Large-Scale Exome Sequencing Study Implicates Both Developmental and Functional Changes in the Neurobiology of Autism.

We present the largest exome sequencing study of autism spectrum disorder (ASD) to date (n = 35,584 total samples, 11,986 with ASD). Using an enhanced analytical framework to integrate de novo and case-control rare variation, we identify 102 risk genes at a false discovery rate of 0.1 or less. Of these genes, 49 show higher frequencies of disruptive de novo variants in individuals ascertained to have severe neurodevelopmental delay, whereas 53 show higher frequencies in individuals ascertained to have ASD; comparing ASD cases with mutations in these groups reveals phenotypic differences. Expressed early in brain development, most risk genes have roles in regulation of gene expression or neuronal communication (i.e., mutations effect neurodevelopmental and neurophysiological changes), and 13 fall within loci recurrently hit by copy number variants. In cells from the human cortex, expression of risk genes is enriched in excitatory and inhibitory neuronal lineages, consistent with multiple paths to an excitatory-inhibitory imbalance underlying ASD.

Cohort profile: Epidemiology and Genetics of Obsessive-compulsive disorder and chronic tic disorders in Sweden (EGOS).

PURPOSE: The EGOS study (Epidemiology and Genetics of Obsessive-compulsive disorder and chronic tic disorders in Sweden) is a large-scale, epidemiological, prospective cohort that is used to identify genetic and environmental risk factors in the etiology of obsessive-compulsive disorder (OCD) and chronic tic disorders (CTD). METHODS: Individuals born between January 1954 and December 1998 with at least two diagnoses of OCD or CTD at different timepoints in the National Patient Register (NPR), and followed between January 1997 and December 2012, represent the EGOS source population (n = 20,374). The Swedish Multi-Generation Registry (MGR) are then used to define family relatedness for all cases and additional phenotypic and demographic data added to the resultant database. To create an epidemiologically valid subset of the source cohort that also includes biospecimens and additional phenotyping, we contact cases from within the source population. To date, 6832 invitations have been sent out and 1853 (27%) have elected to participate in the EGOS biospecimen collection. RESULTS: To date, 1608 biological samples have been collected, of which 1249 are genotyped and 832 supplementary Obsessive-Compulsive Inventory-Revised (OCI-R) and/or Florida Obsessive-Compulsive Inventory (FOCI) have been completed by individuals with OCD and/or CTD, age 16-64 years. DNA samples are genotyped using Infinium Global Screening Array and will undergo whole-exome sequencing in the future. Detailed information is available for each individual through linkage to the Swedish national registers, e.g., identification of additional psychiatric diagnoses, medical diagnoses, birth-related variables, and relevant demographic and social data. CONCLUSION: EGOS benefits from a genetically homogeneous sample with epidemiological ascertainment, minimizing the risk of confounding due to population stratification on ascertainment bias. In addition, this study is built upon clinical diagnoses of OCD and CTD in specialized psychiatric care, which reduces further biases and case misclassification.

Autism genome-wide copy number variation reveals ubiquitin and neuronal genes.

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.

An analysis of candidate autism loci on chromosome 2q24-q33: evidence for association to the STK39 gene.

A susceptibility locus for autism was identified to the chromosome 2q24-q33 region in independent cohorts of families, especially in subsets clinically defined with phrase speech delay (PSD). In the present work, we screened 84 linkage-informative SNPs covering this locus in a cohort of 334 families with autism and in subsets identified with PSD. We observed linkage to autism with the highest non-parametric linkage score (NPL) of 2.79 (P = 0.002) in the PSD subset with at least two affected subjects. In addition, using a set of 109 additional gene-oriented SNPs in this interval we observed that several SNPs encompassing the SLC25A12 gene provided the maximum evidence for linkage (NPL = 3.32, P = 0.0003). Using the transmission disequilibrium test to test for associations, we observed significant over-transmissions of rs2056202 (P = 0.006) within the SLC25A12 gene, rs1807984 (P = 0.007) within the STK39 gene, and rs2305586 (P = 0.009) within the ITGA4 gene. We also found evidence for association between autism and two other SNPs (rs1517342, P = 0.012 and rs971257, P = 0.030) or haplotypes (P = 0.003) of the STK39 gene. STK39 encodes a serine/threonine kinase (SPAK/PASK/STE20-SPS1 homolog) abundantly expressed in the brain with roles in cell differentiation, cell transformation and proliferation, and in regulation of ion transporters. In summary, we have observed further evidence for linkage and association between autism and loci within the 2q24-q33 region, including at STK39, a novel candidate gene for autism.

Identification of rare de novo epigenetic variations in congenital disorders.

