursor protein through the attenuation of the forkheadlike transcription factor 1 (FoxO3a) expression.Therapeutic preservation of neuronal PGC-1alpha expression promotes the nonamyloidogenic processing of amyloid precursor protein precluding the generation of amyloidogenic Abeta peptides.

Mutations in the synaptic scaffolding protein gene SHANK3 are strongly implicated in autism and Phelan–McDermid 22q13 deletion syndrome. The precise location of the mutation within the Shank3 gene is key to its phenotypic outcomes. Here, we report the physiological and behavioral consequences of null and heterozygous mutations in the ankyrin repeat domain in Shank3 mice. Both homozygous and heterozygous mice showed reduced glutamatergic transmission and long-term potentiation in the hippocampus with more severe deficits detected in the homozygous mice. Three independent cohorts were evaluated for magnitude and replicability of behavioral endophenotypes relevant to autism and Phelan–McDermid syndrome. Mild social impairments were detected, primarily in juveniles during reciprocal interactions, while all genotypes displayed normal adult sociability on the three-chambered task. Impaired novel object recognition and rotarod performance were consistent across cohorts of null mutants. Repetitive self-grooming, reduced ultrasonic vocalizations, and deficits in reversal of water maze learning were detected only in some cohorts, emphasizing the importance of replication analyses. These results demonstrate the exquisite specificity of deletions in discrete domains within the Shank3 gene in determining severity of symptoms.

Alzheimer's disease (AD) affects half the US population over the age of 85 and is universally fatal following an average course of 10 years of progressive cognitive disability. Genetic and genome-wide association studies (GWAS) have identified about 33 risk factor genes for common, late-onset AD (LOAD), but these risk loci fail to account for the majority of affected cases and can neither provide clinically meaningful prediction of development of AD nor offer actionable mechanisms. This cohort study generated large-scale matched multi-Omics data in AD and control brains for exploring novel molecular underpinnings of AD. Specifically, we generated whole genome sequencing, whole exome sequencing, transcriptome sequencing and proteome profiling data from multiple regions of 364 postmortem control, mild cognitive impaired (MCI) and AD brains with rich clinical and pathophysiological data. All the data went through rigorous quality control. Both the raw and processed data are publicly available through the Synapse software platform.

PARK8/LRRK2 (leucine-rich repeat kinase 2) was recently identified as a causative gene for autosomal dominant Parkinson's disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Emerging in vitro evidence indicates that aberrant enzymatic activity of LRRK2 protein carrying this mutation can cause neurotoxicity. However, the physiological and pathophysiological functions of LRRK2 in vivo remain elusive. Here we characterize two bacterial artificial chromosome (BAC) transgenic mouse strains overexpressing LRRK2 wild-type (Wt) or mutant G2019S. Transgenic LRRK2-Wt mice had elevated striatal dopamine (DA) release with unaltered DA uptake or tissue content. Consistent with this result, LRRK2-Wt mice were hyperactive and showed enhanced performance in motor function tests. These results suggest a role for LRRK2 in striatal DA transmission and the consequent motor function. In contrast, LRRK2-G2019S mice showed an age-dependent decrease in striatal DA content, as well as decreased striatal DA release and uptake. Despite increased brain kinase activity, LRRK2-G2019S overexpression was not associated with loss of DAergic neurons in substantia nigra or degeneration of nigrostriatal terminals at 12 months. Our results thus reveal a pivotal role for LRRK2 in regulating striatal DA transmission and consequent control of motor function. The PD-associated mutation G2019S may exert pathogenic effects by impairing these functions of LRRK2. Our LRRK2 BAC transgenic mice, therefore, could provide a useful model for understanding early PD pathological events.

Extracellular deposition of the beta/A4 amyloid peptide is a characteristic feature of the brain in patients with Alzheimer disease. beta/A4 amyloid is derived from the amyloid precursor protein (APP), an integral membrane protein that exists as three major isoforms (APP695, APP751, and APP770). Secreted forms of APP found in blood plasma and cerebrospinal fluid arise by proteolytic cleavage of APP within the beta/A4 amyloid domain, precluding the possibility of amyloidogenesis for that population of molecules. In the present study, we have demonstrated that treatment of PC12 cells with phorbol ester produces a severalfold increase in secretion of APP695, APP751, and APP770. This increase is augmented by simultaneous treatment with the protein phosphatase inhibitor okadaic acid. These data indicate that protein phosphorylation regulates intra-beta/A4 amyloid cleavage and APP secretion. These and other results suggest that APP molecules can normally follow either of two processing pathways: regulated secretion or proteolytic degradation unassociated with secretion.

Alzheimer's disease (AD) and related dementias are a major public health challenge and present a therapeutic imperative for which we need additional insight into molecular pathogenesis. We performed a genome-wide association study and analysis of known genetic risk loci for AD dementia using neuropathologic data from 4,914 brain autopsies. Neuropathologic data were used to define clinico-pathologic AD dementia or controls, assess core neuropathologic features of AD (neuritic plaques, NPs; neurofibrillary tangles, NFTs), and evaluate commonly co-morbid neuropathologic changes: cerebral amyloid angiopathy (CAA), Lewy body disease (LBD), hippocampal sclerosis of the elderly (HS), and vascular brain injury (VBI). Genome-wide significance was observed for clinico-pathologic AD dementia, NPs, NFTs, CAA, and LBD with a number of variants in and around the apolipoprotein E gene (APOE). GalNAc transferase 7 (GALNT7), ATP-Binding Cassette, Sub-Family G (WHITE), Member 1 (ABCG1), and an intergenic region on chromosome 9 were associated with NP score; and Potassium Large Conductance Calcium-Activated Channel, Subfamily M, Beta Member 2 (KCNMB2) was strongly associated with HS. Twelve of the 21 non-APOE genetic risk loci for clinically-defined AD dementia were confirmed in our clinico-pathologic sample: CR1, BIN1, CLU, MS4A6A, PICALM, ABCA7, CD33, PTK2B, SORL1, MEF2C, ZCWPW1, and CASS4 with 9 of these 12 loci showing larger odds ratio in the clinico-pathologic sample. Correlation of effect sizes for risk of AD dementia with effect size for NFTs or NPs showed positive correlation, while those for risk of VBI showed a moderate negative correlation. The other co-morbid neuropathologic features showed only nominal association with the known AD loci. Our results discovered new genetic associations with specific neuropathologic features and aligned known genetic risk for AD dementia with specific neuropathologic changes in the largest brain autopsy study of AD and related dementias.

David Whitcomb, Bernie Devlin and colleagues report the results of a genome-wide association study of pancreatitis. They identify common variants at two loci associated with risk of this disease, including one on the X chromosome that shows strong evidence of interaction with alcohol consumption. Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR and SPINK1 variants were associated with pancreatitis risk. We now report two associations at genome-wide significance identified and replicated at PRSS1-PRSS2 (P < 1 × 10−12) and X-linked CLDN2 (P < 1 × 10−21) through a two-stage genome-wide study (stage 1: 676 cases and 4,507 controls; stage 2: 910 cases and 4,170 controls). The PRSS1 variant likely affects disease susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous in males) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men (male hemizygote frequency is 0.26, whereas female homozygote frequency is 0.07).

This study aimed to elucidate the genetic causes underlying early-onset Parkinsonism (EOP) in a consanguineous Iranian family. To attain this, homozygosity mapping and whole-exome sequencing were performed. As a result, a homozygous mutation (c.773G>A; p.Arg258Gln) lying within the NH2 -terminal Sac1-like inositol phosphatase domain of polyphosphoinositide phosphatase synaptojanin 1 (SYNJ1), which has been implicated in the regulation of endocytic traffic at synapses, was identified as the disease-segregating mutation. This mutation impaired the phosphatase activity of SYNJ1 against its Sac1 domain substrates in vitro. We concluded that the SYNJ1 mutation identified here is responsible for the EOP phenotype seen in our patients probably due to deficiencies in its phosphatase activity and consequent impairment of its synaptic functions. Our finding not only opens new avenues of investigation in the synaptic dysfunction mechanisms associated with Parkinsonism, but also suggests phosphoinositide metabolism as a novel therapeutic target for Parkinsonism.

Recent research has uncovered an important role for de novo variation in neurodevelopmental disorders. Using aggregated data from 9,246 families with autism spectrum disorder, intellectual disability, or developmental delay, we found that ∼1/3 of de novo variants are independently present as standing variation in the Exome Aggregation Consortium's cohort of 60,706 adults, and these de novo variants do not contribute to neurodevelopmental risk. We further used a loss-of-function (LoF)-intolerance metric, pLI, to identify a subset of LoF-intolerant genes containing the observed signal of associated de novo protein-truncating variants (PTVs) in neurodevelopmental disorders. LoF-intolerant genes also carry a modest excess of inherited PTVs, although the strongest de novo-affected genes contribute little to this excess, thus suggesting that the excess of inherited risk resides in lower-penetrant genes. These findings illustrate the importance of population-based reference cohorts for the interpretation of candidate pathogenic variants, even for analyses of complex diseases and de novo variation.

22q13 deletion syndrome, also known as Phelan-McDermid syndrome, is a neurodevelopmental disorder characterized by intellectual disability, hypotonia, delayed or absent speech, and autistic features. SHANK3 has been identified as the critical gene in the neurological and behavioral aspects of this syndrome. The phenotype of SHANK3 deficiency has been described primarily from case studies, with limited evaluation of behavioral and cognitive deficits. The present study used a prospective design and inter-disciplinary clinical evaluations to assess patients with SHANK3 deficiency, with the goal of providing a comprehensive picture of the medical and behavioral profile of the syndrome.A serially ascertained sample of patients with SHANK3 deficiency (n = 32) was evaluated by a team of child psychiatrists, neurologists, clinical geneticists, molecular geneticists and psychologists. Patients were evaluated for autism spectrum disorder using the Autism Diagnostic Interview-Revised and the Autism Diagnostic Observation Schedule-G.Thirty participants with 22q13.3 deletions ranging in size from 101 kb to 8.45 Mb and two participants with de novo SHANK3 mutations were included. The sample was characterized by high rates of autism spectrum disorder: 27 (84%) met criteria for autism spectrum disorder and 24 (75%) for autistic disorder. Most patients (77%) exhibited severe to profound intellectual disability and only five (19%) used some words spontaneously to communicate. Dysmorphic features, hypotonia, gait disturbance, recurring upper respiratory tract infections, gastroesophageal reflux and seizures were also common. Analysis of genotype-phenotype correlations indicated that larger deletions were associated with increased levels of dysmorphic features, medical comorbidities and social communication impairments related to autism. Analyses of individuals with small deletions or point mutations identified features related to SHANK3 haploinsufficiency, including ASD, seizures and abnormal EEG, hypotonia, sleep disturbances, abnormal brain MRI, gastroesophageal reflux, and certain dysmorphic features.This study supports findings from previous research on the severity of intellectual, motor, and speech impairments seen in SHANK3 deficiency, and highlights the prominence of autism spectrum disorder in the syndrome. Limitations of existing evaluation tools are discussed, along with the need for natural history studies to inform clinical monitoring and treatment development in SHANK3 deficiency.

Recent advances in genetics and genomics have unveiled numerous cases of autism spectrum disorders (ASDs) associated with rare, causal genetic variations. These findings support a novel view of ASDs in which many independent, individually rare genetic variants, each associated with a very high relative risk, together explain a large proportion of ASDs. Although these rare variants impact diverse pathways, there is accumulating evidence that synaptic pathways, including those involving synaptic cell adhesion, are disrupted in some subjects with ASD. These findings provide insights into the pathogenesis of ASDs and enable the development of model systems with construct validity for specific causes of ASDs. In several neurodevelopmental disorders frequently associated with ASD, including fragile X syndrome, Rett syndrome and tuberous sclerosis, animal models have led to the development of new therapeutic approaches, giving rise to optimism with other causes of ASDs.

