Inherited Rare Variants in Autism: Whole Exome Sequencing in Multiplex and Singleton Families

Background: Autism is a severe neurodevelopmental disorder which aetiology is mainly unknown. Twin and family studies suggest high heritability. To date a few autism risk genes have been identified, most of them found on the basis of overlap with other syndromic neurodevelopmental disorders, or because they are involved in chromosomal rearrangements or copy number variants (CNVs). Whole exome sequencing (WES) represents a powerful technology to identify rare single nucleotide variants (SNVs) that may help to depict the complex genetic architecture of autism. Recently, WES studies suggested novel autism candidate genes through the analysis of rare de novo variants in singleton families. Although these studies represent pioneering insights into the genetics of autism, de novo variants do not explain the whole genetic complexity of the disease.  

Objectives:  In the present study we performed exome sequencing of 10 autism multiplex families with the aim to investigate the role of the inherited pool of rare SNVs and uncover new candidate genes.

Methods: The 10 multiplex families under study include 41 individuals, with 20 parents and 21 probands that fulfil diagnostic criteria for autism spectrum disorder (ASD). Structural variants analysis was performed in the affected individuals with the CytoScan HD array (Affymetrix) to exclude families with reported fully penetrant structural variants.To capture the exome fraction we used the NimbleGen SeqCap EZ Exome Library SR kit. The coding exons targeted corresponded to approximately 34 Mb. On average, individuals had 86% of target covered at >20X and 50% at >75X. Nonsense, frameshift and splice-site mutations were selected for Sanger validation, whereas missense mutations were previously filtered based on pathogenicity predictions. Our selection of inherited variants included only mutations present in both affected siblings. In addition, 20 singletons families have been studied in a second phase of this project. 

Results: Our preliminary data indicate, as recently reported, that a cumulative effect of oligogenic heterozygous variants represents the most plausible genetic model for autism. In our study we found variants in genes already associated with syndromic autism such as NF1 and TSC1, but also genes like SCN1A or ANK2, which have emerged recently as autism genes from exome sequencing studies. Interestingly, the data show that truncating variants may have a predominant role in psychiatric disorders. In fact, we found a correlation between a higher number of truncating SNVs and low Non-verbal IQ performance. Also, we found statistically significant differences between the number of truncating rare SNVs transmitted to both sibs and those that were not transmitted. Protein-protein interaction analysis of the identified SNVs pool, including truncating and potentially damaging missense variants, suggested novel candidate genes for autism. 

Conclusions:  Our data suggests that truncating rare variants may have a major role in the aetiology of autism in multiplex families. This preliminary evidence is currently under study in an additional sample of 20 singleton families.