Trends in Neurosciences
ReviewFeature ReviewA de novo convergence of autism genetics and molecular neuroscience
Introduction
The identification of genes underlying ID and ASD has been most successful for syndromic Mendelian or monogenic disorders – for example, FMR1 (Fragile-X syndrome, [1]), MECP2 (Rett syndrome, [2]), or UBE3A (Angelman syndrome, [3]). Together, however, these syndromes are estimated to account for less than 10% of ASD/ID, suggesting the presence of additional genes and etiologies. Initial population-based studies failed to identify single genes of major effect and few major common risk variants have been replicated, despite the strong observed heritability of these diseases 4, 5, 6, 7. By contrast, targeted and genome-wide microarray studies revealed that large de novo copy number variants (CNVs) were significantly enriched among probands when compared to unaffected siblings and/or controls 8, 9, 10, 11, 12, 13, 14, a finding that echoed the earlier discovery of large chromosomal aberrations in ASD and ID. Both initial and subsequent higher-resolution studies estimate that 8% of sporadic ASD cases carried a de novo CNV, as compared with only 2% of unaffected siblings 11, 12. Furthermore, among children with general developmental delay (DD) and ID, rare large de novo CNVs are thought to account for up to 15% of disease burden [13]. Although individually rare, some of these CNVs were in fact recurrent mutations, mediated by locus-specific genomic instability [14], and many of these same recurrent CNVs observed initially in patients with ID [15] or ASD [16] have been identified in adults with epilepsy [17], bipolar disorder [18], or schizophrenia 19, 20, suggesting overlap in the genetic etiology of these disorders.
The discovery of an aggregate burden of large de novo CNVs and the identification of recurrent events signaled a new paradigm for ASD and ID genetics. Although specific CNVs are individually rare, combined they account for a significant fraction of cases, indicating the presence of considerable locus heterogeneity of ASD and ID. The de novo nature of these CNVs, together with their absence in the general population, suggests they represent a class of highly deleterious and highly penetrant mutations. Their underlying genetic model does not explicitly fit a recessive model of disease because CNVs are primarily present as hemizygous deletions or duplications. These mutations alter the dosage of genes but do not completely abolish their presence. Collectively, these observations support a complex disease/rare variant model for ASD, in which a proportion of etiologic risk is conferred by very rare variants and de novo mutations.
The commoditization of next-generation or ‘massively parallel’ sequencing represents a turning point in human genetics and makes it possible to discover sequence-level variants across nearly all coding regions (‘the exome’) or the whole genome (Box 1). These methods were first applied to confirm point mutations underlying Mendelian disorders [21], and subsequent pilot studies demonstrated that family-based (trio) exome sequencing could discover pathogenic mutations in simplex ID [22] or ASD [23]. In the past year, this paradigm of de novo mutation discovery using exome sequencing of parent–child trios has been expanded to about 1000 ASD or ID families, resulting in the first detailed picture of how de novo coding mutations contribute to these disorders.
In this review, we synthesize the results of recent large-scale exome sequencing studies of ASD and ID 24, 25, 26, 27, 28, 29 and summarize their implications for human neurodevelopmental genetics. There are three themes. (i) Exome sequencing of ASD/ID families has revealed a significant excess of de novo mutations in probands when compared to unaffected siblings and has identified novel candidate genes contributing to the neurological deficits. We note that the strongest effects are observed for de novo loss-of-function (or truncating) mutations (see Glossary), which prematurely truncate the protein due to frameshift and nonsense mutations. (ii) Both CNV and exome sequencing data suggest that no single gene will account for more than 1% of autism cases; rather, rare mutations in hundreds of genes may contribute to ASD or ID. (iii) Analyses of network connectivity further implicate potentially important neurodevelopmental and synaptic pathways in ASD and ID. Collectively, these studies represent a significant step forward for neurodevelopmental disorders providing a springboard for understanding their neurobiological underpinnings. We aim to focus on the molecular convergence revealed by these studies; for readers interested in other aspects of this topic, we suggest excellent reviews on ASD neurobiology [30], de novo mutation 31, 32, and exome sequencing [33]. We emphasize that although this review is focused on the insights gained by considering a de novo/rare variant model of ASD and ID genetics, other genetic etiologies are implicated in ASD as well (for reviews, see 34, 35) and no single etiology is likely to be fully independent of other etiologies or of environmental factors {see [36] for a review; see also the CHARGE (CHildhood Autism Risks from Genetics and Environment) Study [37]}.