Epigenetic alterations offer promise as diagnostic or prognostic markers, but it is not known whether these measures associate with, or predict, clinical state.These questions were addressed in a pilot study with combat veterans with PTSD to determine whether cytosine methylation in promoter regions of the glucocorticoid related NR3C1 and FKBP51 genes would predict or associate with treatment outcome.Veterans with PTSD received prolonged exposure (PE) psychotherapy, yielding responders (n = 8), defined by no longer meeting diagnostic criteria for PTSD, and non-responders (n = 8).Blood samples were obtained at pre-treatment, after 12 weeks of psychotherapy (post-treatment), and after a 3-month follow-up.Methylation was examined in DNA extracted from lymphocytes.Measures reflecting glucocorticoid receptor (GR) activity were also obtained (i.e., plasma and 24 h-urinary cortisol, plasma ACTH, lymphocyte lysozyme IC 50-DEX , and plasma neuropeptide-Y).Methylation of the GR gene (NR3C1) exon 1F promoter assessed at pre-treatment predicted treatment outcome, but was not significantly altered in responders or non-responders at post-treatment or follow-up.In contrast, methylation of the FKBP5 gene (FKBP51) exon 1 promoter region did not predict treatment response, but decreased in association with recovery.In a subset, a corresponding group difference in FKBP5 gene expression was observed, with responders showing higher gene expression at post-treatment than non-responders.Endocrine markers were also associated with the epigenetic markers.These preliminary observations require replication and validation.However, the results support research indicating that some glucocorticoid related genes are subject to environmental regulation throughout life.Moreover, psychotherapy constitutes a form of "environmental regulation" that may alter epigenetic state.Finally, the results further suggest that different genes may be associated with prognosis and symptom state, respectively.

Background Although genetic risk factors for posttraumatic stress disorder (PTSD) in similarly traumatized cohorts can be confounded with risk for type of exposure, the primary risk for exposure to the 9/11 attack on New York City was proximity, allowing study of PTSD risk in a sample that is not confounded by exposure-related risk. Methods Thirty-five Caucasians (15 with PTSD, stratified for exposure, age, and gender) were selected from a population-representative sample of persons exposed to the attack from which longitudinal data had been collected in four previous waves. Whole blood gene expression and cortisol levels were obtained. Results Seventeen probe sets were differentially expressed in PTSD. Identified genes were generally involved in hypothalamic-pituitary-adrenal (HPA) axis, signal transduction, or brain and immune cell function. FKBP5, a modulator of glucocorticoid receptor (GR) sensitivity, showed reduced expression in PTSD, consistent with enhanced GR responsiveness. FKBP5 expression was predicted by cortisol when entered with PTSD severity in regression analysis. Quantitative polymerase chain reaction confirmed significant reductions in FKBP5. Also less expressed in PTSD were STAT5B, a direct inhibitor of GR, and major histocompatibility complex (MHC) Class II. Conclusions Consistent with observations of HPA axis dysfunction in PTSD, several genes involved in glucocorticoid signaling are differentially expressed among those with current PTSD. Although genetic risk factors for posttraumatic stress disorder (PTSD) in similarly traumatized cohorts can be confounded with risk for type of exposure, the primary risk for exposure to the 9/11 attack on New York City was proximity, allowing study of PTSD risk in a sample that is not confounded by exposure-related risk. Thirty-five Caucasians (15 with PTSD, stratified for exposure, age, and gender) were selected from a population-representative sample of persons exposed to the attack from which longitudinal data had been collected in four previous waves. Whole blood gene expression and cortisol levels were obtained. Seventeen probe sets were differentially expressed in PTSD. Identified genes were generally involved in hypothalamic-pituitary-adrenal (HPA) axis, signal transduction, or brain and immune cell function. FKBP5, a modulator of glucocorticoid receptor (GR) sensitivity, showed reduced expression in PTSD, consistent with enhanced GR responsiveness. FKBP5 expression was predicted by cortisol when entered with PTSD severity in regression analysis. Quantitative polymerase chain reaction confirmed significant reductions in FKBP5. Also less expressed in PTSD were STAT5B, a direct inhibitor of GR, and major histocompatibility complex (MHC) Class II. Consistent with observations of HPA axis dysfunction in PTSD, several genes involved in glucocorticoid signaling are differentially expressed among those with current PTSD.

Multiple lines of evidence in schizophrenia, from brain imaging, studies in postmortem brains, and genetic association studies, have implicated oligodendrocyte and myelin dysfunction in this disease. Recent studies suggest that oligodendrocyte and myelin dysfunction leads to changes in synaptic formation and function, which could lead to cognitive dysfunction, a core symptom of schizophrenia. Furthermore, there is accumulating data linking oligodendrocyte and myelin dysfunction with dopamine and glutamate abnormalities, both of which are found in schizophrenia. These findings implicate oligodendrocyte and myelin dysfunction as a primary change in schizophrenia, not only as secondary consequences of the illness or treatment. Strategies targeting oligodendrocyte and myelin abnormalities could therefore provide therapeutic opportunities for patients suffering from schizophrenia.

Each additional copy of the apolipoprotein E4 (APOE4) allele is associated with a higher risk of Alzheimer's dementia, while the APOE2 allele is associated with a lower risk of Alzheimer's dementia, it is not yet known whether APOE2 homozygotes have a particularly low risk. We generated Alzheimer's dementia odds ratios and other findings in more than 5,000 clinically characterized and neuropathologically characterized Alzheimer's dementia cases and controls. APOE2/2 was associated with a low Alzheimer's dementia odds ratios compared to APOE2/3 and 3/3, and an exceptionally low odds ratio compared to APOE4/4, and the impact of APOE2 and APOE4 gene dose was significantly greater in the neuropathologically confirmed group than in more than 24,000 neuropathologically unconfirmed cases and controls. Finding and targeting the factors by which APOE and its variants influence Alzheimer's disease could have a major impact on the understanding, treatment and prevention of the disease.

<h2>ABSTRACT</h2><h3>Objective:</h3> To replicate the factor structure and predictive validity of revised Autism Diagnostic Observation Schedule algorithms in an independent dataset (<i>N</i> = 1,282). <h3>Method:</h3> Algorithm revisions were replicated using data from children ages 18 months to 16 years collected at 11 North American sites participating in the Collaborative Programs for Excellence in Autism and the Studies to Advance Autism Research and Treatment. <h3>Results:</h3> Sensitivities and specificities approximated or exceeded those of the old algorithms except for young children with phrase speech and a clinical diagnosis of pervasive developmental disorders not otherwise specified. <h3>Conclusions:</h3> Revised algorithms increase comparability between modules and improve the predictive validity of the Autism Diagnostic Observation Schedule forautism cases compared to the original algorithms.

To characterize the role of rare complete human knockouts in autism spectrum disorders (ASDs), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a 2-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤ 5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudoautosomal regions on the X chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together, these results provide compelling evidence that rare autosomal and X chromosome complete gene knockouts are important inherited risk factors for ASD.

De novo mutations affect risk for many diseases and disorders, especially those with early-onset. An example is autism spectrum disorders (ASD). Four recent whole-exome sequencing (WES) studies of ASD families revealed a handful of novel risk genes, based on independent de novo loss-of-function (LoF) mutations falling in the same gene, and found that de novo LoF mutations occurred at a twofold higher rate than expected by chance. However successful these studies were, they used only a small fraction of the data, excluding other types of de novo mutations and inherited rare variants. Moreover, such analyses cannot readily incorporate data from case-control studies. An important research challenge in gene discovery, therefore, is to develop statistical methods that accommodate a broader class of rare variation. We develop methods that can incorporate WES data regarding de novo mutations, inherited variants present, and variants identified within cases and controls. TADA, for Transmission And De novo Association, integrates these data by a gene-based likelihood model involving parameters for allele frequencies and gene-specific penetrances. Inference is based on a Hierarchical Bayes strategy that borrows information across all genes to infer parameters that would be difficult to estimate for individual genes. In addition to theoretical development we validated TADA using realistic simulations mimicking rare, large-effect mutations affecting risk for ASD and show it has dramatically better power than other common methods of analysis. Thus TADA's integration of various kinds of WES data can be a highly effective means of identifying novel risk genes. Indeed, application of TADA to WES data from subjects with ASD and their families, as well as from a study of ASD subjects and controls, revealed several novel and promising ASD candidate genes with strong statistical support.

Histone deacetylases (HDACs) compact chromatin structure and repress gene transcription. In schizophrenia, clinical studies demonstrate that HDAC inhibitors are efficacious when given in combination with atypical antipsychotics. However, the molecular mechanism that integrates a better response to antipsychotics with changes in chromatin structure remains unknown. Here we found that chronic atypical antipsychotics downregulated the transcription of metabotropic glutamate 2 receptor (mGlu2, also known as Grm2), an effect that was associated with decreased histone acetylation at its promoter in mouse and human frontal cortex. This epigenetic change occurred in concert with a serotonin 5-HT(2A) receptor-dependent upregulation and increased binding of HDAC2 to the mGlu2 promoter. Virally mediated overexpression of HDAC2 in frontal cortex decreased mGlu2 transcription and its electrophysiological properties, thereby increasing psychosis-like behavior. Conversely, HDAC inhibitors prevented the repressive histone modifications induced at the mGlu2 promoter by atypical antipsychotics, and augmented their therapeutic-like effects. These observations support the view of HDAC2 as a promising new target for schizophrenia treatment.

APOE ɛ4, the most significant genetic risk factor for Alzheimer disease (AD), may mask effects of other loci. We re-analyzed genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) Consortium in APOE ɛ4+ (10 352 cases and 9207 controls) and APOE ɛ4- (7184 cases and 26 968 controls) subgroups as well as in the total sample testing for interaction between a single-nucleotide polymorphism (SNP) and APOE ɛ4 status. Suggestive associations (P<1 × 10(-4)) in stage 1 were evaluated in an independent sample (stage 2) containing 4203 subjects (APOE ɛ4+: 1250 cases and 536 controls; APOE ɛ4-: 718 cases and 1699 controls). Among APOE ɛ4- subjects, novel genome-wide significant (GWS) association was observed with 17 SNPs (all between KANSL1 and LRRC37A on chromosome 17 near MAPT) in a meta-analysis of the stage 1 and stage 2 data sets (best SNP, rs2732703, P=5·8 × 10(-9)). Conditional analysis revealed that rs2732703 accounted for association signals in the entire 100-kilobase region that includes MAPT. Except for previously identified AD loci showing stronger association in APOE ɛ4+ subjects (CR1 and CLU) or APOE ɛ4- subjects (MS4A6A/MS4A4A/MS4A6E), no other SNPs were significantly associated with AD in a specific APOE genotype subgroup. In addition, the finding in the stage 1 sample that AD risk is significantly influenced by the interaction of APOE with rs1595014 in TMEM106B (P=1·6 × 10(-7)) is noteworthy, because TMEM106B variants have previously been associated with risk of frontotemporal dementia. Expression quantitative trait locus analysis revealed that rs113986870, one of the GWS SNPs near rs2732703, is significantly associated with four KANSL1 probes that target transcription of the first translated exon and an untranslated exon in hippocampus (P ⩽ 1.3 × 10(-8)), frontal cortex (P ⩽ 1.3 × 10(-9)) and temporal cortex (P⩽1.2 × 10(-11)). Rs113986870 is also strongly associated with a MAPT probe that targets transcription of alternatively spliced exon 3 in frontal cortex (P=9.2 × 10(-6)) and temporal cortex (P=2.6 × 10(-6)). Our APOE-stratified GWAS is the first to show GWS association for AD with SNPs in the chromosome 17q21.31 region. Replication of this finding in independent samples is needed to verify that SNPs in this region have significantly stronger effects on AD risk in persons lacking APOE ɛ4 compared with persons carrying this allele, and if this is found to hold, further examination of this region and studies aimed at deciphering the mechanism(s) are warranted.

Whole-genome sequencing (WGS) has facilitated the first genome-wide evaluations of the contribution of de novo noncoding mutations to complex disorders. Using WGS, we identified 255,106 de novo mutations among sample genomes from members of 1902 quartet families in which one child, but not a sibling or their parents, was affected by autism spectrum disorder (ASD). In contrast to coding mutations, no noncoding functional annotation category, analyzed in isolation, was significantly associated with ASD. Casting noncoding variation in the context of a de novo risk score across multiple annotation categories, however, did demonstrate association with mutations localized to promoter regions. We found that the strongest driver of this promoter signal emanates from evolutionarily conserved transcription factor binding sites distal to the transcription start site. These data suggest that de novo mutations in promoter regions, characterized by evolutionary and functional signatures, contribute to ASD.

Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden. This study presents a framework to evaluate rare and de novo variation from whole-genome sequencing (WGS). The work suggests that robust results from WGS studies will require large cohorts and strategies that consider the substantial multiple-testing burden.

