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

Researchers in India used advanced genetic testing to look for large DNA changes in 23 children with autism who hadn't received a genetic diagnosis from standard tests. They found many DNA variations but only one potentially important change in a gene called SNAP25-AS1 that affects brain connections. This suggests that these large DNA changes may not be a major cause of autism in children without other medical conditions.

This Indian study used long-read whole genome sequencing to search for structural variants in 23 children with non-syndromic autism who had no genetic diagnosis from standard tests. Using Oxford Nanopore sequencing at ~7x coverage, researchers detected an average of 235,163 structural variants per sample across different variant types. One candidate structural variant was identified - a 2.7 Mb inversion encompassing the SNAP25-AS1 gene, which is involved in synaptic pathways and previously associated with autism. Despite comprehensive sequencing, the study found only modest contribution of structural variants to non-syndromic autism etiology in this cohort.

Limited immediate clinical utility given small sample and single variant identified. May inform future genetic testing strategies but requires validation in larger cohorts. Suggests structural variants have modest role in non-syndromic autism etiology.

Very small sample size (n=23), low genome coverage (~7x), modest read length, and lack of functional validation of identified variants. Single candidate variant found limits generalizability of conclusions.

Despite having heritability estimates of 80%, ~ 50% cases of autism spectrum disorders (ASD) remain without a genetic diagnosis. Structural variants (SVs) detected using long-read whole genome sequencing (lrWGS) 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 lrWGS in whom prior traditional genetic tests did not yield a definitive genetic diagnosis.

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. Samples were sequenced at an average coverage of ~ 7x. 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.

Calls from cuteSV were used as benchmark to identify concordant calls across at least three variant callers. An average whole genome coverage of ~ 7x and N50 read length of 6.65 ± 3.3 kb was obtained across 46 runs (two runs/ sample). On average, a total of approximately 235,163 calls were made across all callers for each sample. The average number of deletions, duplications, insertions, inversions and translocations were 54,787, 3,335, 62,459, 1,286, and 113,296, respectively, were detected across all callers 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. This is the first study from India to assess the role of SVs in the aetiology of non-syndromic ASDs. Despite the small sample size, low-pass genome coverage, and modest N50 read length, the study indicates a modest contribution of SVs in the aetiology of non-syndromic ASD.

Dearth of data supporting the role of SVs in non-syndromic ASDs in other cohorts from around the world further supports our conclusion.