Novel Nonsense Mutation of the Endothelin-BReceptor Gene in a Family WithWaardenburg-Hirschsprung Disease

Petros Syrris,* Nicholas D. Carter, and Michael A. PattonMedical Genetics Unit, St. George’s Hospital Medical School, London, United Kingdom

Waardenburg syndrome (WS) comprisessensorineural hearing loss, hypopigmenta-tion of skin and hair, and pigmentary dis-turbances of the irides. Four types of WShave been classified to date; in WS type IV(WS4), patients additionally have colonicaganglionosis (Hirschsprung disease,HSCR). Mutations in the endothelin-3(EDN3), endothelin-B receptor (EDNRB),and Sox10 genes have been identified ascausative for WS type IV. We screened a fam-ily with a combined WS-HSCR phenotypefor mutations in the EDNRB locus usingstandard DNA mutation analysis and se-quencing techniques. We have identified anovel nonsense mutation at codon 253(CGA→TGA, Arg→STOP). This mutationleads to a premature end of the translationof EDNRB at exon 3, and it is predicted toproduce a truncated and nonfunctional en-dothelin-B receptor. All affected relativeswere heterozygous for the Arg253→STOPmutation, whereas it was not observed inover 50 unrelated individuals used as con-trols. These data confirm the role of EDNRBin the cause of the Waardenburg-Hirsch-sprung syndrome and demonstrate that inWS-HSCR there is a lack of correlation be-tween phenotype and genotype and a vari-able expression of disease even within thesame family. Am. J. Med. Genet. 87:69–71,1999. © 1999 Wiley-Liss, Inc.

KEY WORDS: Waardenburg syndrome;Hirschsprung disease; endo-thelin-B receptor gene; non-sense mutation

INTRODUCTION

Waardenburg syndrome (WS) is an autosomal dom-inant malformation syndrome with an estimated inci-dence of 1/50,000 live births. It comprises congenitalsensorineural hearing loss, pigmentary abnormalitiesof the hair and irides, and dystopia canthorum. Minorcriteria include congenital abnormalities in skin pig-mentation, synophyrys, and hypoplasia of alae nasi[Read and Newton, 1997]. However, WS patients ex-hibit a remarkable degree of phenotypic variability andrarely have all the manifestations of the syndromeeven within families. Four types of WS have been de-scribed based on the presence of certain manifesta-tions. For example, dystopia canthorum is the mostpenetrant trait in Waardenburg syndrome type I (WS1)but it is absent in type II (WS2). A number of mutationsin the PAX3 and the MITF (microphthalmia-associatedtranscription factor) genes cause WS1, WS2, and WS3[Tassabehji et al., 1992, 1994; Hoth et al., 1993].

Waardenburg syndrome type IV (WS4) combines WSand colonic aganglionosis (Hirschsprung disease,HSCR) and is also known as Shah-Waardenburg syn-drome (MIM #277580) [Shah et al., 1981]. Hirsch-sprung disease is a genetic disorder with an incidenceof 1/5,000 live births. It is caused by the absence ofintrinsic ganglion cells in the distal colon, which leadsto intestinal obstruction in new-born infants [Badneret al., 1990]. Mutations in the endothelin-B receptorgene (EDNRB), encoding a G-protein-coupled receptorprotein, and in the gene for its ligand, endothelin-3(EDN3) have been found to cause WS4 [Puffenberger etal., 1994; Chakravarti, 1996; Kusafuka et al., 1996;Amiel et al., 1996; Attie et al., 1995; Edery et al., 1996].More recently, mutations in the Sox10 gene have alsobeen identified in WS-HSCR patients [Pingault et al.,1998].

In this study, we investigated a family with WS4. Wereport a novel nonsense mutation in EDNRB carried ina heterozygous state by all affected individuals.

MATERIALS AND METHODSClinical Analysis

Individuals (II-1 and II-2) were referred to our unitfor consultation regarding their unborn child III-3. The

*Correspondence to: Petros Syrris, Ph.D., Medical GeneticsUnit, St. George’s Hospital Medical School, London, SW17 0RE,UK. E-mail: p.syrris@sghms.ac.uk

Received 7 April 1999; Accepted 30 June 1999

American Journal of Medical Genetics 87:69–71 (1999)

© 1999 Wiley-Liss, Inc.

family is of Afro-Caribbean origin and has a history ofsensorineural deafness, heterochromia, and Hirsch-sprung disease (Fig. 1). In particular, individual II-2 isdeaf in one ear, is short sighted, and has heterochromia(one eye is blue and the other is brown). Her brother(II-4) has bilateral sensorineural deafness with com-plete heterochromia, whereas her sister (II-3) is deaf inone ear and also has blue irides. Her niece (III-4) is deafand has normal brown eyes. III-2 has heterochromiaand long segment Hirschsprung disease, but his hear-ing is normal; III-1 has segmental heterochromia, andIII-3 was also born with heterochromia but no othermanifestations of the disease. Interestingly, synophy-rys, hair or skin hypopigmentation, and dystopia can-thorum are absent in this family.

