RET and EDNRB mutation screening in patients with Hirschsprung disease: Functional studies and its implications for genetic counseling

Abstract

Hirschsprung disease (HSCR) is a major cause of chronic constipation in children. HSCR can be caused by germline mutations in RET and EDNRB. Defining causality of the mutations identified is difficult and almost exclusively based on in silico predictions. Therefore, the reported frequency of pathogenic mutations might be overestimated. We combined mutation analysis with functional assays to determine the frequencies of proven pathogenic RET and EDNRB mutations in HSCR. We sequenced RET and EDNRB in 57 HSCR patients. The identified RET-coding variants were introduced into RET constructs and these were transfected into HEK293 cells to determine RET phosphorylation and activation via ERK. An exon trap experiment was performed to check a possible splice-site mutation. We identified eight rare RET-coding variants, one possible splice-site variant, but no rare EDNRB variants. Western blotting showed that three coding variants p. (Pr270Leu) (Tyr1062Cys)) resulted in reduced ERK activation. Splice-site assays on c.1880-11A4G could not confirm its pathogenicity. Our data suggest that indeed almost half of the identified rare variants are proven pathogenic and that, hence, functional studies are essential for proper genetic counseling.

Functional Studies on Novel RET Mutations and Their Implications for Genetic Counseling for Hirschsprung Disease

“…We confirmed this by immunofluorescence staining, which showed that these two mutant proteins were localized close to the nucleus and the endoplasmic reticulum compared with the RET-WT protein. In support of our results, the extracellular variant p.P270L was previously demonstrated to disrupt the maturation of the RET protein with nonsignificantly lower p-ERK levels (Widowati et al, 2016). Interestingly, a strong reduction of glycosylated RET for p.R77C and p.R67insL was observed in the case of the 1:1 RET wild type and mutant plasmid transfection, even when compared with the results of the truncating variants ( Figure 4 ).…”

“…Missense mutations of the extracellular domain of RET can decrease the level of RET glycosylation (Carlomagno et al, 1996), interfering with its maturation, hindering its transport to the cell membrane and disrupting the binding site of RET for subsequent ligand binding (Widowati et al, 2016; Plaza-Menacho et al, 2006). Although the p-STAT3 and p-ERK levels of p.R77C and p.R67insL were not significantly decreased compared with those of RET-WT, the mutations both led to nonglycosylation of RET and, consequently, their mislocalization due to incorrect folding (Kjaer and Ibanez, 2003).…”

“…To date, most of the RET mutations found in HSCR patients have been reported based only on in silico predictions, and a minority of the RET mutations has been identified in the context of functional consequences via in vitro assays. Furthermore, ∼35% of the RET variants that were functionally tested were ultimately demonstrated to be noncausative variants (Widowati et al, 2016). Notably, RET -CDS mutations are not fully penetrant, and assessments of their functional significance are truly essential, not only for understanding the mechanism of the disease but also for providing accurate genetic counseling and recurrence risk evaluations (Leon et al, 2012).…”

A Novel Zebrafish ret Heterozygous Model of Hirschsprung Disease Identifies a Functional Role for mapk10 as a Modifier of Enteric Nervous System Phenotype Severity

“…Because of the multifactorial etiology of HSCR, disease risk cannot be predicted, even in cases of familial HSCR [51, 59]. For this reason genetic counseling is not usually conducted for HSCR, however, surgical strategies are generalized.…”

Identification of Variants in RET and IHH Pathway Members in a Large Family With History of Hirschsprung Disease

“…23 Candidate variants were validated by Sanger sequencing as previously described. 24 Segregation analysis was performed using family members for which DNA was available (II-2, III-2, IV-1, IV-2, IV-3, IV-4, IV-5, V-1, V-2, V-3, and V-4).…”