Haploinsufficiency of the Shank3 gene, which encodes a scaffolding protein at glutamatergic synapses, is a highly prevalent and penetrant risk factor for autism. Using combined behavioral, electrophysiological, biochemical, imaging, and molecular approaches, we find that Shank3-deficient mice exhibit autism-like social deficits and repetitive behaviors, as well as the significantly diminished NMDA receptor (NMDAR) synaptic function and synaptic distribution in prefrontal cortex. Concomitantly, Shank3-deficient mice have a marked loss of cortical actin filaments, which is associated with the reduced Rac1/PAK activity and increased activity of cofilin, the major actin depolymerizing factor. The social deficits and NMDAR hypofunction are rescued by inhibiting cofilin or activating Rac1 in Shank3-deficient mice and are induced by inhibiting PAK or Rac1 in wild-type mice. These results indicate that the aberrant regulation of synaptic actin filaments and loss of synaptic NMDARs contribute to the manifestation of autism-like phenotypes. Thus, targeting actin regulators provides a strategy for autism treatment.

We systematically analyzed postzygotic mutations (PZMs) in whole-exome sequences from the largest collection of trios (5,947) with autism spectrum disorder (ASD) available, including 282 unpublished trios, and performed resequencing using multiple independent technologies. We identified 7.5% of de novo mutations as PZMs, 83.3% of which were not described in previous studies. Damaging, nonsynonymous PZMs within critical exons of prenatally expressed genes were more common in ASD probands than controls (P < 1 × 10-6), and genes carrying these PZMs were enriched for expression in the amygdala (P = 5.4 × 10-3). Two genes (KLF16 and MSANTD2) were significantly enriched for PZMs genome-wide, and other PZMs involved genes (SCN2A, HNRNPU and SMARCA4) whose mutation is known to cause ASD or other neurodevelopmental disorders. PZMs constitute a significant proportion of de novo mutations and contribute importantly to ASD risk.

Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive cognitive impairment and neurodegeneration. However, despite extensive clinical and genomic studies, the molecular basis of AD development and progression remains elusive.To elucidate molecular systems associated with AD, we developed a large scale gene expression dataset from 1053 postmortem brain samples across 19 cortical regions of 125 individuals with a severity spectrum of dementia and neuropathology of AD. We excluded brain specimens that evidenced neuropathology other than that characteristic of AD. For the first time, we performed a pan-cortical brain region genomic analysis, characterizing the gene expression changes associated with a measure of dementia severity and multiple measures of the severity of neuropathological lesions associated with AD (neuritic plaques and neurofibrillary tangles) and constructing region-specific co-expression networks. We rank-ordered 44,692 gene probesets, 1558 co-expressed gene modules and 19 brain regions based upon their association with the disease traits.The neurobiological pathways identified through these analyses included actin cytoskeleton, axon guidance, and nervous system development. Using public human brain single-cell RNA-sequencing data, we computed brain cell type-specific marker genes for human and determined that many of the abnormally expressed gene signatures and network modules were specific to oligodendrocytes, astrocytes, and neurons. Analysis based on disease severity suggested that: many of the gene expression changes, including those of oligodendrocytes, occurred early in the progression of disease, making them potential translational/treatment development targets and unlikely to be mere bystander result of degeneration; several modules were closely linked to cognitive compromise with lesser association with traditional measures of neuropathology. The brain regional analyses identified temporal lobe gyri as sites associated with the greatest and earliest gene expression abnormalities.This transcriptomic network analysis of 19 brain regions provides a comprehensive assessment of the critical molecular pathways associated with AD pathology and offers new insights into molecular mechanisms underlying selective regional vulnerability to AD at different stages of the progression of cognitive compromise and development of the canonical neuropathological lesions of AD.

Degeneration of the retinal pigment epithelium is a hallmark of geographic atrophy, a type of age-related macular degeneration. Kerur et al. show that this degeneration results from a multistep pathway in which mitochondrial dysfunction in RPE cells, triggered by accumulation of Alu RNA, leads to activation of the noncanonical inflammasome via a cGAS–STING–IRF3 signaling axis. Geographic atrophy is a blinding form of age-related macular degeneration characterized by retinal pigmented epithelium (RPE) death; the RPE also exhibits DICER1 deficiency, resultant accumulation of endogenous Alu-retroelement RNA, and NLRP3-inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human-cell-culture and mouse models is driven by a noncanonical-inflammasome pathway that activates caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-β production and gasdermin D–dependent interleukin-18 secretion. Decreased DICER1 levels or Alu-RNA accumulation triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, interferon-β, and cGAS levels were elevated in the RPE in human eyes with geographic atrophy. Collectively, these data highlight an unexpected role of cGAS in responding to mobile-element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial-damage-induced inflammasome activation, expand the immune-sensing repertoire of cGAS and caspase-4 to noninfectious human disease, and identify new potential targets for treatment of a major cause of blindness.

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.

Late-onset Alzheimer's disease (AD) is heritable with 20 genes showing genome-wide association in the International Genomics of Alzheimer's Project (IGAP). To identify the biology underlying the disease, we extended these genetic data in a pathway analysis.The ALIGATOR and GSEA algorithms were used in the IGAP data to identify associated functional pathways and correlated gene expression networks in human brain.ALIGATOR identified an excess of curated biological pathways showing enrichment of association. Enriched areas of biology included the immune response (P = 3.27 × 10(-12) after multiple testing correction for pathways), regulation of endocytosis (P = 1.31 × 10(-11)), cholesterol transport (P = 2.96 × 10(-9)), and proteasome-ubiquitin activity (P = 1.34 × 10(-6)). Correlated gene expression analysis identified four significant network modules, all related to the immune response (corrected P = .002-.05).The immune response, regulation of endocytosis, cholesterol transport, and protein ubiquitination represent prime targets for AD therapeutics.

Because APOE locus variants contribute to risk of late-onset Alzheimer disease (LOAD) and to differences in age at onset (AAO), it is important to know whether other established LOAD risk loci also affect AAO in affected participants.To investigate the effects of known Alzheimer disease risk loci in modifying AAO and to estimate their cumulative effect on AAO variation using data from genome-wide association studies in the Alzheimer Disease Genetics Consortium.The Alzheimer Disease Genetics Consortium comprises 14 case-control, prospective, and family-based data sets with data on 9162 participants of white race/ethnicity with Alzheimer disease occurring after age 60 years who also had complete AAO information, gathered between 1989 and 2011 at multiple sites by participating studies. Data on genotyped or imputed single-nucleotide polymorphisms most significantly associated with risk at 10 confirmed LOAD loci were examined in linear modeling of AAO, and individual data set results were combined using a random-effects, inverse variance-weighted meta-analysis approach to determine whether they contribute to variation in AAO. Aggregate effects of all risk loci on AAO were examined in a burden analysis using genotype scores weighted by risk effect sizes.Age at disease onset abstracted from medical records among participants with LOAD diagnosed per standard criteria.Analysis confirmed the association of APOE with earlier AAO (P = 3.3 × 10(-96)), with associations in CR1 (rs6701713, P = 7.2 × 10(-4)), BIN1 (rs7561528, P = 4.8 × 10(-4)), and PICALM (rs561655, P = 2.2 × 10(-3)) reaching statistical significance (P < .005). Risk alleles individually reduced AAO by 3 to 6 months. Burden analyses demonstrated that APOE contributes to 3.7% of the variation in AAO (R(2) = 0.256) over baseline (R(2) = 0.221), whereas the other 9 loci together contribute to 2.2% of the variation (R(2) = 0.242).We confirmed an association of APOE (OMIM 107741) variants with AAO among affected participants with LOAD and observed novel associations of CR1 (OMIM 120620), BIN1 (OMIM 601248), and PICALM (OMIM 603025) with AAO. In contrast to earlier hypothetical modeling, we show that the combined effects of Alzheimer disease risk variants on AAO are on the scale of, but do not exceed, the APOE effect. While the aggregate effects of risk loci on AAO may be significant, additional genetic contributions to AAO are individually likely to be small.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) characterized by impairments in social communication and social interaction and the presence of repetitive behaviors and/or restricted interests. ASD has profound etiological and clinical heterogeneity, which has impeded the identification of risk factors and pathophysiological processes underlying the disorder. A constellation of (i) types of genetic variation, (ii) modes of inheritance and (iii) specific genomic loci and genes have all recently been implicated in ASD risk, and these findings are currently being extended with functional analyses in model organisms and genotype–phenotype correlation studies. The overlap of risk loci between ASD and other NDDs raises intriguing questions around the mechanisms of risk. In this review, we will touch upon these aspects of ASD and how they might be addressed.

Genetic loci for Alzheimer's disease (AD) have been identified in whites of European ancestry, but the genetic architecture of AD among other populations is less understood.We conducted a transethnic genome-wide association study (GWAS) for late-onset AD in Stage 1 sample including whites of European Ancestry, African-Americans, Japanese, and Israeli-Arabs assembled by the Alzheimer's Disease Genetics Consortium. Suggestive results from Stage 1 from novel loci were followed up using summarized results in the International Genomics Alzheimer's Project GWAS dataset.Genome-wide significant (GWS) associations in single-nucleotide polymorphism (SNP)-based tests (P < 5 × 10-8) were identified for SNPs in PFDN1/HBEGF, USP6NL/ECHDC3, and BZRAP1-AS1 and for the interaction of the (apolipoprotein E) APOE ε4 allele with NFIC SNP. We also obtained GWS evidence (P < 2.7 × 10-6) for gene-based association in the total sample with a novel locus, TPBG (P = 1.8 × 10-6).Our findings highlight the value of transethnic studies for identifying novel AD susceptibility loci.

Abstract Schizophrenia and bipolar disorder are serious mental illnesses that affect more than 2% of adults. While large-scale genetics studies have identified genomic regions associated with disease risk, less is known about the molecular mechanisms by which risk alleles with small effects lead to schizophrenia and bipolar disorder. In order to fill this gap between genetics and disease phenotype, we have undertaken a multi-cohort genomics study of postmortem brains from controls, individuals with schizophrenia and bipolar disorder. Here we present a public resource of functional genomic data from the dorsolateral prefrontal cortex (DLPFC; Brodmann areas 9 and 46) of 986 individuals from 4 separate brain banks, including 353 diagnosed with schizophrenia and 120 with bipolar disorder. The genomic data include RNA-seq and SNP genotypes on 980 individuals, and ATAC-seq on 269 individuals, of which 264 are a subset of individuals with RNA-seq. We have performed extensive preprocessing and quality control on these data so that the research community can take advantage of this public resource available on the Synapse platform at http://CommonMind.org .

Phelan-McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by psychiatric and neurological features. Most reported cases are caused by 22q13.3 deletions, leading to SHANK3 haploinsufficiency, but also usually encompassing many other genes. While the number of point mutations identified in SHANK3 has increased in recent years due to large-scale sequencing studies, systematic studies describing the phenotype of individuals harboring such mutations are lacking.We provide detailed clinical and genetic data on 17 individuals carrying mutations in SHANK3. We also review 60 previously reported patients with pathogenic or likely pathogenic SHANK3 variants, often lacking detailed phenotypic information.SHANK3 mutations in our cohort and in previously reported cases were distributed throughout the protein; the majority were truncating and all were compatible with de novo inheritance. Despite substantial allelic heterogeneity, four variants were recurrent (p.Leu1142Valfs*153, p.Ala1227Glyfs*69, p.Arg1255Leufs*25, and c.2265+1G>A), suggesting that these are hotspots for de novo mutations. All individuals studied had intellectual disability, and autism spectrum disorder was prevalent (73%). Severe speech deficits were common, but in contrast to individuals with 22q13.3 deletions, the majority developed single words, including 41% with at least phrase speech. Other common findings were consistent with reports among individuals with 22q13.3 deletions, including hypotonia, motor skill deficits, regression, seizures, brain abnormalities, mild dysmorphic features, and feeding and gastrointestinal problems.Haploinsufficiency of SHANK3 resulting from point mutations is sufficient to cause a broad range of features associated with PMS. Our findings expand the molecular and phenotypic spectrum of PMS caused by SHANK3 point mutations and suggest that, in general, speech impairment and motor deficits are more severe in the case of deletions. In contrast, renal abnormalities associated with 22q13.3 deletions do not appear to be related to the loss of SHANK3.

