Long read whole genome sequencing-based discovery of complex structural variants and their role in aetiology of non-syndromic autism spectrum disorder in India

Background: Despite having heritability estimates of 80%, approximately 50% cases of autism spectrum disorders (ASD) remain without a genetic diagnosis. Complex structural variants (SVs) detected using long-read genome sequencing are a relatively new class of variants implicated in neurodevelopmental disorders. Short read sequencing (SRS) and chromosomal microarray (CMA) are unable to resolve these SVs due to their inherent technological limitations. This study was aimed to detect and delineate the role of SVs in children with non-syndromic ASDs using long read whole genome sequencing (lrWGS) in whom prior traditional genetic tests did not yield a definitive genetic diagnosis. Methods: A total of 23 patients with no prior genetic diagnosis from karyotyping, Fragile-X analysis, CMA and short read whole exome sequencing (srWES) were selected for lrWGS using Oxford Nanopore based sequencing platform. All samples were sequenced at an average coverage of ~10x. Contigs generated from high accuracy base calling were aligned against GRCh38/hg38 human reference genome build. SVs were called using five variant callers- Sniffles2, cuteSV, NanoVar SVIM and npInv, and annotated using AnnotSV. Concordant calls across at least three variant callers were filtered and prioritized for downstream analysis. Candidate variants were validated by orthogonal methods. Results: A total of 46 low pass long read sequencing runs were performed for the selected 23 samples (two runs/ sample). The N50 read length of 6.74±3.31 kb was obtained across the runs, and on average, approximately 176,432 calls were made across all callers for each sample. The average number of deletions, duplications, insertions, inversions and translocations were 47,375, 2,498, 62,657, 1084 and 62,817, respectively per sample. Of 23 cases, a candidate SV, an inversion of approximately 2.7 Mb in size encompassing SNAP25-AS1 gene was observed. This gene is likely to be involved in the synaptic pathway and has previously been associated with autism. Conclusion: This is the first study from India to assess the role of complex SVs in the aetiology of non-syndromic ASDs. Despite the small sample size, the study results suggest the limited role of complex SVs in the aetiology of non-syndromic ASD. Dearth of data supporting the role of complex SVs in non-syndromic ASDs in other cohorts from around the world further supports our conclusion. Hence, inclusion of lrWGS in clinical diagnosis for non-syndromic ASD is currently not supported.