Certain human traits such as neurodevelopmental disorders (NDs) and congenital anomalies (CAs) are believed to be primarily genetic in origin. However, even after whole-genome sequencing (WGS), a substantial fraction of such disorders remain unexplained. We hypothesize that some cases of ND-CA are caused by aberrant DNA methylation leading to dysregulated genome function. Comparing DNA methylation profiles from 489 individuals with ND-CAs against 1534 controls, we identify epivariations as a frequent occurrence in the human genome. De novo epivariations are significantly enriched in cases, while RNAseq analysis shows that epivariations often have an impact on gene expression comparable to loss-of-function mutations. Additionally, we detect and replicate an enrichment of rare sequence mutations overlapping CTCF binding sites close to epivariations, providing a rationale for interpreting non-coding variation. We propose that epivariations contribute to the pathogenesis of some patients with unexplained ND-CAs, and as such likely have diagnostic relevance.

Lack of evidence for association of the serotonin transporter gene SLC6A4 with autism.

BACKGROUND: The serotonin transporter (5-HTT) has long been considered likely to play a role in autism. Hyperserotonemia has been consistently found in a proportion of autistic patients, and the use of selective serotonin reuptake inhibitors (SSRIs) can have a positive effect in treating some symptoms of autism. Specific variants of the 5-HTT gene, SLC6A4, especially the insertion-deletion 5-HTTLPR promoter locus, have been found to modulate its expression and transporter function. METHODS: We examined the transmission of the short or long allele of 5-HTTLPR locus to affected individuals, using a large cohort of 352 families. In addition, we screened five single nucleotide polymorphisms (SNPs) in the 5' region of SLC6A4 previously reported to be positively associated with autism, as well as 4 additional SNPs also in the 5' region. RESULTS: No association of the 5-HTTLPR locus with autism was found. Furthermore, no evidence for association of any of the nine SNPs covering the SLC6A4 gene, or any of their haplotypes, was observed in our study. Using obsessive-compulsive behaviors (OCB), severe OCBs or rigid-compulsive subsets of our cohort gave the same negative results. CONCLUSIONS: SLC6A4 variants do not appear to be significantly involved in the liability to autism.

Autism and ultraconserved non-coding sequence on chromosome 7q.

OBJECTIVE: Autism has been linked to a broad region on chromosome 7q that contains a large number of genes involved in transcription and development. This region is also enriched for ultraconserved non-coding elements, defined as human-rodent sequences that are 100% aligned over > or =200 base pairs, which have a high likelihood of being functional. Therefore, as only a few rare coding variants have been detected in the autism candidate genes on 7q examined to date, we decided to screen these ultraconserved elements for possible autism susceptibility alleles. METHODS: We used denaturing high-performance liquid chromatography, and DNA sequencing, to perform variant detection in a total of 146 cases with autism, 96 from the Autism Genetic Resource Exchange and 50 from the Central Valley of Costa Rica, as well as 124 controls from the Polymorphism Discovery Resource Panel. We screened 10 consecutive ultraconserved elements in, or flanking, the genes DLX5/6, AUTS2 and FOXP2 on chromosome 7q. RESULTS: Although we did find several rare variants in autism cases that were not present in controls, we also observed rare variants present in controls and not cases. The most common variant occurred in controls at a frequency of 3.3%. Interestingly, two ultraconserved elements each harbored three independent variants and one ultraconserved element harbored two independent variants, suggesting that ultraconservation is maintained chiefly by a decreased tendency toward fixation, rather than a significantly lower mutation rate. CONCLUSIONS: Our results show that these sequences are unlikely to harbor major autism susceptibility alleles.

Association analysis of the NrCAM gene in autism and in subsets of families with severe obsessive-compulsive or self-stimulatory behaviors.

OBJECTIVES: An autism susceptibility locus (AUTS1, MIM#608636) has been identified in chromosome 7q31. NrCAM is a candidate gene for AUTS1 because it is expressed in the brain and encodes a receptor involved in nervous system development. Polymorphisms in NrCAM have been reported to be associated with autism susceptibility and with substance abuse, implicating NrCAM in reward circuitry. Self-stimulatory, perseverative behavior in autism might be due to defects in reward circuitry. In addition, models of drug addiction have also borrowed from models of obsessive-compulsive behavior designed to reduce anxiety. Thus, our goals were to replicate previous associations of NrCAM with autism, making use of a large cohort, and to clarify whether NrCAM was associated with a specific endophenotype of autism in the repetitive behaviors and stereotyped interests domains. METHODS: We genotyped six NrCAM single nucleotide polymorphisms in 352 families and we tested for association between these polymorphisms and autism in the entire cohort and in two subsets, one with severe obsessive-compulsive behaviors and one with pronounced self-stimulatory behaviors. RESULTS: We found no association between single nucleotide polymorphisms of NrCAM and autism in our large cohort, or in the severe obsessive-compulsive behavior and self-stimulatory behavior subsets. However, we observed a significant overtransmission (21 transmitted vs 6 nontransmitted, chi2=12.054, P=0.0005) of the haplotype G-G-A-G-C-A of rs722519-rs1269622-rs405945-rs6958498-rs401433-rs439587 in the severe obsessive-compulsive behavior subset, likely driven by the G-C haplotype of rs6958498-rs401433, which itself showed significant overtransmission (31 transmitted vs 13 nontransmitted, chi2=8.844, P=0.003). CONCLUSIONS: Overtransmission of particular haplotypes of NrCAM, that may relate to the expression level of NrCAM in the brain, appeared to be associated with autism in the severe obsessive-compulsive behavior subset.

