A De Novo Novel Mutation of the EDNRB Gene in a Taiwanese Boy with Hirschsprung Disease 349J Formos Med Assoc | 2006 • Vol 105 • No 4 EDNRB mutation in Hirschsprung disease Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of ganglion cells in the nerve plexuses of the lower digestive tract. Although mutations in eight different genes ( EDNRB, EDN3, ECE1, SOX10, RET,T GDNF, NTN, SIP1) have been identified in affected individuals, it is now clear that RET and EDNRB are the primary genes implicated in the etiology of HSCR. All eight genes are involved in the early development of the enteric nervous system, and most act through two distinct biochemical pathways mediated by RET aT nd EDNRB. Mutations in RET aT nd EDNRB account for up to 50% and 5% of HSCR cases in the general population, respectively. Interaction between these two signaling pathways could modify RET expression and, therefore, HSCR phenotype. Here, we report the case of a 1-year-old Taiwanese boy who presented with abdominal distension since birth and bilious vomiting after feeding. HSCR (short-segment type) was diagnosed based on X-ray, lower gastrointestinal series and biopsy findings. Mutation analysis revealed a heterozygous T>C missense mutation in exon 1 of the EDNRB gene, that substitutes the highly conserved cysteine-90 residue in the extracellular domain of the G protein-coupled receptor with an arginine residue (C90R). No RET gene mutation was detected in this patient. [J Formos Med Assoc 2006;105(4): 349–354] Key Words: EDNRB gene, Hirschsprung disease, Taiwanese Departments of Emergency Medicine and 1Surgery, National Cheng Kung University Medical College and Hospital, Tainan, Taiwan. Received: March 31, 2005 Revised: May 3, 2005 Accepted: July 5, 2005 Hirschsprung disease (HSCR, OMIM 142623), or aganglionic megacolon, is a congenital disor- der characterized by the absence of enteric gan- glia along a variable length of the intestine. The estimated incidence is approximately 1 in 5000 live births. Molecular genetic analysis has identi- fied several genes that have a role in the develop- ment of HSCR; the major susceptibility gene for this disorder is the RET proto-oncogene.1 Genes encoding functional ligands of the RET-receptor complex, such as the glial cell line-derived neu- rotropic factor (GDNF), neurturin (NTN),2 arte- min (ARTN), persephin (PSPN), and correspond- ing members of the GDNF-family receptor genes (GFR -1-4),2 have also been suggested as putative susceptibility genes associated with *Correspondence to: Dr. Ming-Che Tsai, Department of Emergency Medicine, National Cheng Kung University Hospital, 138, Sheng-Li Road, Tainan 704, Taiwan. E-mail: terence39@yahoo.com HSCR. Waardenburg-Shah syndrome is a dis- order of the embryonic neural crest that combines the clinical features of Waardenburg syndrome and HSCR.3 Patients with Waardenburg-Shah syndrome with megacolon have a homozygous founder mutation in the G-protein-coupled en- dothelin-B receptor gene (EDNRB),4 whereas het- erozygous mutations of EDNRB and endothelin- 3 (EDN3)5 have been identified in individuals with isolated HSCR. Heterozygous mutations of SOX10 have been described in patients with megacolon in Waardenburg-Shah syndrome.6 Mutation of the RET gene accounts for up to 20% of sporadic and 50% of familial cases.7 Mu- tation of the EDNRB gene accounts for 5–10% of all