Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.

De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.

Frequency and Complexity of De Novo Structural Mutation in Autism.

Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement.

FOXP1-related intellectual disability syndrome: a recognisable entity.

BACKGROUND: Mutations in forkhead box protein P1 (FOXP1) cause intellectual disability (ID) and specific language impairment (SLI), with or without autistic features (MIM: 613670). Despite multiple case reports no specific phenotype emerged so far. METHODS: We correlate clinical and molecular data of 25 novel and 23 previously reported patients with FOXP1 defects. We evaluated FOXP1 activity by an in vitro luciferase model and assessed protein stability in vitro by western blotting. RESULTS: Patients show ID, SLI, neuromotor delay (NMD) and recurrent facial features including a high broad forehead, bent downslanting palpebral fissures, ptosis and/or blepharophimosis and a bulbous nasal tip. Behavioural problems and autistic features are common. Brain, cardiac and urogenital malformations can be associated. More severe ID and NMD, sensorineural hearing loss and feeding difficulties are more common in patients with interstitial 3p deletions (14 patients) versus patients with monogenic FOXP1 defects (34 patients). Mutations result in impaired transcriptional repression and/or reduced protein stability. CONCLUSIONS: FOXP1-related ID syndrome is a recognisable entity with a wide clinical spectrum and frequent systemic involvement. Our data will be helpful to evaluate genotype-phenotype correlations when interpreting next-generation sequencing data obtained in patients with ID and/or SLI and will guide clinical management.

Functional impact of global rare copy number variation in autism spectrum disorders.

The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviours. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability. Although ASDs are known to be highly heritable ( approximately 90%), the underlying genetic determinants are still largely unknown. Here we analysed the genome-wide characteristics of rare (<1% frequency) copy number variation in ASD using dense genotyping arrays. When comparing 996 ASD individuals of European ancestry to 1,287 matched controls, cases were found to carry a higher global burden of rare, genic copy number variants (CNVs) (1.19 fold, P = 0.012), especially so for loci previously implicated in either ASD and/or intellectual disability (1.69 fold, P = 3.4 x 10(-4)). Among the CNVs there were numerous de novo and inherited events, sometimes in combination in a given family, implicating many novel ASD genes such as SHANK2, SYNGAP1, DLGAP2 and the X-linked DDX53-PTCHD1 locus. We also discovered an enrichment of CNVs disrupting functional gene sets involved in cellular proliferation, projection and motility, and GTPase/Ras signalling. Our results reveal many new genetic and functional targets in ASD that may lead to final connected pathways.

How interview questions are placed in time influences caregiver description of social communication symptoms on the ADI-R.

BACKGROUND: Caregiver report is crucial for the diagnosis of childhood onset psychiatric disorders, particularly autism. Three experiments were conducted to determine whether caregiver reports of past and current behaviors are affected by question timing and ordering. METHODS: Using the Autism Diagnostic Interview - Revised (ADI-R), two studies systematically varied the order in which caregivers were asked about behaviors. In a third study, descriptions of children's current behaviors at age 5 were compared to retrospective descriptions of behaviors at age 5 collected at age 10. RESULTS: Caregivers, who were first asked about a history of symptoms, described less severe past and present behavior than caregivers reporting current behaviors as well as caregivers reporting current and history of symptoms together. Caregivers retrospectively reported more severe behaviors for age 5 when their children were age 10 than they had when their children were age 5. CONCLUSIONS: Caregivers describe past behaviors differently depending on whether they are asked about current symptoms first. Methods of caregiver reporting can influence interpretations of symptom severity with effects on diagnoses and research findings.

Agenesis of the corpus callosum and autism: a comprehensive comparison.

