Brian J. O'Roak

A general framework for estimating the relative pathogenicity of human genetic variants

Current methods for annotating and interpreting human genetic variation tend to exploit a single information type (for example, conservation) and/or are restricted in scope (for example, to missense changes). Here we describe Combined Annotation–Dependent Depletion (CADD), a method for objectively integrating many diverse annotations into a single measure (C score) for each variant. We implement CADD as a support vector machine trained to differentiate 14.7 million high-frequency human-derived alleles from 14.7 million simulated variants. We precompute C scores for all 8.6 billion possible human single-nucleotide variants and enable scoring of short insertions-deletions. C scores correlate with allelic diversity, annotations of functionality, pathogenicity, disease severity, experimentally measured regulatory effects and complex trait associations, and they highly rank known pathogenic variants within individual genomes. The ability of CADD to prioritize functional, deleterious and pathogenic variants across many functional categories, effect sizes and genetic architectures is unmatched by any current single-annotation method.

Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations

Evidence for the etiology of autism spectrum disorders (ASDs) has consistently pointed to a strong genetic component complicated by substantial locus heterogeneity. We sequenced the exomes of 20 individuals with sporadic ASD (cases) and their parents, reasoning that these families would be enriched for de novo mutations of major effect. We identified 21 de novo mutations, 11 of which were protein altering. Protein-altering mutations were significantly enriched for changes at highly conserved residues. We identified potentially causative de novo events in 4 out of 20 probands, particularly among more severely affected individuals, in FOXP1, GRIN2B, SCN1A and LAMC3. In the FOXP1 mutation carrier, we also observed a rare inherited CNTNAP2 missense variant, and we provide functional support for a multi-hit model for disease risk. Our results show that trio-based exome sequencing is a powerful approach for identifying new candidate genes for ASDs and suggest that de novo mutations may contribute substantially to the genetic etiology of ASDs.

Multiple Recurrent De Novo CNVs, Including Duplications of the 7q11.23 Williams Syndrome Region, Are Strongly Associated with Autism

We have undertaken a genome-wide analysis of rare copy-number variation (CNV) in 1124 autism spectrum disorder (ASD) families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, characterized by a highly social personality. We identify rare recurrent de novo CNVs at five additional regions, including 16p13.2 (encompassing genes USP7 and C16orf72) and Cadherin 13, and implement a rigorous approach to evaluating the statistical significance of these observations. Overall, large de novo CNVs, particularly those encompassing multiple genes, confer substantial risks (OR = 5.6; CI = 2.6-12.0, p = 2.4 × 10(-7)). We estimate there are 130-234 ASD-related CNV regions in the human genome and present compelling evidence, based on cumulative data, for association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin 1.

The contribution of de novo coding mutations to autism spectrum disorder

Whole exome sequencing has proven to be a powerful tool for understanding the genetic architecture of human disease. Here we apply it to more than 2,500 simplex families, each having a child with an autistic spectrum disorder. By comparing affected to unaffected siblings, we show that 13% of de novo missense mutations and 43% of de novo likely gene-disrupting (LGD) mutations contribute to 12% and 9% of diagnoses, respectively. Including copy number variants, coding de novo mutations contribute to about 30% of all simplex and 45% of female diagnoses. Almost all LGD mutations occur opposite wild-type alleles. LGD targets in affected females significantly overlap the targets in males of lower intelligence quotient (IQ), but neither overlaps significantly with targets in males of higher IQ. We estimate that LGD mutation in about 400 genes can contribute to the joint class of affected females and males of lower IQ, with an overlapping and similar number of genes vulnerable to contributory missense mutation. LGD targets in the joint class overlap with published targets for intellectual disability and schizophrenia, and are enriched for chromatin modifiers, FMRP-associated genes and embryonically expressed genes. Most of the significance for the latter comes from affected females.

Rare independent mutations in renal salt handling genes contribute to blood pressure variation.

The effects of alleles in many genes are believed to contribute to common complex diseases such as hypertension. Whether risk alleles comprise a small number of common variants or many rare independent mutations at trait loci is largely unknown. We screened members of the Framingham Heart Study (FHS) for variation in three genes—SLC12A3 (NCCT), SLC12A1 (NKCC2) and KCNJ1 (ROMK)—causing rare recessive diseases featuring large reductions in blood pressure. Using comparative genomics, genetics and biochemistry, we identified subjects with mutations proven or inferred to be functional. These mutations, all heterozygous and rare, produce clinically significant blood pressure reduction and protect from development of hypertension. Our findings implicate many rare alleles that alter renal salt handling in blood pressure variation in the general population, and identify alleles with health benefit that are nonetheless under purifying selection. These findings have implications for the genetic architecture of hypertension and other common complex traits.