Autism spectrum disorder (ASD) affects up to 1 in 59 individuals1. Genome-wide association and large-scale sequencing studies strongly implicate both common variants2-4 and rare de novo variants5-10 in ASD. Recessive mutations have also been implicated11-14 but their contribution remains less well defined. Here we demonstrate an excess of biallelic loss-of-function and damaging missense mutations in a large ASD cohort, corresponding to approximately 5% of total cases, including 10% of females, consistent with a female protective effect. We document biallelic disruption of known or emerging recessive neurodevelopmental genes (CA2, DDHD1, NSUN2, PAH, RARB, ROGDI, SLC1A1, USH2A) as well as other genes not previously implicated in ASD including FEV (FEV transcription factor, ETS family member), which encodes a key regulator of the serotonergic circuitry. Our data refine estimates of the contribution of recessive mutation to ASD and suggest new paths for illuminating previously unknown biological pathways responsible for this condition.

Individuals with autism spectrum disorder (ASD), in addition to the core features of the disease, experience a higher burden of co-occurring medical conditions. This study sought to describe the frequency and distribution of comorbidit conditions in individuals with ASD, and systematically evaluate the possibility that pre- and postnatal exposures (e.g., preterm birth, hypoxia at birth, traumatic brain injury, and fetal alcohol syndrome) associated with ASD may also be linked with distinct comorbidities. We used the SPARK study database, launched by the Simons Foundation Autism Research Initiative (SFARI). Comorbidities considered in the study included neurological, cognitive, psychiatric, and physical conditions. The study sample consisted of 42,569 individuals with ASD and their 11,389 non-ASD siblings (full and half siblings). Majority (74%) of individuals with ASD had at least one comorbidity, and had a greater average number of comorbidities than their non-ASD siblings. Preterm birth and hypoxia at birth were the most common peri-natal exposures in the sample. In logistic regression models adjusted for covariates, these exposures were associated with several distinct comorbidities in ASD cases, including attention and behavior problems, psychiatric and neurological disorders, and growth conditions. A similar pattern of association was also observed in non-ASD siblings. Our findings underscore that individuals with ASD experience a greater burden of comorbidities, which could be partly attributable to the higher rates of perinatal exposures compared to their non-ASD siblings. Study findings, if replicated in other samples, can inform the etiology of comorbidity in ASD.

