Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Abstract

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies

“…Most of the other genes have been identified in rare (familial) syndromic HSCR cases (Amiel et al, 2008). In addition to the rare, coding mutations with large effects, common, non-coding variants with small effects have also been identified (Emison et al, 2010; Jiang et al, 2015). …”

Hirschsprung disease: current perspectives

“…The best example to date lies in the additional susceptibility gene for semaphorin 3A, which results in signaling pathway dysfunction in conjunction with a variant in Ret, leading to malfunction and disease. 20 It is probable that this could function as a viable hypothesis for other identified genetic variations, in keeping with the current concept of a multigenetic etiology for HSCR. Some then lead to syndromic phenotypic expressions of genes identified in associated syndromes (eg, chromosome 21 and ZEB2 genetic variations associated with the Mowat-Wilson syndrome).…”

“…[18][19][20] The reason for the incomplete migration and development of ganglion cells is as yet not fully understood. Early estimates were that genetic variations could be identified in at least 12% of HSCR cases, which is higher than the expected in the normal population.…”

“…It is now understood that the major controlling signaling pathway of the developing enteric neuroblasts appears to be the RET proto-oncogene, with up to 80% of patients with HSCR harboring alteration of at least one RET allele. 20 Most of these are noncoding mutations/polymorphisms within the gene. The RET intronic SNP rs2435357 has been shown to demonstrate significant frequency differences by affecting RET transcription regulation affecting sex, segment length of aganglionosis, and family recurrence.…”

Intestinal Neuronal Dysplasia-Like Submucosal Ganglion Cell Hyperplasia at the Proximal Margins of Hirschsprung Disease Resections

“…The 3 common polymorphisms were identified in genome-wide association study of HSCR patients of European ancestry and replicated independently [35, 36] The prevalence of each of the variables listed was compared between patients with and without proximal IND-SH using Welch Two Sample T-test, Fisher’s Exact test, or Permutation Chi-Squared test.…”