HSCR cases.8,9 Short-segment HSCR occurs CASE REPORT A De Novo Novel Mutation of the EDNRB Gene in a Taiwanese Boy with Hirschsprung Disease Wen-Chau Chen, Shen-Shun Chang,1 Edgar D. Sy,1 Ming-Che Tsai* ©2006 Elsevier & Formosan Medical Association 350 J Formos Med Assoc | 2006 • Vol 105 • No 4 W.C. Chen, et al Physical examination did not show any pig- mentary anomalies or deafness. X-ray examina- tion showed diffuse enlarged bowel gas with absent bowel gas in the rectal area. Lower gas- trointestinal series showed an enlarged cecum, ascending colon and ileum without focal obstruc- tion sign. Suction biopsy was performed and pathology revealed no ganglial neurons in the rectum and sigmoid colon. Acetylcholine ester- ase stain was positive. Under the impression of HSCR (short-segment type), colostomy was ar- ranged. PCR and automated DNA sequencing Genomic DNA was extracted from the peripheral blood (QIAamp Midi Kit; Qiagen, Valencia, CA, USA) of the proband and parents after obtain- ing informed consent. DNA samples were then subjected to mutation screening by amplifica- tion of segments of the RET and EDNRB genes with primers (Tables 1 and 2) synthesized on the basis of intronic sequences from Genbank. in about 25% of RET-caused cases and in more than 95% of EDNRB-related cases.8 Even in fami- lies with apparent monogenic inheritance, there is incomplete penetrance of disease-causing mutations and intra- and interfamily variation of phenotype severity, suggesting that modify- ing genetic, stochastic or environmental factors are involved. In this report, we describe the genetic analysis of the RET and EDNRB genes in a Taiwanese boy with HSCR. Case Report The proband was a 1-year-old Taiwanese boy with HSCR. He presented with abdominal dis- tension since birth and bilious vomiting after feeding. The baby was born at full term with a birth weight of 2665 g. He was the first child of this family. No other family members had a his- tory of the same symptoms/signs. Table 1. Polymerase chain reaction primers used for the amplification of the RET gene from genomic DNA Exon Forward (5’ ➔ 3’) Backward (5’ ➔ 3’) Product size (bp) AT (°C) 1 CGGCGCTTACCTCGCTTCAG TGTCCCGTTTGCTCCAGGAC 622 62 2 CAGTTCTTTTCTAGCCCGTG ATGATTCCCGTGTGTCTCCA 671 52 3 GTTTACACCAGCCCTGGAGC GCTCTGTCTGCCCCACAAGA 631 55 4 CTGTGGAGCGGAGGAGGGGA CTAGGACAGACGGCGCAGAC 534 62 5 CTGACAACACACATCTGGTC CAGAGACACAGGAAGTGCTG 481 55 6 CGTGTTTGCACCAGTGTGAG CACCCAGTCTACTCTGTGCT 401 53 7 GTTCCAGGACTTAGGCTGTG AGCCTTGCAGCTGTACTGCT 449 53 8 CTGGCACTGTCTTTGCTGCC CTCACAAGCCCTCTCCCAAG 468 55 9 CTCCTCTCCCATAAGCCATG GAACTGACAGCCCTGGCAAC 391 55 10 CAGAAAGGCACTGTGACCAA CAGGCTGACAAGTTGTTTGG 554 52 11 GTAAATGGCAGTACCCATGC CACAGCGCCCTATGGAAATG 592 52 12 GCAGAGACAGGCAGCGTTGC CTCGCTCTGCTTCTCTAGGC 458 55 13 CTCTCTGTCTGAACTTGGGC CAGTAGGGAAAGGGAGAAAG 312 52 14 CAGAGCTGCAGCAGTGCTGC CATGCCATGGCAGGGGCATG 508 52 15 CTGCCATGTCACACCCTGAC GTCAGTATGCTGCCAGGGAG 540 57 16 CAGGAGTGTCTACAGCACTC CATTGCAGAGGGCTAGCACT 340 53 17 CGACAGGGTCAGCAGGTGCT CTGGTTTCTCCTGGGGCTGC 341 58 18 CTTTGGAGTTGGAGACAGAG CATGACTCTCTCTCTCTGCA 361 50 19 CTGGTCTCTTGGAGAGGTCA GGTTCAGAGCAGACTTTGGT 411 50 20 CACAGAAACCACGAGTTTGG CTGCTAGGAGGGAAAATCAC 470 50 AT = annealing temperature. 