GDNF and GUCY2C mutation associated with Hirschsprung's disease in a trio pedigree: a case report

Background: Hirschsprung's disease (HSCR) caused by absence or dysfunction of ganglion cells in the submucosal plexus and intermuscular plexus of the intestinal wall. Studies have shown that homozygous mutations of glial cell-line derived neurotrophic factor ( GDNF ) can lead to the occurrence of HSCR. but heterozygous mutation must combine with other related abnormal genes could to cause the HSCR. The other Guanylyl cyclase C ( GUCY2C ) gene is rarely reported as a direct cause of HSCR. Our findings Provides a new perspective for application of mutation screening in the genetic counseling. Case presentation: We report clinical and genetic findings of a trio pedigree with Hirschsprung's disease. Whole exome sequencing and Sanger sequencing were performed for propositus and all members of trio pedigree, respectively. We also collected 74 healthy control samples and used Sanger sequencing to identify that found the mutation. We first described the rare trio pedigree of HSCR caused by two special genes of GDNF (p.Arg93Gln) and GUCY2C (p.Tyr1072Cys). Studies have found that mutations in GDNF alone do not cause HSCR symptom, so is propositus’ mother in this study. Common mutation of GDNF gene mutation inherited from mother of proband, but the rare mutation of GUCY2C from the father. None of the parents showed HSCR phenotype. We provide evidence for the causative role of the two variants. Conclusions: Our study found that GDNF and GUCY2C together lead to the occurrence of HSCR. This finding expands the genotype spectrum and increases knowledge on the genetic molecular mechanisms of the HSCR. Our findings will provide help for the screening of pathogenic genes of HSCR.

HSCR in live births is about 1/5000. There are significant racial differences, with the highest morbidity in Asia, especially in China. The incidence of HSCR in men is 2-4 times that of women. Pathologically, HSCR is characterized by congenital intestinal blockage caused by the absence or dysfunction of ganglion cells in the submucosal plexus and intermuscular plexus of the intestinal wall. The etiology of HSCR mainly focuses on the abnormal proliferation, differentiation and migration of intestinal nerve trunk [2,3]. Intestinal neural crest cell (NCC) migrate and implant into the whole intestine at 5 to 12 weeks of gestation. HSCR is the most common digestive tract malformation in children, and it is a typical developmental disorder of enteric nervous system (ENS) [3]. Most cases of HSCR are sporadic, but there exists the inheritance mode of autosomal dominant, recessive inheritance, with incomplete dominance and diversity phenotypes [4,5]. The main clinical manifestation is complete or incomplete intestinal obstruction. Severe cases are manifested as the failure of passing the first stool within 48 h after birth. Abdominal distention, vomiting, or neonatal enterocolitis, which seriously affects the quality of life of children and even endangers their lives. In recent years, with the development of high-throughput sequencing and the increasing incidence of HSCR, domestic and foreign scholars attach great importance to it. Therefore, the research on the pathogenesis of HSCR has become a hot topic.
Genetic studies have shown that HSCR-related susceptibility genes are mainly derived from two major signaling pathways and one related transcription factors during ENS development [6]. One is the RET/GDNF/GFRA1 signaling pathway. The RET gene encodes a transmembrane receptor and member of the tyrosine protein kinase family of proteins. Binding of ligands such as GDNF (glial cell-line derived neurotrophic factor) and other related molecules, neurturin, artemin and persephin, these molecules are linked to glycosylphosphatidylinositol-anchored coreceptors (GFRalpha1/2/2/4), activation of downstream signaling pathways that play a role in cell differentiation, growth, migration and survival. The encoded receptor is important in development of the nervous system, and the development of organs and tissues derived from the neural crest. This proto-oncogene can undergo oncogenic activation through both cytogenetic rearrangement and activating point mutations [7,8].
Guanylyl cyclase C (GUCY2C) is a transmembrane receptor protein whose expressed mainly in intestinal epithelial cells [11]. Activated GUCY2C can convert guanosine-5'-triphosphate (GTP) into cyclic Guanosine monophosphate (cGMP) and increases the amount of cGMP in cells. Increased cGMP will result in activation of a series of downstream signals, such as including cGMP-dependent protein kinases (PKGs), cyclic nucleotide-gated (CNG) channels and phosphodiesterases(PDEs). These functions are essential for the balance of electrolytes and fluids and tight junctional barrier function of the intestinal epithelium [12]. Abnormal function of GUCY2C gene may lead to autosomal dominant hereditary diarrhea and are predisposed to inflammatory bowel disease [13]. GUCY2C gene is rarely reported as a direct cause of HSCR.
In this study, we collected a trio pedigree with HSCR. The patient showed typical HSCR symptoms.
Through gene sequencing, we identified the pathogenic genes of the family. Scientific guidance was made for future fertility and prenatal screening of the family, so as to provide a richer basis for the pathogenic gene spectrum of HSCR.