The corpus callosum, with its ∼200 million axons, remains enigmatic in its contribution to cognition and behaviour. Agenesis of the corpus callosum is a congenital condition in which the corpus callosum fails to develop; such individuals exhibit localized deficits in non-literal language comprehension, humour, theory of mind and social reasoning. These findings together with parent reports suggest that behavioural and cognitive impairments in subjects with callosal agenesis may overlap with the profile of autism spectrum disorders, particularly with respect to impairments in social interaction and communication. To provide a comprehensive test of this hypothesis, we directly compared a group of 26 adults with callosal agenesis to a group of 28 adults with a diagnosis of autism spectrum disorder but no neurological abnormality. All participants had full-scale intelligence quotient scores >78 and groups were matched on age, handedness, and gender ratio. Using the Autism Diagnostic Observation Schedule together with current clinical presentation to assess autistic symptomatology, we found that 8/26 (about a third) of agenesis subjects presented with autism. However, more formal diagnosis additionally involving recollective parent-report measures regarding childhood behaviour showed that only 3/22 met complete formal criteria for an autism spectrum disorder (parent reports were unavailable for four subjects). We found no relationship between intelligence quotient and autism symptomatology in callosal agenesis, nor evidence that the presence of any residual corpus callosum differentiated those who exhibited current autism spectrum symptoms from those who did not. Relative to the autism spectrum comparison group, parent ratings of childhood behaviour indicated children with agenesis were less likely to meet diagnostic criteria for autism, even for those who met autism spectrum criteria as adults, and even though there was no group difference in parent report of current behaviours. The findings suggest two broad conclusions. First, they support the hypothesis that congenital disruption of the corpus callosum constitutes a major risk factor for developing autism. Second, they quantify specific features that distinguish autistic behaviour associated with callosal agenesis from autism more generally. Taken together, these two findings also leverage specific questions for future investigation: what are the distal causes (genetic and environmental) determining both callosal agenesis and its autistic features, and what are the proximal mechanisms by which absence of the callosum might generate autistic symptomatology?

Individual common variants exert weak effects on the risk for autism spectrum disorders.

While it is apparent that rare variation can play an important role in the genetic architecture of autism spectrum disorders (ASDs), the contribution of common variation to the risk of developing ASD is less clear. To produce a more comprehensive picture, we report Stage 2 of the Autism Genome Project genome-wide association study, adding 1301 ASD families and bringing the total to 2705 families analysed (Stages 1 and 2). In addition to evaluating the association of individual single nucleotide polymorphisms (SNPs), we also sought evidence that common variants, en masse, might affect the risk. Despite genotyping over a million SNPs covering the genome, no single SNP shows significant association with ASD or selected phenotypes at a genome-wide level. The SNP that achieves the smallest P-value from secondary analyses is rs1718101. It falls in CNTNAP2, a gene previously implicated in susceptibility for ASD. This SNP also shows modest association with age of word/phrase acquisition in ASD subjects, of interest because features of language development are also associated with other variation in CNTNAP2. In contrast, allele scores derived from the transmission of common alleles to Stage 1 cases significantly predict case status in the independent Stage 2 sample. Despite being significant, the variance explained by these allele scores was small (Vm< 1%). Based on results from individual SNPs and their en masse effect on risk, as inferred from the allele score results, it is reasonable to conclude that common variants affect the risk for ASD but their individual effects are modest.

Diagnosis of autism spectrum disorders in 2-year-olds: a study of community practice.

BACKGROUND: Longitudinal research studies have demonstrated that experienced clinicians using standardized assessment measures can make a reliable diagnosis of autism spectrum disorders (ASDs) in children under age 3. Limited data are available regarding the sensitivity and specificity of these measures in community settings. The aims of this study were to determine how well a standardized diagnostic observational measure (Autism Diagnostic Observation Schedule - ADOS) functions alone, and with a brief parent measure within a community setting when administered by community clinicians. METHODS: Clinical records for 138 children between the ages of 24 and 36 months of age who were evaluated for possible ASD or social/language concerns at a hospital-based developmental evaluation clinic were examined. Evaluations were conducted by community-based clinical psychologists. Classification results obtained from standardized diagnostic measures were compared with case reviewer diagnosis, by reviewers blind to scores on diagnostic measures, using The Records-based Methodology for ASD Case Definition that was developed by the Metropolitan Atlanta Developmental Disabilities Surveillance Program. RESULTS: When compared with case review diagnosis, the ADOS demonstrated strong sensitivity and specificity for both Autism versus Not Autism and ASD versus Nonspectrum (NS) diagnoses in this young sample. The Social Communication Questionnaire (SCQ), using the lower cutoff of ≥12, had adequate sensitivity when differentiating Autism from Not Autism, but weak sensitivity when differentiating ASD from NS, missing about 80% of the children with pervasive developmental disorder - not otherwise specified. Using either the Modified Checklist for Autism in Toddlers or the SCQ in combination with the ADOS did not result in improved specificity over the ADOS alone and led to a drop in sensitivity when differentiating ASD from NS disorders. CONCLUSIONS: These results demonstrate that following best practice guidelines, the ADOS can be successfully incorporated into clinical practice with relatively good sensitivity and specificity, and worked well with a referred sample of 2-year-olds. A parent questionnaire did not lead to any improvement in diagnostic classification above the ADOS used in isolation.

