HSCR is a complex and heterogeneous disorder with a wide spectrum of mutations responsible for HSCR. RET is considered to be the main participant gene for HSCR (2, 22, 23). The identified mutations of RET mainly include loss-of-function (LOF) mutations and de novo mutations, accounting for > 80% of known pathogenic mutations in HSCR (24). A wide range of identified mutations scattered all over the full coding sequence (CDS) in RET (2). These RET CDS mutations have been account for 20% of the sporadic cases and in up to 50% in familial cases (3).
As yet, over 200 CDS rare variants in HSCR have been described (). These identified rare variants can be divided into two groups: null variants such as nonsense mutation, frame-shift, Indels or canonical ± 1 or 2 splice site variants that produce a truncated protein and variant of uncertain significance (less functional or nonfunctional protein) such as most missense mutations and small in-frame deletions. In general, these variants mainly cause loss-of-function of the RET protein (haploinsufficiency assumed to be mutational mechanism) in HSCR (9, 25–28). RET gene in human contains 21 exons that encodes a tyrosine kinase receptor with a cadherin-like extracellular domain, a cysteine-rich region, and an intercellular tyrosine kinase domain. Based on a functional assessment and location of variants, the RET mutations are classified as follows. First; mutations in coding sequences that code for the extracellular domain of RET that mutation will affect the transport of RET to the plasma membrane during translation of the protein. Second; mutations in the cysteine-rich domain of RET make a covalent dimerization of RET upon ligand activation and diminish its localization at the plasma membrane. Third; mutations targeting the kinase domain of RET that abolished RET tyrosine kinase activity. Fourth; mutations at the C-terminal end that change signaling over alteration of binding proteins, Fifth: mutations in regulatory sequences (promoter and intron 1) that reduce RET transcription. These mutations affect protein function and accordingly lead to HSCR (29).
In HSCR, two types of variants (rare and common) in RET have been observed which have a major role in the manifestation of the disease. The common variants in RET have been mainly identified in the commonest form of HSCR (S-HSCR and sporadic forms), and the rare variants in CDS of RET are often found in more severe forms of HSCR (L/TCA-HSCR and familial) (3). Unlike common variants, rare destructive variants normally have a profound effect on the risk of developing the disease and have higher penetrance. Rare damaging variants are negatively selected, and they have to be newly (de novo) generated and accumulated to exert its destructive effect on a population level (30). Since, enormous amount of genomic data has been usually generated by NGS technology (especially WES service) planning an experiment with 2 or more subjects may help to determine significant disease-causing variants, and this would probably ease the difficulty of interpreting such rare variants (31). Regarding HSCR, more than 17 HSCR susceptibility genes and three signaling pathways have been identified concerning the etiology of the disease. A comprehensive understanding of the genetics of an inherited rare complex disease is a major challenge requiring further efforts. In the present study, we performed WES on a proband (IV-V) of an extended family with variable expressivity. We found a novel nonsense mutation (c.942C > A, p.Y314X) as a null variant at exon 5 of the RET gene that is associated with a wide range of phenotypes and incomplete penetrance.
This mutation, p.Y314X, may lead to a truncated protein without protein kinase critical domain as mentioned above. In general, nonsense variants are associated with severe pathogenic impact in the genes with loss of function mechanism. This devastating variant is located in a gene with an autosomal dominant mode of inheritance, which may lead to protein loss of function. The patient IV-V with the identified causative variant had other phenotypes such as single kidney, absent of peritoneum and pigmentation of the face in addition to HSCR, in which these manifestations explain variable expression in the disease. However, this variant has been segregated in her mother (III-IV), grandmother (II-II) and aunt (III-V) of the patient (IV-V), these members of the family don’t have shown HSCR. The single kidney and diarrhea were manifestations in III-IV and II-II, respectively. Also, chronic constipation and premature ovarian failure were observed in III-V. Furthermore, in the other members of the family (II-IV and III-XII) with chronic constipation and without HSCR, the novel variant p.Y314X was detected. This phenomenon described by incomplete or reduced penetrance. In a meta-analysis study of RET gene in Hirschsprung disease by Puri P and Tomuschat C, it is suggested that carrying one pathogenic RET mutation maybe share a little role in genotype-phenotype correlation on its own and that these variabilities can be more dependent upon genotypes at different loci and/or environmental factors (32, 33). As mentioned, HSCR as a complex disease is influenced by many mutations, each of which might have only a small effect (34, 35). Also, RET mutations include rare high penetrant variants (at coding sequence) and common low-penetrant variants (at introns and the promoter regions) that seem to act in a synergistic way predisposing to HSCR phenotype (8, 36–38). Another explanation could be that the majority of HSCR cases without RET rare and common variants or the presence of additive phenotypes in the family history of this study might be explained by yet unidentified mutational events in the known HSCR genes or unknown genes, acting alone or in combination. In addition to modifying genes, environmental and epigenetic factors can be described as variable expression and incomplete penetrance in multifactorial diseases such as HSCR (39). Ultimately, other members in the pedigree of family (II-III, II-V, II-VII, III-VIII and III-X) died at the birth with manifestation/congenital malformations such as closed anus.
Also, we identified some rare benign and VUS variants (heterozygous mode) in other HSCR related genes (Table 2). As mentioned above, several variants (common and rare variants) with additive effects contribute to Hirschsprung disease as a complex phenotype that could be observed with other phenotypes. So, the impact or pathogenicity of these variants should be followed by a burden test in control and patients, and functional studies in zebrafish models.
Also, approximately 18% of cases with HSCR co-occurs with other congenital malformations (40). These are often Gastrointestinal anomalies (intestinal malrotation, imperforate anus), genitourinary anomalies (cryptorchidism, inguinal hernia, hypospadias, renal malformation), cardiac anomalies (such as atrial septal defect, ventricular septal defect, patent ductus arteriosus, tetralogy of Fallot), and central nervous system anomalies (intellectual disability and microcephaly) (41). In this study, in addition to HSCR, the history of the family was shown other manifestations/malformations such as single kidney, closed anus, premature ovarian failure, chronic constipation, and so on. However, RET mutations (rare or/and common variants) plays a major role in both isolated and syndromic HSCR. Various authors have described HSCR occurrence does not seem to depend on the RET genotype alone but 1 or more other genes can be contributed in HSCR expression (42, 43). Other studies revealed that the common gene networks such as RET/GDNF signaling pathway participates in the development of the enteric nervous system and also kidneys formation. Subsequently, RET mutations can be recognized in a variety of congenital abnormalities with isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT) and HSCR together (44). For example, in one case report with total colonic aganglionosis as well as right renal agenesis and oligomeganephronia, gene study in this patient revealed a heterozygous p.S811F mutation in exon 14 of RET. Also, in our study, the proband IV-V and her mother (III-IV) indicated a single kidney and HCSR together with identified p.Y314X mutation in exon 5.