Clinico-epidemiological and trio-based genetic analysis of Bangladeshi Hirschsprung disease patients

Abstract

Hirschsprung disease (HSCR), also known as colonic aganglionosis, is the leading cause of functional intestinal obstruction. It is a complex rare congenital disease with an incidence of one in 3500-5000 live births and is characterized by the absence of enteric ganglia in the distal colon due to the failure of neural crest cells to fully colonize the hindgut during embryonic development. Genetic studies over the last three decades have identified at least 30 genes associated with HSCR. Many of these genes converge into several biological pathways that often crosstalk to orchestrate the dynamic process of enteric nervous system (ENS) development and HSCR pathogenesis. Yet, variants in these genes can explain no more than 30% of HSCR patients, indicating the existence of other HSCR-associated genetic lesions as well as biological processes that are yet to be discovered. In this study, we applied whole exome sequencing (WES) and genomewide single nucleotide polymorphism (SNP) array to 24 sporadic HSCR patients and their unaffected parents from Bangladesh. This enabled us to comprehensively examine the genetic profile of each patient by taking together different inheritance models and different types of genetic variants (rare variants, common risk variant, and rare copy number variants). We found that using this approach, we could explain as much as 50% of patients by the known HSCR-associated genes or biological processes. However, every patient has unique genetic profile which demonstrates the complex polygenic nature of this disease. Using geneset analysis, this study also provided additional evidence of several previously implicated biological processes relevant to HSCR that include – RET signaling, EDNRB signaling, Hedgehog/Notch signaling, and cell-matrix interaction. Importantly, for the first time, we demonstrated enrichment of microtubule-related genes in HSCR patients. This finding was also supported by the demonstration of excess of rare damaging variants in these genes in a separate large cohort of Chinese HSCR patients. Finally, using CRISPR/Cas9 genome editing in zebrafish, we confirmed one of these genes - DYNC1H1 – to be necessary for ENS development. In summary, our study shows that the genetic diagnostic yield of HSCR patients can be increased by a comprehensive genetic profile obtained by different inheritance models and variants. We also show that microtubule-associated genes including DYNC1H1 are potential HSCR candidate genes - opening a new area of further investigation. These findings might also have implications in future adoption of WES into clinical setting.