Abstract
Hirschsprung disease (HSCR) is a genetic disorder characterized by the absence of ganglion cells in the gut. RET is considered to be the main susceptibility gene. In our previous screening of 83 HSCR patients, targeted exome sequencing identified nine rare variants of RET, most of which were new discoveries. Here, we performed in vitro arrays with functional studies to investigate their effects. Two variants (p.R77C and p.R67insL) were demonstrated to disrupt the glycosylation of RET and affect its subcellular localization. Three nonsense mutations (p.W85X, p.E252X, and p.Y263X) could not produce detectable RET full-length protein, and the other three mutations (p.R770X, p.Q860X, and p.V778Afs*1) were translated into truncated proteins of predicted sizes. One canonical splice acceptor site mutation (c.2802-2 A > G) was verified to affect gene regulation through aberrant splicing. In addition, we explored the effects of read-through reagents on RET nonsense mutations and showed that G418 significantly increased the full-length RET protein expression of p.Y263X in a dose-dependent manner, together with a mild recovery of p-ERK and p-STAT3. Our data provide a functional analysis of novel RET mutations and suggest that all of the rare variants detected from patients with clinically severe HSCR are indeed pathogenic. Thus, our findings have implications for proper genetic counseling.
Highlights
Hirschsprung disease (HSCR, MIM 142623), or congenital megacolon, was first described in 1888 by Dr Harald Hirschsprung (1830–1916), a Danish pediatrician (Sergi, 2015)
We report nine rare RET mutations found in 87 HSCR patients, including one missense mutation, one splice site mutation, one in-frame insertion, one frameshift mutation, and five nonsense mutations (Jiang et al, 2017b)
Functional studies demonstrated that these identified mutations all disrupted RET biological functions via various mechanisms: 1) two extracellular domain mutations, namely, p.R77C and p.R67insL, disrupted protein glycosylation, and the lack of glycosylation of the RET protein resulted in cellular mislocalization (Kjaer and Ibanez, 2003); and 2) five nonsense mutations, one frameshift mutation, and a splice site mutation, all of which produced truncated proteins, had abnormal RET function and activity
Summary
Hirschsprung disease (HSCR, MIM 142623), or congenital megacolon, was first described in 1888 by Dr Harald Hirschsprung (1830–1916), a Danish pediatrician (Sergi, 2015). The average incidence of HSCR is 1 in 5,000 live births with a sex ratio of approximately four males/one female. The incidence varies significantly between different ethnic groups, and the highest incidence, namely, 1 in 3,571 live births, has been reported in Asians (Kenny et al, 2010). HSCR is defined as the absence of ganglion cells in the myenteric and submucosal plexuses of the intestinal wall with concomitant hypertrophy of parasympathetic nerve fibers (Sergi, 2015; Schriemer et al, 2016).
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