Molecular Cytogenetic Analysis and Resequencing of Contactin Associated Protein-Like 2 in Autism Spectrum Disorders

Autism spectrum disorders (ASD) are a group of related neurodevelopmental syndromes with complex genetic etiology. We identified a de novo chromosome 7q inversion disrupting Autism susceptibility candidate 2 (AUTS2) and Contactin Associated Protein-Like 2 (CNTNAP2) in a child with cognitive and social delay. We focused our initial analysis on CNTNAP2 based on our demonstration of disruption of Contactin 4 (CNTN4) in a patient with ASD; the recent finding of rare homozygous mutations in CNTNAP2 leading to intractable seizures and autism; and in situ and biochemical analyses reported herein that confirm expression in relevant brain regions and demonstrate the presence of CNTNAP2 in the synaptic plasma membrane fraction of rat forebrain lysates. We comprehensively resequenced CNTNAP2 in 635 patients and 942 controls. Among patients, we identified a total of 27 nonsynonymous changes; 13 were rare and unique to patients and 8 of these were predicted to be deleterious by bioinformatic approaches and/or altered residues conserved across all species. One variant at a highly conserved position, I869T, was inherited by four affected children in three unrelated families, but was not found in 4010 control chromosomes (p = 0.014). Overall, this resequencing data demonstrated a modest nonsignificant increase in the burden of rare variants in cases versus controls. Nonetheless, when viewed in light of two independent studies published in this issue of AJHG showing a relationship between ASD and common CNTNAP2 alleles, the cytogenetic and mutation screening data suggest that rare variants may also contribute to the pathophysiology of ASD, but place limits on the magnitude of this contribution.

De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes

Megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndromes are sporadic overgrowth disorders associated with markedly enlarged brain size and other recognizable features. We performed exome sequencing in 3 families with MCAP or MPPH, and our initial observations were confirmed in exomes from 7 individuals with MCAP and 174 control individuals, as well as in 40 additional subjects with megalencephaly, using a combination of Sanger sequencing, restriction enzyme assays and targeted deep sequencing. We identified de novo germline or postzygotic mutations in three core components of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. These include 2 mutations in AKT3, 1 recurrent mutation in PIK3R2 in 11 unrelated families with MPPH and 15 mostly postzygotic mutations in PIK3CA in 23 individuals with MCAP and 1 with MPPH. Our data highlight the central role of PI3K-AKT signaling in vascular, limb and brain development and emphasize the power of massively parallel sequencing in a challenging context of phenotypic and genetic heterogeneity combined with postzygotic mosaicism.

Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1

Epileptic encephalopathies are a devastating group of epilepsies with poor prognosis, of which the majority are of unknown etiology. We perform targeted massively parallel resequencing of 19 known and 46 candidate genes for epileptic encephalopathy in 500 affected individuals (cases) to identify new genes involved and to investigate the phenotypic spectrum associated with mutations in known genes. Overall, we identified pathogenic mutations in 10% of our cohort. Six of the 46 candidate genes had 1 or more pathogenic variants, collectively accounting for 3% of our cohort. We show that de novo CHD2 and SYNGAP1 mutations are new causes of epileptic encephalopathies, accounting for 1.2% and 1% of cases, respectively. We also expand the phenotypic spectra explained by SCN1A, SCN2A and SCN8A mutations. To our knowledge, this is the largest cohort of cases with epileptic encephalopathies to undergo targeted resequencing. Implementation of this rapid and efficient method will change diagnosis and understanding of the molecular etiologies of these disorders.

Copy number variation detection and genotyping from exome sequence data

While exome sequencing is readily amenable to single-nucleotide variant discovery, the sparse and nonuniform nature of the exome capture reaction has hindered exome-based detection and characterization of genic copy number variation. We developed a novel method using singular value decomposition (SVD) normalization to discover rare genic copy number variants (CNVs) as well as genotype copy number polymorphic (CNP) loci with high sensitivity and specificity from exome sequencing data. We estimate the precision of our algorithm using 122 trios (366 exomes) and show that this method can be used to reliably predict (94% overall precision) both de novo and inherited rare CNVs involving three or more consecutive exons. We demonstrate that exome-based genotyping of CNPs strongly correlates with whole-genome data (median r(2) = 0.91), especially for loci with fewer than eight copies, and can estimate the absolute copy number of multi-allelic genes with high accuracy (78% call level). The resulting user-friendly computational pipeline, CoNIFER (copy number inference from exome reads), can reliably be used to discover disruptive genic CNVs missed by standard approaches and should have broad application in human genetic studies of disease.

Disruptive CHD8 mutations define a subtype of autism early in development.

Autism spectrum disorder (ASD) is a heterogeneous disease in which efforts to define subtypes behaviorally have met with limited success. Hypothesizing that genetically based subtype identification may prove more productive, we resequenced the ASD-associated gene CHD8 in 3,730 children with developmental delay or ASD. We identified a total of 15 independent mutations; no truncating events were identified in 8,792 controls, including 2,289 unaffected siblings. In addition to a high likelihood of an ASD diagnosis among patients bearing CHD8 mutations, characteristics enriched in this group included macrocephaly, distinct faces, and gastrointestinal complaints. chd8 disruption in zebrafish recapitulates features of the human phenotype, including increased head size as a result of expansion of the forebrain/midbrain and impairment of gastrointestinal motility due to a reduction in postmitotic enteric neurons. Our findings indicate that CHD8 disruptions define a distinct ASD subtype and reveal unexpected comorbidities between brain development and enteric innervation.