Although there is considerable evidence for a strong genetic component to idiopathic autism, several genomewide screens for susceptibility genes have been performed with limited concordance of linked loci, reflecting either numerous genes of weak effect and/or sample heterogeneity. Because decreasing sample heterogeneity would increase the power to identify genes, the effect on evidence for linkage of restricting a sample of autism-affected relative pairs to those with delayed onset (at age >36 mo) of phrase speech (PSD, for phrase speech delay) was studied. In the second stage of a two-stage genome screen for susceptibility loci involving 95 families with two or more individuals with autism or related disorders, a maximal multipoint heterogeneity LOD score (HLOD) of 1.96 and a maximal multipoint nonparametric linkage (NPL) score of 2.39 was seen on chromosome 2q. Restricting the analysis to the subset of families (n=49) with two or more individuals having a narrow diagnosis of autism and PSD generated a maximal multipoint HLOD score of 2.99 and an NPL score of 3.32. The increased scores in the restricted sample, together with evidence for heterogeneity in the entire sample, indicate that the restricted sample comprises a population that is more genetically homogeneous, which could therefore increase the likelihood of positional cloning of susceptibility loci. Although there is considerable evidence for a strong genetic component to idiopathic autism, several genomewide screens for susceptibility genes have been performed with limited concordance of linked loci, reflecting either numerous genes of weak effect and/or sample heterogeneity. Because decreasing sample heterogeneity would increase the power to identify genes, the effect on evidence for linkage of restricting a sample of autism-affected relative pairs to those with delayed onset (at age >36 mo) of phrase speech (PSD, for phrase speech delay) was studied. In the second stage of a two-stage genome screen for susceptibility loci involving 95 families with two or more individuals with autism or related disorders, a maximal multipoint heterogeneity LOD score (HLOD) of 1.96 and a maximal multipoint nonparametric linkage (NPL) score of 2.39 was seen on chromosome 2q. Restricting the analysis to the subset of families (n=49) with two or more individuals having a narrow diagnosis of autism and PSD generated a maximal multipoint HLOD score of 2.99 and an NPL score of 3.32. The increased scores in the restricted sample, together with evidence for heterogeneity in the entire sample, indicate that the restricted sample comprises a population that is more genetically homogeneous, which could therefore increase the likelihood of positional cloning of susceptibility loci. Autism/autistic disorder (MIM 209850) is a development disorder characterized by three classes of symptoms, including impairments in communication and reciprocal social interactions and repetitive or stereotyped behaviors and interests (Rapin and Katzman Rapin and Katzman, 1998Rapin I Katzman R Neurobiology of autism.Ann Neurol. 1998; 43: 7-14Crossref PubMed Scopus (201) Google Scholar). Twin studies have indicated that genetic factors play an important role in the etiology of autism (Folstein and Rutter Folstein and Rutter, 1977Folstein S Rutter M Infantile autism: a genetic study of 21 twin pairs.J Child Psychol Psychiatry. 1977; 18: 297-321Crossref PubMed Scopus (892) Google Scholar; Ritvo et al. Ritvo et al., 1985Ritvo ER Freeman BJ Mason-Brothers A Mo A Ritvo AM Concordance for the syndrome of autism in 40 pairs of afflicted twins.Am J Psychiatry. 1985; 142: 74-77PubMed Google Scholar; Steffenburg et al. Steffenburg et al., 1989Steffenburg S Gillberg C Hellgren L Andersson L Gillberg IC Jakobsson G Bohman M A twin study of autism in Denmark, Finland, Iceland, Norway and Sweden.J Child Psychol Psychiatry. 1989; 30: 405-416Crossref PubMed Scopus (624) Google Scholar; Bailey et al. Bailey et al., 1995Bailey A Le Couteur A Gottesman I Bolton P Simonoff E Yuzda E Rutter M Autism as a strongly genetic disorder: evidence from a British twin study.Psychol Med. 1995; 25: 63-77Crossref PubMed Scopus (1781) Google Scholar). The concordance rate for monozygotic twins is much higher than that of dizygotic twins. In addition, family studies indicate that the recurrence risk to siblings, estimated from multiple studies at 1%–3%, is profoundly higher than the risk to the general population, which is ∼.5–2/1,000 (Bolton et al. Bolton et al., 1994Bolton P Macdonald H Pickles A Rios P Goode S Crowson M Bailey A Rutter M A case-control family history study of autism.J Child Psychol Psychiatry. 1994; 35: 877-900Crossref PubMed Scopus (803) Google Scholar; Szatmari et al. Szatmari et al., 1998Szatmari P Jones MB Zwaigenbaum L MacLean JE Genetics of autism: overview and new directions.J Autism Dev Disord. 1998; 28: 351-368Crossref PubMed Scopus (214) Google Scholar). The mode of inheritance of autism appears complex, and latent-class analyses suggest that 3–10 genes may underlie the disorder (Pickles et al. Pickles et al., 1995Pickles A Bolton P Macdonald H Bailey A Le Couteur A Sim CH Rutter M Latent-class analysis of recurrence risks for complex phenotypes with selection and measurement error: a twin and family history study of autism.Am J Hum Genet. 1995; 57: 717-726PubMed Google Scholar), although an interpretation of at least one genomewide linkage analysis has argued for >10 genes underlying the disorder (Risch et al. Risch et al., 1999Risch N Spiker D Lotspeich L Nouri N Hinds D Hallmayer J Kalaydjieva L et al.A genomic screen of autism: evidence for a multilocus etiology.Am J Hum Genet. 1999; 65: 493-507Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Several genomewide screens for autism-susceptibility genes have recently been published (International Molecular Genetic Study of Autism Consortium [IMGSAC] International Molecular Genetic Study of Autism Consortium, 1998International Molecular Genetic Study of Autism Consortium A full genome screen for autism with evidence for linkage to a region on chromosome 7q: International Molecular Genetic Study of Autism Consortium.Hum Mol Genet. 1998; 7: 571-578Crossref PubMed Scopus (476) Google Scholar; Barrett et al. Barrett et al., 1999Barrett S Beck JC Bernier R Bisson E Braun TA Casavant TL Childress D et al.An autosomal genomic screen for autism: collaborative linkage study of autism.Am J Med Genet. 1999; 88: 609-615Crossref PubMed Scopus (307) Google Scholar; Philippe et al. Philippe et al., 1999Philippe A Martinez M Guilloud-Bataille M Gillberg C Rastam M Sponheim E Coleman M Zappella M Aschauer H Van Maldergem L Penet C Feingold J Brice A Leboyer M van Malldergerme L Genome-wide scan for autism susceptibility genes: Paris Autism Research International Sibpair Study.Hum Mol Genet. 1999; 8: 805-812Crossref PubMed Scopus (418) Google Scholar; Risch et al. Risch et al., 1999Risch N Spiker D Lotspeich L Nouri N Hinds D Hallmayer J Kalaydjieva L et al.A genomic screen of autism: evidence for a multilocus etiology.Am J Hum Genet. 1999; 65: 493-507Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar; see also Ashley-Koch et al. Ashley-Koch et al., 1999Ashley-Koch A Wolpert CM Menold MM Zaeem L Basu S Donnelly SL Ravan SA Powell CM Qumsiyeh MB Aylsworth AS Vance JM Gilbert JR Wright HH Abramson RK DeLong GR Cuccaro ML Pericak-Vance MA Genetic studies of autistic disorder and chromosome 7.Genomics. 1999; 61: 227-236Crossref PubMed Scopus (163) Google Scholar; Auranen et al. Auranen et al., 2000Auranen M Nieminen T Majuri S Vanhala R Peltonen L Jarvela I Analysis of autism susceptibility gene loci on chromosomes 1p, 4p, 6q, 7q, 13q, 15q, 16p, 17q, 19q and 22q in Finnish multiplex families.Mol Psychiatry. 2000; 5: 320-322Crossref PubMed Scopus (47) Google Scholar). In these screens, there was little overlap in the highest linkage peaks. The first screen, by the IMGSAC (International Molecular Genetic Study of Autism Consortium, 1998International Molecular Genetic Study of Autism Consortium A full genome screen for autism with evidence for linkage to a region on chromosome 7q: International Molecular Genetic Study of Autism Consortium.Hum Mol Genet. 1998; 7: 571-578Crossref PubMed Scopus (476) Google Scholar), identified a peak on chromosome 7, where there appears to be the greatest concordance of findings (reviewed in Folstein and Mankoski Folstein and Mankoski, 2000Folstein SE Mankoski RE Chromosome 7q: where autism meets language disorder?.Am J Hum Genet. 2000; 67: 278-281Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, even in those studies with some evidence for linkage to this region, there are discrepancies in peak localization. Furthermore, the evidence for linkage observed by the IMGSAC has decreased at this location with additional families (IMGSAC International Molecular Genetic Study of Autism Consortium, 2000International Molecular Genetic Study of Autism Consortium (2000) Search for autism susceptibility loci: genome screen follow-up and fine mapping of a candidate region on chromosome 7q. Presented at Collegium Internationale Neuro-Psychopharamacologium 22nd Congress, Brussels, July 9–13Google Scholar). The next highest peak in the IMGSAC study and the highest peaks in other studies—for example, on chromosome 16p (IMGSAC International Molecular Genetic Study of Autism Consortium, 1998International Molecular Genetic Study of Autism Consortium A full genome screen for autism with evidence for linkage to a region on chromosome 7q: International Molecular Genetic Study of Autism Consortium.Hum Mol Genet. 1998; 7: 571-578Crossref PubMed Scopus (476) Google Scholar), 13 (Barrett et al. Barrett et al., 1999Barrett S Beck JC Bernier R Bisson E Braun TA Casavant TL Childress D et al.An autosomal genomic screen for autism: collaborative linkage study of autism.Am J Med Genet. 1999; 88: 609-615Crossref PubMed Scopus (307) Google Scholar), 2q, 7q, 16p, and 19p (Philippe et al. Philippe et al., 1999Philippe A Martinez M Guilloud-Bataille M Gillberg C Rastam M Sponheim E Coleman M Zappella M Aschauer H Van Maldergem L Penet C Feingold J Brice A Leboyer M van Malldergerme L Genome-wide scan for autism susceptibility genes: Paris Autism Research International Sibpair Study.Hum Mol Genet. 1999; 8: 805-812Crossref PubMed Scopus (418) Google Scholar), and 1p (Risch et al. Risch et al., 1999Risch N Spiker D Lotspeich L Nouri N Hinds D Hallmayer J Kalaydjieva L et al.A genomic screen of autism: evidence for a multilocus etiology.Am J Hum Genet. 1999; 65: 493-507Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar)—showed less reproducibility between studies. These issues have led to the suggestion that autism may involve extensive genetic heterogeneity (Lamb et al. Lamb et al., 2000Lamb JA Moore J Bailey A Monaco AP Autism: recent molecular genetic advances.Hum Mol Genet. 2000; 9: 861-868Crossref PubMed Scopus (127) Google Scholar) and/or many interacting genes of weak effect (Risch et al. Risch et al., 1999Risch N Spiker D Lotspeich L Nouri N Hinds D Hallmayer J Kalaydjieva L et al.A genomic screen of autism: evidence for a multilocus etiology.Am J Hum Genet. 1999; 65: 493-507Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). If so, it may prove very difficult, with the methods of linkage analysis and positional cloning, to use genomewide linkage analysis to identify susceptibility loci with the typical sample size of 50–200 families. Regardless of the actual number of genes involved in autism, decreasing sample heterogeneity will increase the likelihood of identifying genes. Sample heterogeneity may be reduced by use of narrower inclusion criteria or by identication of traits that are shared among subgroups of families. Identifying traits that tend to be shared by family members—particularly by affected sibling pairs—is a means of identifying useful subgroups of families and may identify traits that have greater genetic components. Two studies, both with <50 sibships, have been published that identify Autism Diagnostic Interview (ADI) (Lord et al. Lord et al., 1994Lord C Rutter M Le Couteur A Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders.J Autism Dev Disord. 1994; 24: 659-685Crossref PubMed Scopus (6298) Google Scholar) components, which showed increased familiality. In one study (Spiker et al. Spiker et al., 1994Spiker D Lotspeich L Kraemer HC Hallmayer J McMahon W Petersen PB Nicholas P Pingree C Wiese-Slater S Chiotti C et al.Genetics of autism: characteristics of affected and unaffected children from 37 multiplex families.Am J Med Genet. 1994; 54: 27-35Crossref PubMed Scopus (80) Google Scholar), familiality was observed in the repetitive behavior domain, and in the second study (MacLean et al. MacLean et al., 1999MacLean JE Szatmari P Jones MB Bryson SE Mahoney WJ Bartolucci G Tuff L Familial factors influence level of functioning in pervasive developmental disorder.J Am Acad Child Adolesc Psychiatry. 1999; 38: 746-753Abstract Full Text PDF PubMed Scopus (63) Google Scholar), familiality was observed in impairments in nonverbal communication and verbal/nonverbal status. In a recent analysis of 136 sibling pairs with autism, strong evidence was found for concordance between affected sibling pairs for the severity of repetitive behaviors, the level of deficits in nonverbal communication, the presence of phrase speech, and the age at onset of phrase speech (J. M. Silverman, C. J. Smith, J. Schmeidler, E. Hollander, B. A. Lawlor, M. Fitzgerald, J. D. Buxbaum, K. Delaney, P. Galvin, unpublished data). Any of these clinical traits might be useful to subcategorize families for linkage analysis. Families in which affected members demonstrate greater deficits in these domains may be at greater genetic risk for autism and/or may be genetically more homogeneous. On the basis of studies of traits in nonaffected members of autistic probands' families, the use of language has been proposed as a means to segregate families with autism for linkage (Folstein et al. Folstein et al., 1999Folstein SE Santangelo SL Gilman SE Piven J Landa R Lainhart J Hein J Wzorek M Predictors of cognitive test patterns in autism families.J Child Psychol Psychiatry. 1999; 40: 1117-1128Crossref PubMed Google Scholar). More recently, because of the presence of a gene involved in language disorder on 7q (Fisher et al. Fisher et al., 1998Fisher SE Vargha-Khadem F Watkins KE Monaco AP Pembrey ME Localisation of a gene implicated in a severe speech and language disorder.Nat Genet. 1998; 18: 168-170Crossref PubMed Scopus (382) Google Scholar), near the peak of linkage to autism observed in several studies, it has been suggested that grouping families in subsets on the basis of language may increase evidence for linkage to this region (Folstein and Mankoski Folstein and Mankoski, 2000Folstein SE Mankoski RE Chromosome 7q: where autism meets language disorder?.Am J Hum Genet. 2000; 67: 278-281Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In one sample, restricting analyses to families with phrase-speech delay (PSD) and including as affected those parents who, through self-reporting, had delayed onset of speech, trouble learning to read, or persistent spelling difficulties, did in fact increase evidence for linkage to 7q (Folstein and CLSA Folstein and for CLSA, 2000Folstein SE for CLSA Autism LOD on chromosome 7q was increased subsetting the sample on language acquisition.Am J Hum Genet. 2000; 67: S49Google Scholar). On the basis of evidence for familiality of age at onset of phrase speech (MacLean et al. MacLean et al., 1999MacLean JE Szatmari P Jones MB Bryson SE Mahoney WJ Bartolucci G Tuff L Familial factors influence level of functioning in pervasive developmental disorder.J Am Acad Child Adolesc Psychiatry. 1999; 38: 746-753Abstract Full Text PDF PubMed Scopus (63) Google Scholar; J. M. Silverman, C. J. Smith, J. Schmeidler, E. Hollander, B. A. Lawlor, M. Fitzgerald, J. D. Buxbaum, K. Delaney, P. Galvin, unpublished data), restricting analyses to families with PSD may be useful to decrease heterogeneity, thereby increasing the power to identify susceptibility loci throughout the genome. In the current study, Autism Diagnostic Interview–Revised (ADI-R) (Lord et al. Lord et al., 1994Lord C Rutter M Le Couteur A Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders.J Autism Dev Disord. 1994; 24: 659-685Crossref PubMed Scopus (6298) Google Scholar) criteria were used to identify 95 families with affected relative pairs with autism, borderline autism, or Asperger disorder (table 1). To be considered affected with autism, an individual had to satisfy the prespecified cutoff scores in all three symptom areas of the ADI-R and had to present with evidence for onset of symptoms at age <36 mo (Lord et al. Lord et al., 1994Lord C Rutter M Le Couteur A Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders.J Autism Dev Disord. 1994; 24: 659-685Crossref PubMed Scopus (6298) Google Scholar). Individuals who failed to meet the ADI-R algorithm criteria for autism by no more than one point in the social domain and either, but not both, the communication or the repetitive-behavior domain were given a research diagnosis of borderline autism. A diagnosis of borderline autism was also given to individuals in which all three domains were above threshold but the onset criterion for autism was not met. A similar research category has been used in another study (IMGSAC International Molecular Genetic Study of Autism Consortium, 1998International Molecular Genetic Study of Autism Consortium A full genome screen for autism with evidence for linkage to a region on chromosome 7q: International Molecular Genetic Study of Autism Consortium.Hum Mol Genet. 1998; 7: 571-578Crossref PubMed Scopus (476) Google Scholar). A research diagnosis of Asperger disorder was given to those with neither autism nor borderline autism but who met DSM-IV criteria for Asperger disorder. At least one individual in each family had to satisfy ADI-R criteria for autism. In 84 families, a second individual also met ADI-R criteria for autism (families in class I), whereas in 8 families a second individual met criteria for borderline autism, and in 3 families a second individual met DSM-IV criteria for Asperger disorder (families in class II). Family studies suggest that genetic liability extends to other pervasive developmental disorders and to Asperger disorder, and it has been argued that including such individuals will not introduce significant genetic heterogeneity (IMGSAC International Molecular Genetic Study of Autism Consortium, 1998International Molecular Genetic Study of Autism Consortium A full genome screen for autism with evidence for linkage to a region on chromosome 7q: International Molecular Genetic Study of Autism Consortium.Hum Mol Genet. 1998; 7: 571-578Crossref PubMed Scopus (476) Google Scholar).Table 1Demographics of Family GroupsNo. of Families with PSD/Total No. of FamiliesClass IClasses I and IIFamilies49/8457/95Individuals genotyped231/383273/439Autistic males82/13490/145Autistic females16/3516/35Borderline autistic males1/18/8Borderline autistic females0/01/1Males with Asperger disorder0/00/1Females with Asperger disorder0/00/2Sib pairs46/8054/91Sib trios1/21/2First-cousin pairs2/22/2Note.