351J Formos Med Assoc | 2006 • Vol 105 • No 4 EDNRB mutation in Hirschsprung disease For polymerase chain reaction (PCR) amplifi- cation, approximately 200 ng of genomic DNA, 12.8 pmol of each primer, 10 μmol dNTP and 1.25 U of Taq (Qiagen) were used in a total vol- ume of 50 μL. The amplification conditions were 94° C for 5 minutes, followed by 40 cycles of 94° C for 45 seconds, annealing temperature for 45 seconds and 72° C for 45 seconds, and extension at 72° C for 10 minutes. PCR products were puri- fied by QIAquick columns (Qiagen) and sequenced with both forward and backward primers (377 ABI Advanced Biotechnologies, Columbia, MD, USA). Automated DNA sequencing of the EDNRB gene revealed a heterozygous T to C transition of codon 90 in exon 1 (Figure), which predicted a substitution of cysteine by arginine (C90R). This mutation was confirmed with backward primer for exon 1. Neither of the parents had this mu- tation. The mutation created a new restriction en- zyme site (AciI). The mutation was absent in 100 normal unrelated Taiwanese controls by restric- tion fragment analysis, indicating that it was not a polymorphism. No mutation was detected in the RET gene of this patient. Discussion HSCR is a frequent neurocristopathy10 character- ized by the absence of submucosal and myenteric plexus in a variable length of the gastrointestinal tract. In the vast majority of cases (80%), the agan- glionic tract involves the rectum and the sigmoid colon only (short-segment HSCR), while in 20% of cases, it extends towards the proximal end of the colon.11 Although 80% of cases are sporadic, pedigree and segregation analyses suggested the involvement of one or several dominant genes with low penetrance in HSCR.12 A major HSCR gene has been mapped to chromosome 10q11.2, and the disease has been ascribed to mutations in the RET proto-oncogene,T 1,13–16 which encodes a re- ceptor tyrosine kinase. However, the lack of geno- type–phenotype correlations, the low penetrance and the sex-dependent effect of RET mutations supported the existence of one or more modifier gene(s) in familial HSCR.7,17 Puffenberger et al reported evidence that HSCR type 2 (HSCR2; OMIM 600155), an apparently multigenic dis- order, is due to mutations in EDNRB.4 Endothelin (EDN) is a potent vasoactive pep- tide, which can induce a wide range of cellular and physiologic responses.18 In mammalian cells, there are at least two EDN receptor subtypes, EDNRA and EDNRB,19 both of which belong to the superfamily of rhodopsin-like G-protein- coupled receptors (GPCRs)20 that contain seven Table 2. PCR primers used for the amplification of the EDNRB gene from genomic DNA Exon Forward (5’ ➔ 3’) Backward (5’ ➔ 3’) Product size (bp) AT (°C) 1 CTCTGCTTGTCTCTAGGCTC GATTCAGTAGGTCTGGGGTG 881 55 2,3 GTGATACAATTCAGAGGGCA CACTGAGATCAAGGGGATTC 734 50 4 CAGTAAGTGTGGCCTGAAAG GTGAAGTGGAACCGAAGTGA 562 50 5 GATCTAGGGAGAATCAGAAC GAAGTACTGAAGCTGGCTGA 643 50 6 GCACAGAAGCTACAATGACT CTACCAAAAACAGGGAACAG 530 50 7 CAAAGAAAGTCAGAACCCTG TCCATGCCGTAAACAGCTCA 407 50 AT = annealing temperature. Figure. Automated DNA sequenc- ing of the EDNRB gene revealed a T to C transition: (A) sequence from the proband; (B) sequence from a normal control. BB A 352 J Formos Med Assoc | 2006 • Vol 105 • No 4 W.C. Chen, et al transmembrane domains. The extracellular and transmembrane domains of GPCRs are involved in ligand binding, whereas the intracellular do- mains are involved in G protein coupling and subsequent effector regulation. To determine when EDNRB signaling