Case Presentation Participants and clinical diagnosis
A 15-year-old girl came to our hospital because of defecation difficulties. According to their parents' complaints and relevant diagnostic data, the little girl was hospitalized in XiangYang City Central Hospital for many times due to defecation difficulties and stricture of anus since she was born. At that time, HSCR was diagnosed by experienced doctors in XiangYang City Central Hospital. We identify HSCR as a congenital disease. It has been described a correlation between the occurrence of HSCR and anorectal malformations [14,15]. The anal expansion operation was performed in the newborn, But the difficulty of defecating has not been eliminated. The patient's father was 41 years old and his mother was 39 years old. They were all healthy and had no HSCR related symptoms. But the mother dictates that she must pay great attention to diet, otherwise constipation will occur easily. They all live in FanCheng District, Xiangyang City, Hubei Province. This study was approved by the Ethics Committee of Xiangyang No.1 People's Hospital, Hubei province. After obtaining informed consent from participates, we collected the trio pedigree with HSCR and age matched 74 healthy control peripheral blood samples from each member. 5 ml venous blood samples were collected and stored in an Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulant tube in a refrigerator at − 20℃.

DNA extraction and Genomic analysis
Genomic DNAs (gDNAs) of the family (one affected and two unaffected members) and 74 healthy control samples were extracted from peripheral blood using TIANGEN Blood DNA extraction kit (article number: DP304-03).
Whole exome sequencing (WES) was performed on propositus(Ⅱ-1) in our trio pedigree ( Fig. 1) with a mean target coverage of 100×. BGI Exome V4 kit was used to exome capture and sequencing. Firstly, we used cutadapt (v1.15) to trim adaptor sequences at the tail of sequencing reads, and then aligned sequencing reads to human reference genome (UCSC hg19) with BWA (v0.7.15). Duplicated reads were marked by Picard (v2.4.1). Qualimap [16] (v2.2.1) was used to calculate base quality metrics, genome mapping rate, and the coverage of targeted regions. Base quality score recalibration, indel realignment and variants (SNVs & InDels) calling were performed following the best practice protocol of the Genome Analysis Toolkit (GATK, v3.8). Variant filtering was done by a finely tuned in house script. Pass-filter variants were annotated using Pubvar variant annotation engine (www.pubvar.com) and VEP [17].
In genetic analysis, we separately identified variants that fit the dominant and recessive inheritance models. Variants met anyone of the following criteria were excluded from genetic analysis: maximum population frequency was large than 0.01, genotype confidence was low, or predicted as benign by all five algorithms (SIFT[18], PolyPhen 2[19], MetaSVM [20], MCAP [21] and MutationTaster [22]). The pathogenic evidences of candidate disease-causing variants were scored by InterVar [23] (1.0.8) according to American College of Medical Genetics and Genomics(ACMG) guidelines [24].
The other is the rare mutation c.3215A > G (NM_004963, rs35179392, p.Tyr1072Cys) of GUCY2C gene. Both genes sites have very low frequency of mutation in the 1000 Genomes, gnomAD exomes and East Asian gnomAD exomes individuals(≤ 0.004). Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to design the primers contain the rs771970145 and rs35179392 (Table 1). Next, PCR was used to amplify and sanger sequence was performed on three people(Ⅰ-1,Ⅰ-2,Ⅱ-1) of the pedigree to affirm GDNF and GUCY2C mutation. We found that the rare mutation of GUCY2C gene was carried by the father of the patient, but the common mutation of GDNF gene was carried by mother (Fig. 2, Table 2).
We identified DGNF and GUCY2C were heterozygous mutations, respectively. According to the evidence classification of ACMG pathogenicity, GDNF belongs to PS1 + PS4 + PM1 + PM2 + PP3, GUCY2C belongs to PM2 + PP2 + PP3. These two mutations were classified as variants of uncertain significance, but computer projections predicted them to be damaging or probably damaging by SIFT, Polyphen2, MetaSVM, MCAP and Mutation Taster (Table 3).
In order to confirm the mutation of mutant gene in healthy population, 74 healthy control samples were collected from physical examination center of XiangYang City First People's Hospital to detect mutant gene. We successfully amplified GDNF rs771970145 gene of 74 samples and GUCY2C rs35179392 of 64 samples by PCR and sanger sequence were used. No rare allele carrier was found at either locus of each sample (Fig. 2, The full sanger sequence results in the Additional file).

