The use of high throughput sequencing for the identification of variants contributing to Hirschsprung disease in the Maltese

Abstract

Hirschsprung disease (HSCR) is a rare congenital disorder, affecting 1 in 5000 live births, and is characterized by the absence of neurons in the distal section of the colon. New-borns present with symptoms within the first days of life, and these commonly include failure to pass meconium, abdominal pain, abdominal distension, constipation and vomiting. The condition can present as an isolated trait or in conjunction with other symptoms (syndromic HSCR). To date, the inheritance of variants within a number of genes has been linked with the development of HSCR; however 70% of patients do not have a genetic explanation. The aim of this study is to use high throughput sequencing to identify causative variants in Maltese HSCR patients. Three nuclear families, consisting of a proband, parents and a sibling, were recruited. All three probands were tested using gene panel high throughput sequencing (RET, NRTN, NRG1, EDNRB, EDN3 and GDNF). Two families were then chosen to be tested by whole genome sequencing; all sequencing was carried out on Illumina platforms. Multiple filtering strategies based on different modes of inheritance were applied to short-list the most interesting variants; databases were then used to assess their biological significance to the disease. In the first family, an accumulation of single nucleotide polymorphisms (SNPs) in the SEMA3 gene cluster (rs1228877 MAMI MAF 0.372; rs1228937 MAMI MAF 0.381; rs11766001 MAMI MAF 0.233; rs80227144 MAMI MAF 0.0573; rs12707682 MAMI MAF 0.215; rs7811476 MAMI MAF 0.246; rs6468008 MAMI MAF 0.225; rs17560655 MAMI MAF 0.176), known to increase HSCR risk, was observed. However, in the second family no obvious HSCR cause was found. It is a possibility that HSCR in these families is not caused by a coding sequence variant but by aberrant epigenetic changes, further studies are required.