Section snippets
An increase in de novo loss-of-function mutations
Both de novo CNVs and single nucleotide variants (SNVs) can have, in principle, similarly disruptive effects on genes. Crucially, however, the detection of de novo SNVs yields gene-level specificity, thus allowing individual pathogenic genes and neurobiological pathways to be identified. Moreover, a small subset of the de novo mutations (∼4% for unaffected and ∼9% of affected 26, 27) are disruptive (e.g., frameshift, premature stop codon, splice-donor defect) with respect to the protein's
Candidate genes yet few recurrent hits
Many strong neurobiological candidates have emerged from the genes disrupted by de novo mutations in these studies, including mutations in previously identified ASD/ID genes. Several mutations were identified in neurexin 1 (NRXN1) and neuroligin 1 (NLGN1); both are central components of the neurexin–neuroligin synaptic cell adhesion complex [42]. Numerous de novo mutations were identified in genes or loci linked to Mendelian disorders, many of which have features of ASD or ID. These loci and
Large-scale resequencing of candidate genes
Given the low rate of recurrence among genes with de novo mutations, estimates of overall locus heterogeneity for ASD have yielded between 300 and 1000 genes that could confer increased ASD risk when subjected to de novo mutation (Figure 1). Even if exome sequencing prices continue to fall, the cost to confirm the association for a significant fraction of these genes remains impractically high, especially if thousands or tens of thousands of samples are required, as has been suggested by CNV
Novel candidates and their neurobiology
Many of the top genes from recent exome studies are novel candidates for ASD and ID, including the strongest overall association: CHD8, an ATP-dependent chromodomain helicase that directly regulates CTNNB1 [45] as well as the p53 pathway [46]. The CHD8 protein has known binding activity with another chromodomain helicase, CHD7 [47], which is the key protein in CHARGE (Coloboma of the eye, Heart defects, Atresia, Retardation of growth, Genital and/or urinary abnormalities, and Ear abnormalities
Protein interaction networks converge on common pathways
Knowledge of molecular-level interaction between proteins has enabled the development of transcriptional networks [67] and protein–protein interaction (PPI) networks enriched for mutation in ASD and ID cases. These networks provide a powerful method to unify the landscape of mutations observed in genetically heterogeneous human disorders by leveraging regulatory interactions between genes and/or physical interactions between proteins. For example, Iossifov et al. found that 14/59 genes
Comparing and contrasting mutations in ASD and ID
In examining data presented in this review, several observations regarding the genetic or etiologic differences between ASD and ID diagnoses emerge, although the significant imbalance in the number of available exomes for ASD (n = 593) and ID (n = 151) advises caution in these comparisons. First, whereas the statistical significance of the clustering of the PPI network does not depend on the inclusion of the two ID studies (Table S1), some genes in the network have been observed only in ID studies
Concluding remarks and future directions
New sequencing technology and the establishment of large well-phenotyped family-based cohorts, such as the SSC, have enabled the systematic discovery of mutations that underlie the genetic etiology of ASD and ID. As a measurable indicator of progress, we note that in 2005, approximately 10% of the genetic etiology of autism was understood. Within 7 years advances in genomics technology facilitated the rapid discovery of de novo SNVs and CNVs leading to the discovery of disruptive genetic
Glossary
- CNV (copy number variant)
- loss or insertion of DNA, typically larger than 50 bp and often up to several megabases.
- Connected component
- a set of connected nodes that are part of a PPI network and can represent a pathway, complex protein structure, or cellular function.
- GC bias
- the tendency for sequencing reactions to produce fewer reads in regions of the genome with a high fraction of GC base pairs.
- Hidden species problem
- a method for estimating an unknown number of classes (species) from a distribution
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