A genome-wide scan for common alleles affecting risk for autism.

Although autism spectrum disorders (ASDs) have a substantial genetic basis, most of the known genetic risk has been traced to rare variants, principally copy number variants (CNVs). To identify common risk variation, the Autism Genome Project (AGP) Consortium genotyped 1558 rigorously defined ASD families for 1 million single-nucleotide polymorphisms (SNPs) and analyzed these SNP genotypes for association with ASD. In one of four primary association analyses, the association signal for marker rs4141463, located within MACROD2, crossed the genome-wide association significance threshold of P < 5 × 10(-8). When a smaller replication sample was analyzed, the risk allele at rs4141463 was again over-transmitted; yet, consistent with the winner's curse, its effect size in the replication sample was much smaller; and, for the combined samples, the association signal barely fell below the P < 5 × 10(-8) threshold. Exploratory analyses of phenotypic subtypes yielded no significant associations after correction for multiple testing. They did, however, yield strong signals within several genes, KIAA0564, PLD5, POU6F2, ST8SIA2 and TAF1C.

A novel approach of homozygous haplotype sharing identifies candidate genes in autism spectrum disorder.

Autism spectrum disorder (ASD) is a highly heritable disorder of complex and heterogeneous aetiology. It is primarily characterized by altered cognitive ability including impaired language and communication skills and fundamental deficits in social reciprocity. Despite some notable successes in neuropsychiatric genetics, overall, the high heritability of ASD (~90%) remains poorly explained by common genetic risk variants. However, recent studies suggest that rare genomic variation, in particular copy number variation, may account for a significant proportion of the genetic basis of ASD. We present a large scale analysis to identify candidate genes which may contain low-frequency recessive variation contributing to ASD while taking into account the potential contribution of population differences to the genetic heterogeneity of ASD. Our strategy, homozygous haplotype (HH) mapping, aims to detect homozygous segments of identical haplotype structure that are shared at a higher frequency amongst ASD patients compared to parental controls. The analysis was performed on 1,402 Autism Genome Project trios genotyped for 1 million single nucleotide polymorphisms (SNPs). We identified 25 known and 1,218 novel ASD candidate genes in the discovery analysis including CADM2, ABHD14A, CHRFAM7A, GRIK2, GRM3, EPHA3, FGF10, KCND2, PDZK1, IMMP2L and FOXP2. Furthermore, 10 of the previously reported ASD genes and 300 of the novel candidates identified in the discovery analysis were replicated in an independent sample of 1,182 trios. Our results demonstrate that regions of HH are significantly enriched for previously reported ASD candidate genes and the observed association is independent of gene size (odds ratio 2.10). Our findings highlight the applicability of HH mapping in complex disorders such as ASD and offer an alternative approach to the analysis of genome-wide association data.

Setting Families Up for Success: A Pilot Study of a Toolkit to Enhance the Autism Spectrum Disorder Diagnostic Evaluation Process.

Families of children with autism spectrum disorder (ASD) face challenges engaging in services following diagnosis. This study: (1) developed and implemented a toolkit to tailor ASD evaluation feedback to families' needs, and (2) evaluated caregiver and provider perceptions of the toolkit. Focus groups with providers (N = 11) informed toolkit development. Seven providers participated in pilot training and implementation. Provider and caregiver toolkit perceptions were assessed using interviews, surveys, and a fidelity checklist. Toolkit strategies reflect focus group themes. Provider and caregiver ratings suggest the initial feasibility, acceptability, and utility of the toolkit. This toolkit may be feasible to implement in community settings and may increase caregiver satisfaction, though further refinements are needed to support service connection.

