smMIP based NGS assay for diagnosis of 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.