—Families were recruited if they had at least two members affected with a suspected diagnosis of autism, pervasive development disorder, or Asperger disorder. With written informed consent from the parents, all potentially affected individuals were subsequently assessed by the Autism Diagnostic Interview-Revised (ADI-R) (Lord et al. Lord et al., 1994Lord C Rutter M Le Couteur A Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders.J Autism Dev Disord. 1994; 24: 659-685Crossref PubMed Scopus (6298) Google Scholar). Most assessments were conducted by ADI raters (including C.J.S.) trained by Dr. Catherine Lord, at the University of Chicago. Additional raters were trained by C.J.S., who has had extensive training with Dr. Lord and her group and has been credentialed as an off-site trainer and as a “reliable rater” against which another’s reliability can be assessed. All raters achieved reliability of >90% with either Dr. Lord’s group or C.J.S. The families include those recruited by the Autism Genetic Research Exchange (AGRE), by the Seaver Autism Research Center, and collaboratively by AGRE and the Seaver Center. ADI-R worksheets were reviewed, and diagnosis confirmed, by C.J.S. PSD was defined as onset of phrase speech at age >36 mo and corresponded to question 13 on the ADI-R. For a family to be categorized as having PSD, two or more members had PSD. Open table in a new tab Note.— Families were recruited if they had at least two members affected with a suspected diagnosis of autism, pervasive development disorder, or Asperger disorder. With written informed consent from the parents, all potentially affected individuals were subsequently assessed by the Autism Diagnostic Interview-Revised (ADI-R) (Lord et al. Lord et al., 1994Lord C Rutter M Le Couteur A Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders.J Autism Dev Disord. 1994; 24: 659-685Crossref PubMed Scopus (6298) Google Scholar). Most assessments were conducted by ADI raters (including C.J.S.) trained by Dr. Catherine Lord, at the University of Chicago. Additional raters were trained by C.J.S., who has had extensive training with Dr. Lord and her group and has been credentialed as an off-site trainer and as a “reliable rater” against which another’s reliability can be assessed. All raters achieved reliability of >90% with either Dr. Lord’s group or C.J.S. The families include those recruited by the Autism Genetic Research Exchange (AGRE), by the Seaver Autism Research Center, and collaboratively by AGRE and the Seaver Center. ADI-R worksheets were reviewed, and diagnosis confirmed, by C.J.S. PSD was defined as onset of phrase speech at age >36 mo and corresponded to question 13 on the ADI-R. For a family to be categorized as having PSD, two or more members had PSD. A genetic screen was performed in two stages. In the first stage, 35 families were genotyped with 382 ABI PRISM markers (Linkage Marker Set MD-10, Applied Biosystems) across the autosomes (∼10-cM density), using a microcapillary-based ABI PRISM 310 Genetic Analyzer. Using two-point analyses, we observed the strongest evidence for linkage on chromosome 2q, at marker D2S364 (heterogeneity LOD [HLOD] under dominant, 2.25) (table 2). An adjacent marker also provided evidence for linkage (D2S335, HLOD under dominant, 1.20). These markers, along with flanking markers, were then analyzed in the entire cohort (n=95). Evidence suggestive of linkage was observed in the entire sample on distal chromosome 2 by both parametric (maximal multipoint HLOD scores [MMHLS] of 1.96) and nonparametric (nonparametric linkage [NPL] score of 2.39) methods (fig. 1float-anchor , top left, and table 3, bottom right). The estimate of families with linkage (α, table 3), as well as the divergence between LOD scores and HLOD scores (table 3), is suggestive of genetic heterogeneity in the sample.Table 2Two-Point Linkage Results of Genome ScanDominantRecessiveChromosome and MarkerPosition (cM)LODHLODαLODHLODαNPL ScoreP1: D1S2842273.5−.661.17.61−2.061.24.461.40.082: D2S3197.6.021.20.66−3.41.55.311.69.04 D2S112141.6−3.57.39.38−4.74.44.281.04.14 D2S335175.9−1.481.20.58−5.98.54.261.59.05 D2S364186.21.802.25.82−1.281.65.462.45.01 D2S325204.5−3.26.67.47−5.52.65.291.52.06 D2S2382213.5−2.68.35.37−4.80.66.301.26.103: D3S128591.2−4.65.32.33−9.10.12.121.05.14 D3S1278129.7−5.50.45.34−6.76.64.271.44.08 D3S1267139.1−3.67.96.47−7.77.67.251.91.03 D3S1292146.6−9.74.39.27−10.67.86.251.24.10 D3S1279169.6−4.19.34.32−8.07.46.201.01.155: D5S40611.9−.671.21.60−2.291.46.461.65.05 D5S63019.7−3.37.62.41−6.03.73.261.30.096: D6S30914.1−2.73.56.44−4.44.85.321.65.05 D6S264179.1−2.64.56.16−3.56.90.391.12.137: D7S50278.7−1.95.45.42−4.83.47.031.30.09 D7S684147.2−4.23.75.45−6.09.63.281.53.068: D8S55021.3−4.18.74.46−6.69.64.271.59.05 D8S270103.7−4.08.31.33−6.97.38.221.12.139: D9S181759.3−2.37.44.43−6.94.10.121.04.14 D9S17570.3−2.21.51.46−5.82.31.211.25.10 D9S28394.9−1.751.09.56−3.981.09.381.72.04 D9S158161.71.051.66.801.161.89.741.41.0711: D11S98767.5−5.03.47.35−6.91.61.281.13.13 D11S131473.6−3.12.65.43−5.35.74.301.44.07 D11S93780.0−4.27.73.43−7.51.36.211.19.1117: D17S186864.2−1.39.69.51−3.33.87.371.10.1319: D19S21620.0−4.72.41.36−5.31.35.321.06.14 D19S22642.3−2.60.50.40−5.68.61.251.13.1220: D20S11521.2−2.56.54.47−4.89.52.291.04.15Note.—Thirty-five first-stage families were genotyped with 382 autosomal ABI PRISM markers. Two-point LOD and HLOD (Smith Smith, 1963Smith CAB Testing for heterogeneity of the recombination fraction values in human genetics.Ann Hum Genet. 1963; 27: 175-182Crossref PubMed Scopus (220) Google Scholar; Durner and Greenberg Durner and Greenberg, 1992Durner M Greenberg DA Effect of heterogeneity and assumed mode of inheritance on lod scores.Am J Med Genet. 1992; 42: 271-275Crossref PubMed Scopus (23) Google Scholar) scores were calculated under dominant or recessive models (Vieland et al. Vieland et al., 1992bVieland VJ Hodge SE Greenberg DA Adequacy of single-locus approximations for linkage analyses of oligogenic traits.Genet Epidemiol. 1992; 9: 45-59Crossref PubMed Scopus (87) Google Scholar; Durner et al. Durner et al., 1999Durner M Vieland VJ Greenberg DA Further evidence for the increased power of LOD scores compared with nonparametric methods.Am J Hum Genet. 1999; 64: 281-289Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) at each marker, with penetrance set at 50% (Greenberg et al. Greenberg et al., 1998Greenberg DA Abreu P Hodge SE The power to detect linkage in complex disease by means of simple LOD-score analyses.Am J Hum Genet. 1998; 63: 870-879Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and with disease-allele frequencies arbitrarily set at .1 for recessive inheritance and .006 for dominant inheritance. For HLOD analyses, the proportion of families with linkage (α) was also estimated. In addition, two-point NPL scores were determined, along with associated P values. All genotyped family members were included in the analyses, and any individual not receiving a research diagnosis of autism, borderline autism, or Asperger disorder was defined as phenotypically unknown. All analyses were done with GENEHUNTER (Kruglyak et al. Kruglyak et al., 1996Kruglyak L Daly MJ Reeve-Daly MP Lander ES Parametric and nonparametric linkage analysis: a unified multipoint approach.Am J Hum Genet. 1996; 58: 1347-1363PubMed Google Scholar). All markers demonstrating a LOD, HLOD, or NPL score >1.0 are shown. Of the 35 families in the first-stage screen, 22 had PSD and 27 were in class 1. Open table in a new tab Table 3Multipoint Analyses of Chromosome 2qWith PSDTotalFamilies in Class I175.4 cM; 2.99 HLOD; α = .69181.1 cM; 1.97 HLOD; α = .50Dominant174.0 cM; 1.52 LOD183.3 cM; −3.49 LOD;174.4 cM; 2.71 HLOD; α = .50174.0 cM; 1.47 HLOD; α = .27Recessive174.4 cM; −1.88 LOD;172.6 cM; −13.21 LOD;NPL176.8 cM; Z-score = 3.32; P = 3.8 × 10−4176.8 cM; Z-score = 2.47; P = 6.2 × 10−3Families in Classes I and II179.0 cM; 2.62 HLOD; α = .61183.3 cM; 1.96 HLOD; α = .48Dominant181.2 cM; −.30 LOD183.3 cM; −4.54 LOD175.4 cM; 2.46 HLOD; α = .42174.0 cM; 1.38 HLOD; α = .24Recessive174.0 cM; −5.06 LOD;174.0 cM; −18.05 LOD;NPL176.8 cM; Z-score = 3.17; P = 7.1 × 10−4176.8 cM; Z-score = 2.39; P = 7.8 × 10−3Note.—Multipoint LOD and HLOD scores were calculated under dominant or recessive models, as detailed in the legend to figure 1. The peak score is indicated, along with its position and, for HLOD, the estimated fraction of families with linkage (α). Multipoint NPL scores are also indicated, along with the position of the peak, and associated P values. Open table in a new tab Note.— Thirty-five first-stage families were genotyped with 382 autosomal ABI PRISM markers. Two-point LOD and HLOD (Smith Smith, 1963Smith CAB Testing for heterogeneity of the recombination fraction values in human genetics.Ann Hum Genet. 1963; 27: 175-182Crossref PubMed Scopus (220) Google Scholar; Durner and Greenberg Durner and Greenberg, 1992Durner M Greenberg DA Effect of heterogeneity and assumed mode of inheritance on lod scores.Am J Med Genet. 1992; 42: 271-275Crossref PubMed Scopus (23) Google Scholar) scores were calculated under dominant or recessive models (Vieland et al. Vieland et al., 1992bVieland VJ Hodge SE Greenberg DA Adequacy of single-locus approximations for linkage analyses of oligogenic traits.Genet Epidemiol. 1992; 9: 45-59Crossref PubMed Scopus (87) Google Scholar; Durner et al. Durner et al., 1999Durner M Vieland VJ Greenberg DA Further evidence for the increased power of LOD scores compared with nonparametric methods.Am J Hum Genet. 1999; 64: 281-289Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) at each marker, with penetrance set at 50% (Greenberg et al. Greenberg et al., 1998Greenberg DA Abreu P Hodge SE The power to detect linkage in complex disease by means of simple LOD-score analyses.Am J Hum Genet. 1998; 63: 870-879Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and with disease-allele frequencies arbitrarily set at .1 for recessive inheritance and .006 for dominant inheritance. For HLOD analyses, the proportion of families with linkage (α) was also estimated. In addition, two-point NPL scores were determined, along with associated P values. All genotyped family members were included in the analyses, and any individual not receiving a research diagnosis of autism, borderline autism, or Asperger disorder was defined as phenotypically unknown. All analyses were done with GENEHUNTER (Kruglyak et al. Kruglyak et al., 1996Kruglyak L Daly MJ Reeve-Daly MP Lander ES Parametric and nonparametric linkage analysis: a unified multipoint approach.Am J Hum Genet. 1996; 58: 1347-1363PubMed Google Scholar). All markers demonstrating a LOD, HLOD, or NPL score >1.0 are shown. Of the 35 families in the first-stage screen, 22 had PSD and 27 were in class 1. Note.— Multipoint LOD and HLOD scores were calculated under dominant or recessive models, as detailed in the legend to figure 1. The peak score is indicated, along with its position and, for HLOD, the estimated fraction of families with linkage (α). Multipoint NPL scores are also indicated, along with the position of the peak, and associated P values. In an attempt to narrow diagnostic criteria to reduce heterogeneity, families in which there were two or more members with autism (families in class I) and onset of phrase speech at age >36 mo were examined for evidence of linkage. In the 49 families meeting these criteria, there was increased evidence for linkage (MMHLS of 2.99 and NPL score of 3.32), compared with the results for the entire cohort (fig. 1, bottom right, and table 3, top left). Further, exploratory analyses were performed to identify the determinants in the family groups that contributed to the increased evidence for linkage. Linkage analysis only in families in which there were two individuals meeting ADI-R criteria for autism (families in class I, n=84), without a requirement for PSD, had little effect on observed MMHLS and NPL scores (fig. 1, top right, and table 3, top right), compared with analyses done on the entire sample. In contrast, linkage analysis in the 57 families in classes I and II in which the affected family members had PSD demonstrated more dramatic increases in MMHLS and NPL scores (fig. 1, bottom left, and table 3, bottom left), compared with analyses done in the entire sample. These data indicate that limiting analyses to families with PSD had a greater effect on the evidence for linkage than limiting the study to families in class I. Evidence for heterogeneity decreased as more restrictive criteria were applied. Thus α, the estimate of families with linkage, increased from 48% to 69%, using multipoint HLOD analyses under the dominant model, and increased from 24% to 50%, using these analyses under the recessive model (table 3). With evidence for greater homogeneity in the families in class I with PSD, we performed traditional multipoint LOD score analyses in this sample (that is, we did not allow for heterogeneity). Under a dominant model, maximal LOD score was 1.52 at 166.4 cM (table 3, top left). At this position, the LOD score was −1.88 under a recessive model. In all other subsets of the data, LOD scores were below zero in both dominant and recessive models. The positive LOD score in the most restricted sample suggests that there is increased homogeneity in this subset of families. To test for heterogeneity, we made use of the predivided sample test (Morton Morton, 1956Morton NE The detection and estimation of linkage between the genes for elliptocytosis and the Rh blood type.Am J Hum Genet. 1956; 8: 80-96PubMed Google Scholar). LOD or HLOD scores were compared between the entire sample, the most restricted sample, and all families excluded from the most restricted sample. For both the MMHLS and maximal multipoint LOD score analyses, these comparisons provided significant evidence for heterogeneity (χ2=6.31; df=1, P=.0120, and χ2=7.71, df=1; P=.0054, respectively). The data in this study provide evidence suggesting linkage to autism for a region on chromosome 2. It has recently been reported that the IMGSAC, which had previously found its highest LOD score on chromosome 7q (IMGSAC International Molecular Genetic Study of Autism Consortium, 1998International Molecular Genetic Study of Autism Consortium A full genome screen for autism with evidence for linkage to a region on chromosome 7q: International Molecular Genetic Study of Autism Consortium.Hum Mol Genet. 1998; 7: 571-578Crossref PubMed Scopus (476) Google Scholar), has found that, after additional families were genotyped (for a total of 173), the highest MLS scores were on chromosomes 9 (MLS 2.61), 16 (MLS 2.55), and 2 (MLS 2.33) (IMGSAC International Molecular Genetic Study of Autism Consortium, 2000International Molecular Genetic Study of Autism Consortium (2000) Search for autism susceptibility loci: genome screen follow-up and fine mapping of a candidate region on chromosome 7q. Presented at Collegium Internationale Neuro-Psychopharamacologium 22nd Congress, Brussels, July 9–13Google Scholar). The peak on chromosome 2 was localized to the same position as the peak we observed. In a recent scan involving 75 families, there is also weak evidence for linkage in the same region (∼180 cM) (Barrett et al. Barrett et al., 1999Barrett S Beck JC Bernier R Bisson E Braun TA Casavant TL Childress D et al.An autosomal genomic screen for autism: collaborative linkage study of autism.Am J Med Genet. 1999; 88: 609-615Crossref PubMed Scopus (307) Google Scholar). In addition, in an unpublished study involving 99 families, there was a peak (MLS 1.30 at 198.6 cM) on chromosome 2 that overlapped with our peak (Bass et al. Bass et al., 2000Bass MP Menold MM Joyner KL Wolpert CM Donnelly SL Ravan SA McClain C vonWendt L Gilbert JR Wright HH Abramson RK DeLong GR Cuccaro ML Pericak-Vance MA Association analysis of GI candidate genes in autistic disorder.Am J Hum Genet. 2000; 67: S49Google Scholar). Although slightly distal, in a scan involving 51 families, two-point MLS analysis yielded scores of 0.98 and 0.65 at markers D2S382 (at 210 cM) and D2S364 (at 231.5 cM) (Philippe et al. Philippe et al., 1999Philippe A Martinez M Guilloud-Bataille M Gillberg C Rastam M Sponheim E Coleman M Zappella M Aschauer H Van Maldergem L Penet C Feingold J Brice A Leboyer M van Malldergerme L Genome-wide scan for autism susceptibility genes: Paris Autism Research International Sibpair Study.Hum Mol Genet. 1999; 8: 805-812Crossref PubMed Scopus (418) Google Scholar). In the current analyses, there was increased evidence for linkage when the analyses were restricted to those families in which affected family members demonstrate PSD. Because the current analyses involved parametric analyses under two models as well as grouping the data in subsets, the results can only be taken as suggestive and require replication in a separate group of families before evidence for linkage will be compelling. Several approaches have been considered in the autism research community as means to improve the chances of finding genes by positional cloning. These approaches include narrowing the definition of affectedness to define more genetically homogeneous groups and/or increasing information by defining individuals with a broader autism phenotype (Le Couteur et al. Le Couteur et al., 1996Le Couteur A Bailey A Goode S Pickles A Robertson S Gottesman I Rutter M A broader phenotype of autism: the clinical spectrum in twins.J Child Psychol Psychiatry. 1996; 37: 785-801Crossref PubMed Scopus (329) Google Scholar; Piven et al. Piven et al., 1997Piven J Palmer P Jacobi D Childress D Arndt S Broader autism phenotype: evidence from a family history study of multiple-incidence autism families.Am J Psychiatry. 1997; 154: 185-190PubMed Google Scholar) as affected. In the current study, the increase in LOD and HLOD scores in the restricted sample with PSD and the evidence for heterogeneity in the entire sample indicates that narrower inclusion criteria may result in decreased heterogeneity and may thereby increase power to detect linkage in autism. Because it is likely that autism-susceptibility genes result in a broader autism phenotype in unaffected family members, a further increase in power might be achieved in this restricted sample by including as affected those individuals with a broader autism phenotype. In summary, the current results, when combined with results from other groups, indicate that there may be an autism-susceptibility gene on chromosome 2q. Furthermore, these results suggest that, if narrower inclusion criteria are used, it may be possible to define a genetically more-homogeneous group that could facilitate the positional cloning of putative autism-susceptibility genes. We thank Dr. Susan Hodge for insights into the use of the predivided sample test. We also thank Cure Autism Now and the Autism Genetic Resource Exchange for providing DNA samples. These studies were supported by grants from the Seaver Autism Research Center (to K.L.D.) and Cure Autism Now (to J.D.B.). Erratum et al.The American Journal of Human GeneticsAugust, 2001In BriefIn the June 2001 issue of the Journal, in the report “Evidence for a Susceptibility Gene for Autism on Chromosome 2 and for Genetic Heterogeneity,” by Buxbaum et al. (68:1514–1520), there was an error in the Acknowledgments: the second sentence should read “We gratefully acknowledge the resources provided by the AGRE Consortium and the participating AGRE families.” In addition, a list of the consortium members should have been provided. The members of the AGRE Scientific Advisory Board are Daniel H. Full-Text PDF Open Archive

