smMIP based NGS assay for diagnosis of Lysosomal Storage Disorders in India. Single molecule molecular inversion probe based targeted sequencing assay for diagnosis of 23 common lysosomal storage disorders in India

Introduction: The present diagnostic pathway for lysosomal storage disorders (LSDs) is a sequential process involving biochemical screening, followed by enzyme assays and gene sequencing. In many cases, diagnosis of LSDs is difficult and can take up to 5 years as there are overlapping clinical features. Moreover, this diagnostic pathway is expensive due to its iterative nature. Here, we describe a novel low-cost and high-throughput sequencing assay using small molecule molecular inversion probes (smMIPs) to identify causative SNV and CNV in 23 genes associated with 23 common LSDs in India. Methods: A total of 903 smMIPs were designed to target exon and exon-intron boundaries using MIPgen and hg19 genome build that were pooled to create a sequencing library. We assessed the diagnostic accuracy of the assay in a cohort of 49 samples with previously known genetic diagnoses. Subsequently, the diagnostic yield was assessed in a cohort of 175 LSD patients with a clinical suspicion of LSD but no genetic diagnosis. Illumina MiSeq platform was used for sequencing at a mean coverage of ~200x. Data was analysed using a custom bioinformatics pipeline that included python and bash scripts. Results: The average percentage of coding region of the 23 genes covered by the 903 smMIPs was 99.17%. A 98% concordance of results with previously diagnosed cases was observed (n=48/49) whereby the discordant sample arose as a result of region containing the variant not being covered by the smMIP probe due to low sequence complexity. Furthermore, a diagnostic yield of 80.6% (n=140/174) was obtained in the 2nd cohort. Importantly, 7 patients with Niemann-Pick disease type C and one patient with neuronal ceroid lipofuscinosis type 6 were identified, which cannot be readily detected by enzyme testing. Lastly, single as well as multi-exon heterozygous and homozygous deletions were detected accurately. Conclusion: Overall, the assay was robust in calling SNVs and CNVs. The proposed assay could improve the LSD diagnosis rate at an affordable cost.