A phenotypic spectrum of autism is attributable to the combined effects of rare variants, polygenic risk and sex.

The genetic etiology of autism spectrum disorder (ASD) is multifactorial, but how combinations of genetic factors determine risk is unclear. In a large family sample, we show that genetic loads of rare and polygenic risk are inversely correlated in cases and greater in females than in males, consistent with a liability threshold that differs by sex. De novo mutations (DNMs), rare inherited variants and polygenic scores were associated with various dimensions of symptom severity in children and parents. Parental age effects on risk for ASD in offspring were attributable to a combination of genetic mechanisms, including DNMs that accumulate in the paternal germline and inherited risk that influences behavior in parents. Genes implicated by rare variants were enriched in excitatory and inhibitory neurons compared with genes implicated by common variants. Our results suggest that a phenotypic spectrum of ASD is attributable to a spectrum of genetic factors that impact different neurodevelopmental processes.

Oligogenic Effects of 16p11.2 Copy-Number Variation on Craniofacial Development.

A copy-number variant (CNV) of 16p11.2 encompassing 30 genes is associated with developmental and psychiatric disorders, head size, and body mass. The genetic mechanisms that underlie these associations are not understood. To determine the influence of 16p11.2 genes on development, we investigated the effects of CNV on craniofacial structure in humans and model organisms. We show that deletion and duplication of 16p11.2 have "mirror" effects on specific craniofacial features that are conserved between human and rodent models of the CNV. By testing dosage effects of individual genes on the shape of the mandible in zebrafish, we identify seven genes with significant effects individually and find evidence for others when genes were tested in combination. The craniofacial phenotypes of 16p11.2 CNVs represent a model for studying the effects of genes on development, and our results suggest that the associated facial gestalts are attributable to the combined effects of multiple genes.

Paternally inherited cis-regulatory structural variants are associated with autism.

The genetic basis of autism spectrum disorder (ASD) is known to consist of contributions from de novo mutations in variant-intolerant genes. We hypothesize that rare inherited structural variants in cis-regulatory elements (CRE-SVs) of these genes also contribute to ASD. We investigated this by assessing the evidence for natural selection and transmission distortion of CRE-SVs in whole genomes of 9274 subjects from 2600 families affected by ASD. In a discovery cohort of 829 families, structural variants were depleted within promoters and untranslated regions, and paternally inherited CRE-SVs were preferentially transmitted to affected offspring and not to their unaffected siblings. The association of paternal CRE-SVs was replicated in an independent sample of 1771 families. Our results suggest that rare inherited noncoding variants predispose children to ASD, with differing contributions from each parent.

Combining information from multiple sources in the diagnosis of autism spectrum disorders.

BACKGROUND: Standard case criteria are proposed for combined use of the Autism Diagnostic Interview-Revised and Autism Diagnostic Observation Schedule to diagnose autism and to define the broader category of autism spectrum disorders. METHOD: Single and combined Autism Diagnostic Interview-Revised and Autism Diagnostic Observation Schedule algorithms were compared to best estimate diagnoses in four samples: U.S. (n = 960) and Canadian (n = 232) participants 3 years and older, U.S. participants younger than 36 months (n = 270), and U.S. participants older than 36 months with profound mental retardation (n = 67). RESULTS: Sensitivities and specificities of 80% and higher were obtained when strict criteria for an autism diagnosis using both instruments were applied in the U.S. samples, and 75% or greater in the Canadian sample. Single-instrument criteria resulted in significant loss of specificity. Specificity was poor in the sample with profound mental retardation. Lower sensitivity and specificity were also obtained when proposed criteria for broader spectrum disorders were applied. CONCLUSIONS: The Autism Diagnostic Interview-Revised and Autism Diagnostic Observation Schedule make independent, additive contributions to the judgment of clinicians that result in a more consistent and rigorous application of diagnostic criteria.

Does bilateral damage to the human amygdala produce autistic symptoms?