Autistic disorder (OMIM 209850) is a disease with a significant genetic component of a complex nature.1 Cytogenetic abnormalities in the Prader-Willi/Angelman syndrome critical region (15q11–13) have been described in several individuals with autism.1 For this reason, markers across this region have been screened for evidence of linkage and association, and a marker (155CA-2) in the γ-aminobutyric acid type-A receptor β3 subunit gene (GABRB3) has been associated in one study2 but not others.3–5 We completed an association analysis with 155CA-2 using the transmission disequilibrium test (TDT) in a set of 80 autism families (59 multiplex and 21 trios). We also used four additional markers (69CA, 155CA-1, 85CA, and A55CA-1) localized within 150 kb of 155CA-2. The use of multi-allelic TDT (MTDT) (P < 0.002), as well as the TDT (P < 0.004), demonstrated an association between autistic disorder and 155CA-2 in these families. Meiotic segregation distortion could be excluded as a possible cause for these results since no disequilibrium was observed in unaffected siblings. These findings support a role for genetic variants within the GABA receptor gene complex in 15q11–13 in autistic disorder.

HER4 is a member of the epidermal growth factor receptor family and has an essential function in heart and neural development. Identification of two HER4 isoforms, HER4 JM-a and JM-b, which differ in their extracellular juxtamembrane region and in their susceptibility to cleavage after phorbol ester stimulation, showed that the juxtamembrane region of the receptor is critical for proteolysis. We now demonstrate that phorbol ester and pervanadate are effective stimuli for HER4 JM-a processing and that the HER4 JM-b isoform does not undergo cleavage in response to any of the stimuli studied. We also show that HER4 JM-a is not cleaved in cells lacking the metalloprotease tumor necrosis factor-α-converting enzyme (TACE) and that reexpression of TACE in these cells restores constitutive and regulated processing of HER4 JM-a. Moreover, we show that the sequence specific to the HER4 JM-a juxtamembrane region is sufficient to confer susceptibility to phorbol 12-myristate 13-acetate-induced cleavage of the HER2 receptor. In conclusion, we provide evidence that TACE is essential for the regulated shedding of the HER4 JM-a receptor.

Autism is a psychiatric disorder with estimated heritability of 90%. One-third of autistic individuals experience seizures. A susceptibility locus for autism was mapped near a cluster of voltage-gated sodium channel genes on chromosome 2. Mutations in two of these genes, SCN1A and SCN2A, result in the seizure disorder GEFS+. To evaluate these sodium channel genes as candidates for the autism susceptibility locus, we screened for variation in coding exons and splice sites in 117 multiplex autism families. A total of 27 kb of coding sequence and 3 kb of intron sequence were screened. Only six families carried variants with potential effects on sodium channel function. Five coding variants and one lariat branchpoint mutation were each observed in a single family, but were not present in controls. The variant R1902C in SCN2A is located in the calmodulin binding site and was found to reduce binding affinity for calcium-bound calmodulin. R542Q in SCN1A was observed in one autism family and had previously been identified in a patient with juvenile myoclonic epilepsy. The effect of the lariat branchpoint mutation was tested in cultured lymphoblasts. Additional population studies and functional tests will be required to evaluate pathogenicity of the coding and lariat site variants. SNP density was 1/kb in the genomic sequence screened. We report 38 sodium channel SNPs that will be useful in future association and linkage studies.

The major component of amyloid plaque cores and cerebrovascular amyloid deposits found in Alzheimer disease is the beta/A4 peptide, which is derived from the Alzheimer amyloid protein precursor (APP). Recent evidence suggests that abnormalities in beta/A4 peptide production or beta/A4 peptide aggregation may underlie cerebral amyloidosis. In the present study, treatment of cells with phorbol dibutyrate, which activates protein kinase C, and/or okadaic acid, which inhibits protein phosphatases 1 and 2A, reduced beta/A4 peptide production by 50-80%. These effects were observed with APP695 and APP751 expressed in stably transfected CHO cells, as well as with endogenous APP in human glioma (Hs 683) cells. Phorbol dibutyrate also decreased beta/A4 peptide production in cells expressing various mutant forms of APP associated with familial Alzheimer disease, one of which was reported to manifest greatly increased beta/A4 peptide production in cultured cells. Mastoparan and mastoparan X, compounds which can activate phospholipase C and hence protein kinase C, also decreased beta/A4 peptide production in CHO cells stably transfected with APP695. A model is presented in which decreases in beta/A4 peptide production can be achieved by accelerating the metabolism of APP through a nonamyloidgenic secretory pathway.

Amyloid deposition is a neuropathological hallmark of Alzheimer's disease. The principal component of amyloid deposits is beta amyloid peptide (Abeta), a peptide derived by proteolytic processing of the amyloid precursor protein (APP). APP is axonally transported by the fast anterograde component. Several studies have indicated that Abeta deposits occur in proximity to neuritic and synaptic profiles. Taken together, these latter observations have suggested that APP, axonally transported to nerve terminals, may be processed to Abeta at those sites. To examine the fate of APP in the CNS, we injected [35S]methionine into the rat entorhinal cortex and examined the trafficking and processing of de novo synthesized APP in the perforant pathway and at presynaptic sites in the hippocampal formation. We report that both full-length and processed APP accumulate at presynaptic terminals of entorhinal neurons. Finally, we demonstrate that at these synaptic sites, C-terminal fragments of APP containing the entire Abeta domain accumulate, suggesting that these species may represent the penultimate precursors of synaptic Abeta.

Mutations in the PTEN gene are associated with a broad spectrum of disorders, including Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, and Lhermitte-Duclos disease. In addition, PTEN mutations have been described in a few patients with autism spectrum disorders (ASDs) and macrocephaly. In this study, we screened the PTEN gene for mutations and deletions in 88 patients with ASDs and macrocephaly (defined as >or=2 SD above the mean). Mutation analysis was performed by direct sequencing of all exons and flanking regions, as well as the promoter region. Dosage analysis of PTEN was carried out using multiplex ligation-dependent probe amplification (MLPA). No partial or whole gene deletions were observed. We identified a de novo missense mutation (D326N) in a highly conserved amino acid in a 5-year-old boy with autism, mental retardation, language delay, extreme macrocephaly (+9.6 SD) and polydactyly of both feet. Polydactyly has previously been described in two patients with Lhermitte-Duclos disease and CS and is thus likely to be a rare sign of PTEN mutations. Our findings suggest that PTEN mutations are a relatively infrequent cause of ASDs with macrocephaly. Screening of PTEN mutations is warranted in patients with autism and pronounced macrocephaly, even in the absence of other features of PTEN-related tumor syndromes.

FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with β1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound–healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.

The principal component of Alzheimer's amyloid plaques, Aβ, derives from proteolytic processing of the Alzheimer's amyloid protein precursor (APP). FE65 is a brain-enriched protein that binds to APP. Although several laboratories have characterized the APP-FE65 interaction <i>in vitro</i>, the possible relevance of this interaction to Alzheimer's disease has remained unclear. We demonstrate here that APP and FE65 co-localize in the endoplasmic reticulum/Golgi and possibly in endosomes. Moreover, FE65 increases translocation of APP to the cell surface, as well as both αAPP_s and Aβ secretion. The dramatic (4-fold) FE65-dependent increase in Aβ secretion suggests that agents which inhibit the interaction of FE65 with APP might reduce Aβ secretion in the brain and therefore be useful for preventing or slowing amyloid plaque formation.

Schizophrenia is a psychiatric disorder with onset in late adolescence and unclear etiology characterized by both positive and negative symptoms, as well as cognitive deficits. To identify copy number variations (CNVs) that increase the risk of schizophrenia, we performed a whole-genome CNV analysis on a cohort of 977 schizophrenia cases and 2,000 healthy adults of European ancestry who were genotyped with 1.7 million probes. Positive findings were evaluated in an independent cohort of 758 schizophrenia cases and 1,485 controls. The Gene Ontology synaptic transmission family of genes was notably enriched for CNVs in the cases (P = 1.5 x 10(-7)). Among these, CACNA1B and DOC2A, both calcium-signaling genes responsible for neuronal excitation, were deleted in 16 cases and duplicated in 10 cases, respectively. In addition, RET and RIT2, both ras-related genes important for neural crest development, were significantly affected by CNVs. RET deletion was exclusive to seven cases, and RIT2 deletions were overrepresented common variant CNVs in the schizophrenia cases. Our results suggest that novel variations involving the processes of synaptic transmission contribute to the genetic susceptibility of schizophrenia.

Human brain ageing is associated with reductions in a variety of nicotinic receptors subtypes, whereas changes in age-related disorders including Alzheimer's disease or Parkinson's disease are more selective. In Alzheimer's disease, in the cortex there is a selective loss of the alpha4 (but not alpha3 or 7) subunit immunoreactivity and of nicotine or epibatidine binding but not alpha-bungarotoxin binding. Epibatidine binding is inversely correlated with clinical dementia ratings and with the level of Abeta1-42, but not related to plaque or tangle densities. In contrast, alpha-bungarotoxin binding is positively correlated with plaque densities in the entorhinal cortex. In human temporal cortex loss of acetylcholinesterase catalytic activity is positively correlated with decreased epibatidine binding and in a transgenic mouse model over expressing acetylcholinesterase, epibatidine binding is elevated. In Parkinson's disease, loss of striatal nicotine binding appears to occur early but is not associated with a loss of alpha4 subunit immunoreactivity. Tobacco use in normal elderly individuals is associated with increased alpha4 immunoreactivity in the cortex and lower densities of amyloid-beta plaques, and with greater numbers of dopaminergic neurons in the substantia nigra pars compacta. These findings indicate an early involvement of the alpha4 subunit in beta-amyloidosis but not in nigro-striatal dopaminergic degeneration.