Discussion And Conclusion
Most HSCR is caused by abnormal RET gene. About the RET/GDNF/GFRA1 Signalling Pathway, The RET proto-oncogene was identified as the causative gene for HSCR. In addition, there are human papillary thyroid carcinoma and multiple endocrine neoplasia (MEN) types 2A and 2B [25][26][27][28]. One of the ligands for RET is GDNF, which connect to RET via GDNF family receptor α 1(GFRα1). When the ligand and receptor have associated, RET was recruited to cholesterol-rich areas on the cell membrane where it undergoes autophosphorylation, and activation of downstream effector pathways [29]. It was found that mice with homozygous mutation of GDNF gene showed deficiency of kidney and ENS, had similar phenotypes with RET knockout mice. [30,31]. These studies show that RET/GDNF/GFRA1 signaling Pathway played a crucial role in the development of ENS and renal agenesis or dysplasia.
In our case, the GDNF mutation was inherited from the patient's phenotypically normal mother. But she is prone to constipation. One possibility is that is a chance occurrence, the GDNF mutation does not contribute to the patient's phenotype. We consider this to be unlikely for several reasons: First, this change was not found in 74 control samples from the same race and area, but also not found in other 203 cases [32]. From these, we argue against the existence of missense polymorphisms. Second, Arg93Gln substitutes, different charge properties of two amino acids. Thirdly, this change is found in the mature peptide between the presumptive proteolytic processing side and the seven canonical TGF-B cysteine residues. The last is that heterozygous mutation of GDNF gene lead to the haploinsufficiency. In the study of a large population of mice heterozygous for GDNF null mutation, there are obvious absence of intestinal ganglion cells. This Gdnf +/− mutant cohort recapitulates complex features characteristic of HSCR, including dominant inheritance, incomplete penetrance, and variable severity of symptoms, HSCR-like phenotypes. As many as one fifth Gdnf +/− mutant mice die shortly after birth [33]. Moreover, this residue is conserved in human, mouse and rat. The mutation of the same amino acid arginine site has been reported many times before in HSCR patients [32,34,35].
Genetic variation in the GDNF promoter affects its expression and modifies the severity of HSCR in rats carrying Ednrb mutations [36]. In addition, Michael A. et al. found in 33 stillborn fetuses that had bilateral or unilateral renal agenesis existed the mutations of gene RET, GDNF and GFRA1 [37]. It may be that in many cases GDNF homozygous mutations cause early fetal demise, GDNF heterozygous mutations Increased risk of non cystic fibrosis meconium ileus (CF-MI). From these studies, we boldly infer that a heterozygous mutation will not sufficiently lead to HSCR and related phenotypes, but will certainly associate with it and increase the possibility of HSCR occurrence. The other gene GUCY2C mutation was inherited from the patient's phenotypically normal father. We focused on the role of GUCY2C in intestinal secretion. When GUCY2C gene is over activated, resulting in elevated levels of cGMP, that elicits a signaling cascade, activated Type Ⅱ cGMP dependent protein kinase(PKGII) and the cystic fibrosis transmembrane conductance regulator(CFTR), Eventually leading to secretory diarrhea [38,39]. On the other hand, It has also been reported that the compound heterozygous mutation of GUCY2C is related to non CF-MI [40]. There are research findings when the activity of GUCY2C is reduced or lost, the expression of cGMP will not be up-regulated, and the downstream protein kinase PKGII will not be activated, ultimately causing intestinal secretion dysfunction and non CF-MI [41]. Based on previous studies and our findings, we boldly speculate that GUCY2C activity in a balanced state plays a very important role in intestinal secretion. Its over activation will lead to diarrhea, on the contrary, it will lead to non CF-MI. Different domain mutations have different effects on the function of proteins, which lead to different disease phenotypes. Whether CUCY2C plays a role in the development of intestinal neurons remains to be further studied. Heterozygous for GDNF causes absence or dysfunction of intestinal neuron cell and Heterozygous for GUCY2C causes abnormal intestinal mucosal secretion. Our case suggests that under certain conditions, monogenic diseases may be caused by heterozygous mutations of two genes.
In conclusion, our study found that GDNF and GUCY2C together lead to the occurrence of HSCR. This finding expands the genotype spectrum and increases knowledge on the genetic molecular mechanisms of the HSCR. We provide a new perspective for the pathogenesis of HSCR. We should pay attention to this mutation in pre-pregnancy screening and prenatal diagnosis.     Figure 2 The Sanger sequence of two pathogenic mutations, PCR was used to amplify and sanger sequence was performed. a: GDNF and GUCY2C was sequenced in three people(Ⅰ-1,Ⅰ-2,Ⅱ-1) of the pedigree to affirm mutation. The mother carry the GDNF mutation and the father carry the GUCY2C mutation. b: 74 healthy control samples were collected to sequence GDNF and GUCY2C mutation site. No rare allele carrier was found.

Supplementary Files
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