A leading neurological hypothesis for autism postulates amygdala dysfunction. This hypothesis has considerable support from anatomical and neuroimaging studies. Individuals with bilateral amygdala lesions show impairments in some aspects of social cognition. These impairments bear intriguing similarity to those reported in people with autism, such as impaired recognition of emotion in faces, impaired theory of mind abilities, failure to fixate eyes in faces, and difficulties in regulating personal space distance to others. Yet such neurological cases have never before been assessed directly to see if they meet criteria for autism spectrum disorders (ASD). Here we undertook such an investigation in two rare participants with developmental-onset bilateral amygdala lesions. We administered a comprehensive clinical examination, as well as the Autism Diagnostic Observation Schedule (ADOS), the Social Responsiveness Scale (SRS), together with several other standardized questionnaires. Results from the two individuals with amygdala lesions were compared with published norms from both healthy populations as well as from people with ASD. Neither participant with amygdala lesions showed any evidence of autism across the array of different measures. The findings demonstrate that amygdala lesions in isolation are not sufficient for producing autistic symptoms. We suggest instead that it may be abnormal connectivity between the amygdala and other structures that contributes to autistic symptoms at a network level.

What Happens Next? Follow-Up From the Children's Toddler School Program.

This study was a follow-up of a group of 29 children diagnosed with autism spectrum disorders at age 2 who attended an inclusive toddler program until age 3. Children ranged in age from 4 to 12 years at the time of the parent survey and follow-up testing. The majority of children were placed in a special education (noninclusive) preschool class, but among the children who were in elementary school at the time of follow-up, 63% were in general education classroom placement. Diagnoses of autism spectrum disorders remained stable, socialization skills remained a weakness, and child-related parental stress remained high despite average cognitive and language skills in the majority of children. Social skill development and support remained a service need.

Between a ROC and a hard place: decision making and making decisions about using the SCQ.

BACKGROUND: The Social Communication Questionnaire (SCQ), formerly the Autism Screening Questionnaire (ASQ), is based on a well-validated parent interview, the Autism Diagnostic Interview (ADI). It has shown promise as a screening measure for autism spectrum disorders (ASDs) in a research-referred older sample, though recent studies with younger children reported lower sensitivities when using the suggested cutoff of > or = 15 to differentiate ASDs from children with nonspectrum disorders (NS). METHODS: Diagnostic discrimination of the SCQ was evaluated alone and in combination with the ADOS (Autism Diagnostic Observation Schedule) in a clinical and research-referred sample of 590 children and adolescents (2 to 16 years), with best estimate consensus diagnoses of autism, pervasive developmental disorder, not otherwise specified (PDD-NOS) and non-ASD disorders. The SCQ was completed before the evaluation in most cases. Performance of the SCQ was also compared with the Autism Diagnostic Interview - Revised (ADI-R). RESULTS: Absolute scores and sensitivity in the younger children and specificity for all groups were lower than reported in the original study. Using receiver operating curves (ROC) to examine the area under the curve (AUC), the SCQ was more similar to the ADI-R total score in differentiating ASD from NS disorders in the older (8-10, >11) than younger age groups (<5, 5-7). Lowering the cutoff score in the 2 younger groups improved sensitivity, with specificity remaining relatively low in all groups. Using the SCQ in combination with the ADOS resulted in improved specificity. Diagnostic discrimination was best using the ADI-R and ADOS in combination. CONCLUSIONS: Those interested in using the SCQ should consider adjusting cutoff scores according to age and purpose, and using it in combination with another measure. Sensitivity or specificity may be prioritized for research or screening depending on goals.

The Role of the Autism Diagnostic Observation Schedule in the Assessment of Autism Spectrum Disorders in School and Community Settings.

Autism diagnostic practices among school and clinical psychologists, particularly those using the Autism Diagnostic Observation Schedule (ADOS), were examined using national survey results (N = 132). School and clinical psychologists were similar in following the Best Practice Guidelines for screening, diagnosis and assessment, School psychologists were more likely to include a school or home observation and teacher report than clinical psychologists but evaluated significantly fewer children with autism spectrum disorders per year compared to clinical psychologists. School psychologists who were ADOS users were more likely to consider themselves autism experts and include a review of records than ADOS non-users. Perceived advantages of the ADOS included its strength in capturing ASD-specific behaviors and the standardized structure provided for observation, while diagnostic discrimination and required resources were the most commonly identified disadvantages.