WHOLE GENOME SEQUENCING ANALYSIS OF JAPANESE ASD TRIOS: UNRAVELING PATHOGENIC VARIANTS

Autism spectrum disorder (ASD) is a genetically and phenotypically heterogeneous neurodevelopmental disorder with a strong genetic basis. De novo variants, spontaneously arising and not inherited, play a pivotal role in ASD pathogenesis. Advancements in sequencing technology have expanded our ability to detect these genetic variants. With advancements in sequencing technology, our group embarked on whole genome sequencing (WGS), a method now in the limelight. This report details our initial WGS analysis on a Japanese ASD trio sample.

Detect de novo variants of various sizes and types. Ascertain pathogenicity of de novo and other variants. Characterize the clinical phenotypes of ASD patients bearing these pathogenic variants.

DNA samples was obtained from 57 patients with autism spectrum disorder and their parents. This study was approved by the ethics committee of Nagoya University and each participating institute. Written informed consent was procured from all participants. Preliminary screening ensured the absence of pathogenic copy number variants (CNVs) as identified by array comparative hybridization (aCGH). We performed short-read WGS on the MGI T7 platform and detected rare single nucleotide variants (SNVs) or insertion– deletion variants (INDELs) and rare CNVs. This was followed by a trio analysis to identify de novo, inherited homozygous, and hemizygous variants.

On average, 60 de novo SNVs and INDELs were detected per individual. Of these, 8 de novo variants localized to exonic regions linked to known ASD genes. We revealed a potential novel loss-of-function variant in BSN, a presynaptic gene. Nine exonic homozygous and hemizygous variants appeared as candidates. Within the rare CNVs encompassing coding regions, we identified 4 de novo, 15 homozygous, and 3 hemizygous variants. This includes a de novo duplication of TRIM49 in a patient with ASD and intellectual disability (ID), a homozygous loss of ARHGAP11B, the human-specific gene in a patient with ASD and ID (both parents exhibited a heterozygous loss of this region). Additionally, 10 rare CNVs, inherited either paternally or maternally, were located in genes previously associated with ASD/DD (developmental disorder), encompassing a maternal duplication of RHEB— a component of the mTOR pathway— in a patient diagnosed with ASD, ID, and a brain tumor. A maternal 15q11.2 duplication was also detected in another patient with ASD and ID. Furthermore, while estimating clinical significance was challenging, CNVs encompassing non-coding regions and regulatory elements related to known ASD/DD genes were also identified in several patients. Notably, ASD patients with discernible pathogenic variants often exhibited comorbidities, such as ID, epilepsy, and distinct physical traits. Utilizing WGS enhanced the detection rate of variants that elucidated the underlying pathogenesis of ASD.

Our trio-based WGS strategy facilitated the detection of minute CNVs, overlooked by aCGH. This approach yielded a more precise evaluation of the pathogenic and clinical implications when contrasted with parental genetic information. While our current short-read methodology had its limitations with certain sequences, the advent and adoption of long-read sequencing promises more accurate analyses in forthcoming studies.