Autism/autistic disorder (MIM number 209850) is a complex, largely genetic psychiatric disorder. The authors recently mapped a susceptibility locus for autism to chromosome region 2q24-q33 (MIM number 606053). In the present study, genes across the 2q24-q33 interval were analyzed to identify an autism susceptibility gene in this region.Mutation screening of positional candidate genes was performed in two stages. The first stage involved identifying, in unrelated subjects showing linkage to 2q24-q33, genetic variants in exons and flanking sequence within candidate genes and comparing the frequency of the variants between autistic and unrelated nonautistic subjects. Two single nucleotide polymorphisms (SNPs) that showed evidence for divergent distribution between autistic and nonautistic subjects were identified, both within SLC25A12, a gene encoding the mitochondrial aspartate/glutamate carrier (AGC1). In the second stage, the two SNPs in SLC25A12 were further genotyped in 411 autistic families, and linkage and association tests were carried out in the 197 informative families.Linkage and association were observed between autistic disorder and the two SNPs, rs2056202 and rs2292813, found in SLC25A12. Using either a single affected subject per family or all affected subjects, evidence for excess transmission was found by the Transmission Disequilibrium Test for rs2056202, rs2292813, and a two-locus G*G haplotype. Similar results were observed using TRANSMIT for the analyses. Evidence for linkage was supported by linkage analysis with the two SNPs, with a maximal multipoint nonparametric linkage score of 1.57 and a maximal multipoint heterogeneity lod score of 2.11. Genotype relative risk could be estimated to be between 2.4 and 4.8 for persons homozygous at these loci.A strong association of autism with SNPs within the SLC25A12 gene was demonstrated. Further studies are needed to confirm this association and to decipher any potential etiological role of AGC1 in autism.

The metabolic fate of the Alzheimer beta/A4 amyloid precursor protein (APP) includes intraamyloid proteolysis that leads to the production of secreted N-terminal and cell-associated C-terminal fragments. The cellular sites at which this processing occurs are not known. We have examined the route of APP processing in metabolically labeled PC12 cells. The lysosomotropic drug chloroquine exerted inhibitory effects on the degradation of mature APP holoprotein. In addition, recovery of a C-terminal fragment resulting from normal intraamyloid cleavage was significantly increased in the presence of chloroquine, suggesting that further degradation of the C-terminal fragment was inhibited. Chloroquine had virtually no effect on APP maturation (N- and O-glycosylation and tyrosine sulfation) or secretion. Treatment with either monensin (which inhibits distal Golgi function) or brefeldin A (which causes resorption of the Golgi into the endoplasmic reticulum and fusion of the trans-Golgi network with the endosomal system) prevented normal APP maturation and abolished APP secretion and recovery of C-terminal fragments, indicating that intact Golgi function is necessary for APP maturation and processing. Our results suggest that a substantial proportion of APP is degraded in an intracellular acidic compartment but that the coupled APP cleavage/secretion event occurs in a chloroquine-insensitive compartment. The observations are consistent with the existence of multiple cellular routes for the trafficking and proteolysis of APP.

Various first messengers linked to phospholipase C, including acetylcholine and interleukin 1, regulate the production both of the secreted form of the amyloid protein precursor (APP) and of amyloid beta-protein. We have now identified intracellular signals which are responsible for mediating these effects. We show that activation of phospholipase C may affect APP processing by either of two pathways, one involving an increase in protein kinase C and the other an increase in cytoplasmic calcium levels. The effects of calcium on APP processing appear to be independent of protein kinase C activation. The observed effects of calcium on APP processing may be of therapeutic utility.

Research during the past decade has seen significant progress in the understanding of the genetic architecture of autism spectrum disorders (ASDs), with gene discovery accelerating as the characterization of genomic variation has become increasingly comprehensive. At the same time, this research has highlighted ongoing challenges. Here we address the enormous impact of high-throughput sequencing (HTS) on ASD gene discovery, outline a consensus view for leveraging this technology, and describe a large multisite collaboration developed to accomplish these goals. Similar approaches could prove effective for severe neurodevelopmental disorders more broadly. Research during the past decade has seen significant progress in the understanding of the genetic architecture of autism spectrum disorders (ASDs), with gene discovery accelerating as the characterization of genomic variation has become increasingly comprehensive. At the same time, this research has highlighted ongoing challenges. Here we address the enormous impact of high-throughput sequencing (HTS) on ASD gene discovery, outline a consensus view for leveraging this technology, and describe a large multisite collaboration developed to accomplish these goals. Similar approaches could prove effective for severe neurodevelopmental disorders more broadly.

Increasingly, mutations in genes causing Mendelian disease will be supported by individual and small families only; however, exome sequencing studies have thus far focused on syndromic phenotypes characterized by low locus heterogeneity. In contrast, retinitis pigmentosa (RP) is caused by >50 known genes, which still explain only half of the clinical cases. In a single, one-generation, nonsyndromic RP family, we have identified a gene, dehydrodolichol diphosphate synthase (DHDDS), demonstrating the power of combining whole-exome sequencing with rapid in vivo studies. DHDDS is a highly conserved essential enzyme for dolichol synthesis, permitting global N-linked glycosylation. Zebrafish studies showed virtually identical photoreceptor defects as observed with N-linked glycosylation-interfering mutations in the light-sensing protein rhodopsin. The identified Lys42Glu variant likely arose from an ancestral founder, because eight of the nine identified alleles in 27,174 control chromosomes were of confirmed Ashkenazi Jewish ethnicity. These findings demonstrate the power of exome sequencing linked to functional studies when faced with challenging study designs and, importantly, link RP to the pathways of N-linked glycosylation, which promise new avenues for therapeutic interventions.

Autism spectrum disorders (ASD) are etiologically heterogeneous, with hundreds of rare, highly penetrant mutations and genomic imbalances involved, each contributing to a very small fraction of cases. In this issue of Molecular Autism, Soorya and colleagues evaluated 32 patients with Phelan-McDermid syndrome, caused by either deletion of 22q13.33 or SHANK3 mutations, using gold-standard diagnostic assessments and showed that 84% met criteria for ASD, including 75% meeting criteria for autism. This study and prior studies demonstrate that this syndrome appears to be one of the more penetrant causes of ASD. In this companion review, we show that in samples ascertained for ASD, SHANK3 haploinsufficiency is one of the more prevalent monogenic causes of ASD, explaining at least 0.5% of cases. We note that SHANK3 haploinsufficiency remains underdiagnosed in ASD and developmental delay, although with the increasingly widespread use of chromosomal microarray analysis and targeted sequencing of SHANK3, the number of cases is bound to rise.

Autism spectrum disorders (ASDs) comprise a constellation of highly heritable neuropsychiatric disorders. Genome-wide studies of autistic individuals have implicated numerous minor risk alleles but few common variants, suggesting a complex genetic model with many contributing loci. To assess commonality of biological function among rare risk alleles, we compared functional knowledge of genes overlapping inherited structural variants in idiopathic ASD subjects relative to healthy controls. In this study we show that biological processes associated with synapse function and neurotransmission are significantly enriched, with replication, in ASD subjects versus controls. Analysis of phenotypes observed for mouse models of copy-variant genes established significant and replicated enrichment of observable phenotypes consistent with ASD behaviors. Most functional terms retained significance after excluding previously reported ASD loci. These results implicate several new variants that involve synaptic function and glutamatergic signaling processes as important contributors of ASD pathophysiology and suggest a sizable pool of additional potential ASD risk loci.

Haploinsufficiency of SHANK3, due to either hemizygous gene deletion (termed 22q13 deletion syndrome or Phelan-McDermid syndrome) or to gene mutation, accounts for about 0.5% of the cases of autism spectrum disorder (ASD) and/or developmental delay, and there is evidence for a wider role for SHANK3 and glutamate signaling abnormalities in ASD and related conditions. Therapeutic approaches that reverse deficits in SHANK3-haploinsufficiency may therefore be broadly beneficial in ASD and in developmental delay.We observed that daily intraperitoneal injections of human insulin-like growth factor 1 (IGF-1) over a 2-week period reversed deficits in hippocampal α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) signaling, long-term potentiation (LTP), and motor performance that we had previously reported in Shank3-deficient mice. Positive effects were observed with an IGF-1 peptide derivative as well.We observed significant beneficial effects of IGF-1 in a mouse model of ASD and of developmental delay. Studies in mouse and human neuronal models of Rett syndrome also show benefits with IGF-1, raising the possibility that this compound may have benefits broadly in ASD and related conditions, even with differing molecular etiology. Given the extensive safety data for IGF-1 in children with short stature due to primary IGF-1 deficiency, IGF-1 is an attractive candidate for controlled clinical trials in SHANK3-deficiency and in ASD.

Copy number variation (CNV) is an important determinant of human diversity and plays important roles in susceptibility to disease. Most studies of CNV carried out to date have made use of chromosome microarray and have had a lower size limit for detection of about 30 kilobases (kb). With the emergence of whole-exome sequencing studies, we asked whether such data could be used to reliably call rare exonic CNV in the size range of 1-30 kilobases (kb), making use of the eXome Hidden Markov Model (XHMM) program. By using both transmission information and validation by molecular methods, we confirmed that small CNV encompassing as few as three exons can be reliably called from whole-exome data. We applied this approach to an autism case-control sample (n = 811, mean per-target read depth = 161) and observed a significant increase in the burden of rare (MAF ≤1%) 1-30 kb CNV, 1-30 kb deletions, and 1-10 kb deletions in ASD. CNV in the 1-30 kb range frequently hit just a single gene, and we were therefore able to carry out enrichment and pathway analyses, where we observed enrichment for disruption of genes in cytoskeletal and autophagy pathways in ASD. In summary, our results showed that XHMM provided an effective means to assess small exonic CNV from whole-exome data, indicated that rare 1-30 kb exonic deletions could contribute to risk in up to 7% of individuals with ASD, and implicated a candidate pathway in developmental delay syndromes.

Rapid advances in sequencing technologies set the stage for the large-scale medical sequencing efforts to be performed in the near future, with the goal of assessing the importance of rare variants in complex diseases. The discovery of new disease susceptibility genes requires powerful statistical methods for rare variant analysis. The low frequency and the expected large number of such variants pose great difficulties for the analysis of these data. We propose here a robust and powerful testing strategy to study the role rare variants may play in affecting susceptibility to complex traits. The strategy is based on assessing whether rare variants in a genetic region collectively occur at significantly higher frequencies in cases compared with controls (or vice versa). A main feature of the proposed methodology is that, although it is an overall test assessing a possibly large number of rare variants simultaneously, the disease variants can be both protective and risk variants, with moderate decreases in statistical power when both types of variants are present. Using simulations, we show that this approach can be powerful under complex and general disease models, as well as in larger genetic regions where the proportion of disease susceptibility variants may be small. Comparisons with previously published tests on simulated data show that the proposed approach can have better power than the existing methods. An application to a recently published study on Type-1 Diabetes finds rare variants in gene IFIH1 to be protective against Type-1 Diabetes.

We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD.

Mutations in the synaptic gene SHANK3 lead to a neurodevelopmental disorder known as Phelan-McDermid syndrome (PMS). PMS is a relatively common monogenic and highly penetrant cause of autism spectrum disorder (ASD) and intellectual disability (ID), and frequently presents with attention deficits. The underlying neurobiology of PMS is not fully known and pharmacological treatments for core symptoms do not exist. Here, we report the production and characterization of a Shank3-deficient rat model of PMS, with a genetic alteration similar to a human SHANK3 mutation. We show that Shank3-deficient rats exhibit impaired long-term social recognition memory and attention, and reduced synaptic plasticity in the hippocampal-medial prefrontal cortex pathway. These deficits were attenuated with oxytocin treatment. The effect of oxytocin on reversing non-social attention deficits is a particularly novel finding, and the results implicate an oxytocinergic contribution in this genetically defined subtype of ASD and ID, suggesting an individualized therapeutic approach for PMS.

Autism spectrum disorders are a group of highly heritable neurodevelopmental disorders with a complex genetic etiology. The International Molecular Genetic Study of Autism Consortium previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we performed a high-density association analysis in AUTS1 and AUTS5, testing more than 3000 single nucleotide polymorphisms (SNPs) in all known genes in each region, as well as SNPs in non-genic highly conserved sequences. SNP genotype data were also used to investigate copy number variation within these regions. The study sample consisted of 127 and 126 families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Further investigation of the strongest association results was conducted in an independent European family sample containing 390 affected individuals. Association and copy number variant analysis highlighted several genes that warrant further investigation, including IMMP2L and DOCK4 on chromosome 7. Evidence for the involvement of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family.