Neural plasticity and the brain renin-angiotensin system.

The brain renin-angiotensin system mediates several classic physiologies including body water balance, maintenance of blood pressure, cyclicity of reproductive hormones and sexual behaviors, and regulation of pituitary gland hormones. In addition, angiotensin peptides have been implicated in neural plasticity and memory. The present review initially describes the extracellular matrix (ECM) and the roles of cell adhesion molecules (CAMs), matrix metalloproteinases, and tissue inhibitors of metalloproteinases in the maintenance and degradation of the ECM. It is the ECM that appears to permit synaptic remodeling and thus is critical to the plasticity that is presumed to underlie mechanisms of memory consolidation and retrieval. The interrelationship among long-term potentiation (LTP), CAMs, and synaptic strengthening is described, followed by the influence of angiotensins on LTP. There is strong support for an inhibitory influence by angiotensin II (AngII) and a facilitory role by angiotensin IV (AngIV), on LTP. Next, the influences of AngII and IV on associative and spatial memories are summarized. Finally, the impact of sleep deprivation on matrix metalloproteinases and memory function is described. Recent findings indicate that sleep deprivation-induced memory impairment is accompanied by a lack of appropriate changes in matrix metalloproteinases within the hippocampus and neocortex as compared with non-sleep deprived animals. These findings generally support an important contribution by angiotensin peptides to neural plasticity and memory consolidation.

Linkage and association of the mitochondrial aspartate/glutamate carrier SLC25A12 gene with autism.

OBJECTIVE: 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. METHOD: 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. RESULTS: 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. CONCLUSIONS: 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.

Ethanol-induced impairment of spatial memory and brain matrix metalloproteinases.

The formation of spatial memory appears to be dependent upon an intact hippocampus capable of the specific biochemical changes associated with synaptic remodeling. Hippocampal damage results in the disruption of synaptic remodeling and the acquisition of spatial memory tasks. Ethanol also disrupts normal hippocampal functioning and spatial memory. The present investigation established a dose-response relationship between ethanol treatment and impairment of spatial memory as measured using the circular water maze task. Intraperitoneal ethanol doses of 1.5 and 2 g/kg significantly increased the latency and distance swam to find the submerged pedestal as compared with a 1 g/kg dose, and 0.15 M NaCl vehicle control treatments. On days 2, 4, and 6 of acquisition animals were sacrificed and brain tissues were retained from the hippocampus, prefrontal neocortex, and cerebellum for measurement of matrix metalloproteinases (MMPs). The results indicated that ethanol treatment interfered with MMP-9, but not MMP-2, activity in the hippocampus, and to a lesser degree in the prefrontal cortex. No changes in the cerebellum were measured. Elevations in MMP activity appear to be a prerequisite to reconfiguration of extracellular matrix cell adhesion molecules thought to be important in the process of synaptic plasticity, which in turn appears to be necessary for memory consolidation. Thus, ethanol-induced impairment in the acquisition of spatial memory tasks may, in part, be due to disruption of brain MMP activity.

Most genetic risk for autism resides with common variation.

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.

A large-scale screen for coding variants in SERT/SLC6A4 in autism spectrum disorders.

In the current study we explored the hypothesis that rare variants in SLC6A4 contribute to autism susceptibility and to rigid-compulsive behaviors in autism. We made use of a large number of unrelated cases with autism spectrum disorders (approximately 350) and controls (approximately 420) and screened for rare exonic variants in SLC6A4 by a high-throughput method followed by sequencing. We observed no difference in the frequency of such variants in the two groups, irrespective of how we defined the rare variants. Furthermore, we did not observe an association of rare coding variants in SLC6A4 with rigid-compulsive traits scores in the cases. These results do not support a significant role for rare coding variants in SLC6A4 in autism spectrum disorders, nor do they support a significant role for SLC6A4 in rigid-compulsive traits in these disorders.

Multiplex ligation-dependent probe amplification for genetic screening in autism spectrum disorders: efficient identification of known microduplications and identification of a novel microduplication in ASMT.