is required during em- bryogenesis, Shin et al determined that Ednrb is required during a restricted period of neural crest development between embryonic days 10 and 12.5.21 They concluded that EDNRB is required for the migration of both melanoblasts and enteric neuroblasts. Arai et al demonstrated that the human genome contains a single copy of the EDNRB gene,22 which spans 24 kb and comprises 7 exons and 6 introns that encode a 442 amino acid protein expressed in brain, kidney, lung, heart and endothelial cells.23 Inagaki et al showed that this protein is also ex- pressed in the human colon, particularly in the myenteric plexus, mucosal layer, ganglion and blood vessels of the submucosa.24 Recently, muta- tions in the EDNRB gene have been identified in HSCR patients,8 including deletion/insertion mutations,25,26 non-sense mutations,26–28 splicing mutations29 and several missense mutations (Table 3).4,5,9,25,30–35 The mutation was dosage sensitive in that homozygotes and heterozygotes had a 74% and a 21% risk, respectively, of developing HSCR.4 Other analyses of patients in the extended Mennonite pedigree showed that HSCR is a multigenic disorder. For all clinical forms of HSCR, there is a greater incidence of megacolon in males than in females, and the same is true for the spe- cific EDNRB mutation.12 The C90R mutation seems to be significant in the pathogenesis of HSCR for several reasons: (1) it was absent in 100 normal controls, making the hypothesis of a coincidental polymorphism very unlikely; (2) it led to substitution of a hydrophilic amino acid with a polar side chain (cysteine) by Table 3. Endothelin-B receptor gene mutations in Hirschsprung disease (HSCR)/Waardenburg syndrome (WS) Mutation Location Phenotype (segment length) Genotype Reference # 169 G>A G57S EC HSCR (S) Heterozygous 30 268 T>C C90R EC HSCR Heterozygous Present case 325 T>C C109R TM I HSCR (S) Heterozygous 31 548 C>G A183G TM III HSCR/WS Homozygous 29 556 G>A G186R TM III HSCR (L)/WS Homozygous 33 601 C>T R201X IL II ABCD syndrome Homozygous 28 678 G>T W226C TM IV HSCR (S&L) Heterozygous 08 707 C>T R253X EL II HSCR (L)/WS Heterozygous 26 801+2 T>C Splicing mutation EL II HSCR (S) Heterozygous 28 824 G>A W275X TM V HSCR (S) Heterozygous 25 828 G>T W276C TM V HSCR (L&S) Heterozygous/homozygous 03 874 T>C F292L TM V HSCR (L)/WS Heterozygous 34 878insT Y293L (PTC+ 6 aa) TM V HSCR (S) Heterozygous 25 914 G>A S305N IL III HSCR (S) Heterozygous 24 928 G>A A310T IL III HSCR (S) Heterozygous 32 955 C>T R319W IL III HSCR (S) Heterozygous 30 1122 G>A M374I TM VII HSCR Heterozygous 04 1132delA N378I (PTC+ 12 aa) TM VII HSCR (S) Heterozygous 24 1148 C>T P383L TM VII HSCR (S) Heterozygous 30 1170 C>A S390R C HSCR (L) Heterozygous 31 ABCD = albinism, black lock, cell migration disorder of the neurocytes of the gut, and deafness; C = carboxyl-terminal region and adjacent to TM7; EC = extracellular domain; EL = extracellular loop; IL = intracellular loop; L = long-segment; PTC = premature termination of codon; S = short-segment; TM = transmembrane domain. 353J Formos Med Assoc | 2006 • Vol 105 • No 4 EDNRB mutation in Hirschsprung disease a basic hydrophilic amino acid (arginine); (3) this region is highly conserved between species, which probably indicates an important functional role in EDNRB signaling. 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