BACKGROUND: It has previously been shown that specific microdeletions and microduplications, many of which also associated with cognitive impairment (CI), can present with autism spectrum disorders (ASDs). Multiplex ligation-dependent probe amplification (MLPA) represents an efficient method to screen for such recurrent microdeletions and microduplications. METHODS: In the current study, a total of 279 unrelated subjects ascertained for ASDs were screened for genomic disorders associated with CI using MLPA. Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (Q-PCR) and/or direct DNA sequencing were used to validate potential microdeletions and microduplications. Methylation-sensitive MLPA was used to characterize individuals with duplications in the Prader-Willi/Angelman (PWA) region. RESULTS: MLPA showed two subjects with typical ASD-associated interstitial duplications of the 15q11-q13 PWA region of maternal origin. Two additional subjects showed smaller, de novo duplications of the PWA region that had not been previously characterized. Genes in these two novel duplications include GABRB3 and ATP10A in one case, and MKRN3, MAGEL2 and NDN in the other. In addition, two subjects showed duplications of the 22q11/DiGeorge syndrome region. One individual was found to carry a 12 kb deletion in one copy of the ASPA gene on 17p13, which when mutated in both alleles leads to Canavan disease. Two subjects showed partial duplication of the TM4SF2 gene on Xp11.4, previously implicated in X-linked non-specific mental retardation, but in our subsequent analyses such variants were also found in controls. A partial duplication in the ASMT gene, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes and previously suggested to be involved in ASD susceptibility, was observed in 6-7% of the cases but in only 2% of controls (P = 0.003). CONCLUSION: MLPA proves to be an efficient method to screen for chromosomal abnormalities. We identified duplications in 15q11-q13 and in 22q11, including new de novo small duplications, as likely contributing to ASD in the current sample by increasing liability and/or exacerbating symptoms. Our data indicate that duplications in TM4SF2 are not associated with the phenotype given their presence in controls. The results in PAR1/PAR2 are the first large-scale studies of gene dosage in these regions, and the findings at the ASMT locus indicate that further studies of the duplication of the ASMT gene are needed in order to gain insight into its potential involvement in ASD. Our studies also identify some limitations of MLPA, where single base changes in probe binding sequences alter results. In summary, our studies indicate that MLPA, with a focus on accepted medical genetic conditions, may be an inexpensive method for detection of microdeletions and microduplications in ASD patients for purposes of genetic counselling if MLPA-identified deletions are validated by additional methods.

Mutation screening of the PTEN gene in patients with autism spectrum disorders and macrocephaly.

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.

Family-based association study of TPH1 and TPH2 polymorphisms in autism.

The TPH1 and TPH2 genes encode the rate-limiting enzymes that control serotonin biosynthesis, and serotonin is clearly altered in autism. In the current study, eight SNPs in the TPH1 gene region and eight SNPs within the TPH2 gene were examined by family-based association tests in a large cohort of 352 families with autism and in clinically defined subsets of these families with either severe obsessive-compulsive behaviors (sOCB) or self-stimulatory behaviors (SSB). We found no evidence for association between autism and single SNPs or haplotypes of the TPH1 and TPH2 genes in the cohort of all families or in the sOCB and SSB subsets. In particular, we failed to replicate the association between autism and variants of the TPH2 gene, rs4341581 (TRANSMIT P = 1; PDT P = 0.323; FBAT P = 0.446) and rs11179000 (TRANSMIT P = 0.174; PDT P = 0.293; FBAT P = 0.374). Furthermore, no evidence for linkage was observed between autism and SNPs in the TPH1 and TPH2 genes (although linkage at the TPH2 locus was observed in the SSB subset). Thus, it appears unlikely that the TPH1 and TPH2 genes play a significant role in the susceptibility to autism or to autism endophenotypes including sOCB and SSB.

Multiplexed variation scanning for 1,000 amplicons in hundreds of patients using mismatch repair detection (MRD) on tag arrays.

Identification of the genetic basis of common disease may require comprehensive sequence analysis of coding regions and regulatory elements in patients and controls to find genetic effects caused by rare or heterogeneous mutations. In this study, we demonstrate how mismatch repair detection on tag arrays can be applied in a case-control study. Mismatch repair detection allows >1,000 amplicons to be screened for variations in a single laboratory reaction. Variation scanning in 939 amplicons, mostly in coding regions within a linkage peak, was done for 372 patients and 404 controls. In total, >180 Mb of DNA was scanned. Several variants more prevalent in patients than in controls were identified. This study demonstrates an approach to the discovery of susceptibility genes for common disease: large-scale direct sequence comparison between patients and controls. We believe this approach can be scaled up to allow sequence comparison in the whole-genome coding regions among large sets of cases and controls at a reasonable cost in the near future.