Mutation Analysis

A sample of venous blood was taken from individualsII-1, II-2, III-1, III-2, and III-3; no other members of thefamily were available for blood sampling. DNA was ex-tracted by conventional methods, and all exons of theEDNRB gene were amplified by polymerase chain re-action (PCR) using primers described before [Kusafukaet al., 1997]. Single strand conformational polymor-phism (SSCP) analysis on nondenaturing acrylamidegels was performed at two different temperatures (4°Cand room temperature). PCR products showing an ab-errant SSCP pattern were sequenced using an ABIPRISM 377 DNA sequencer (PE Applied Biosystems,Foster City, CA) according to the manufacturers’ in-structions.

RESULTS

Mutational analysis of the EDNRB gene in this fam-ily demonstrated the presence of a novel nonsense mu-tation in exon 3 (Fig. 2). All affected members of thefamily were heterozygous for a C→T transition at

codon 253 (CGA→TGA). This mutation results in thesubstitution of an arginine with a termination codon(Arg→STOP). Unaffected individual II-1 was homozy-gous for the wild-type codon (CGA), whereas theArg253→STOP mutation was not found in over 50 un-related individuals used as controls. No other muta-tions were found in the remaining six EDNRB exons.

DISCUSSION

The endothelin-B receptor gene encodes a heptaheli-cal receptor that is involved in the G-protein-mediatedintracellular signaling pathway [Sakurai et al., 1992].In this study we report a nonsense mutation in exon 3of the EDNRB gene that has not been previously ob-served. It causes a premature termination of the trans-lation of EDNRB and, therefore, the production of atruncated protein. Codon 253 is thought to be part of anextracellular loop of the EDNRB molecule between thefourth and fifth transmembrane domains [Sakamoto etal., 1993]. Therefore, the Arg253→STOP mutation re-sults in a complete loss of more than half the EDNRBprotein. It is predicted that this truncated form of theEDNRB receptor would be nonfunctional. However, allaffected individuals who were heterozygous for theArg253→STOP mutation had a variety of manifesta-tions ranging from heterochromia to Hirschsprung dis-ease, which has also been the case with other reportedmutations at the EDNRB locus [Puffenberger et al.,1994].

It has been well documented that the WS-HSCR syn-drome is characterized by incomplete penetrance andvariable phenotypic manifestations even between indi-viduals carrying the same mutation. For example,Puffenberger et al. [1994] first reported a W276C mu-tation in the EDNRB gene, which showed a lack ofpenetrance in both heterozygotes and homozygotes andvariable phenotypic features accompanied by a greaterincidence of megacolon in male than female patients.In our family, heterochromia was the principal mani-festation of the syndrome followed by sensorineuraldeafness. Interestingly, the only member of the family

Fig. 1. Pedigree of the WS-HSCR family. Individuals II-1, II-2, III-1,III-2, and III-3 were investigated for mutations in the EDNRB gene.

Fig. 2. Sequence analysis of exon 3 of the EDNRB gene demonstratingthe presence of the CGA→TGA mutation at codon 253 (Arg→STOP) inindividual III-2 compared with the wild-type sequence of a normal control.

70 Syrris et al.

with intestinal aganglionosis is male (III-2). It is spec-ulated that such a phenotypic heterogeneity would in-dicate the involvement of additional factors in the de-velopment of WS or HSCR. Therefore, it is reasonableto assume that unidentified mutations in genes in-volved in cell signaling pathways may lead to WS orHSCR phenotype, too. Indeed, another gene (endothe-lin-converting enzyme 1, ECE-1) was recently impli-cated in the cause of Hirschsprung disease [Hofstra etal., 1999].

REFERENCESAmiel J, Attie T, Jan D, Pelet A, Edery P, Bidaud C, Lacombe D, Tam P,

Simeoni J, Flori E, Nihoul-Fekete C, Munnich A, Lyonnet S. 1996.Heterozygous endothelin receptor B (EDNRB) mutations in isolatedHirschsprung disease. Hum Mol Genet 5:355–357.

Attie T, Till M, Pelet A, Amiel J, Edery P, Boutrand L, Munnich A, LyonnetS. 1995. Mutation of the endothelin-receptor B gene in Waardenburg-Hirschsprung disease. Hum Mol Genet 4:2407–2409.

Badner JA, Sieber WK, Garver KL, Chakravarti A. 1990. A genetic studyof Hirschsprung’s disease. Am J Hum Genet 46:568–580.

Chakravarti A. 1996. Endothelin receptor-mediated signalling in Hirsch-sprung’s disease. Hum Mol Genet 5:303–307.

Edery P, Attie T, Amiel J, Pelet A, Eng C, Hofstra RM, Martelli H, BidaudC, Munnich A, Lyonnet S. 1996. Mutation of the endothelin-3 gene inthe Waardenburg-Hirschsprung disease (Shah-Waardenburg syn-drome). Nat Genet 12:442–444.

Hofstra RMW, Valdenaire O, Arch E, Osinga J, Kroes H, Loffler B-M,Hamosh A, Meijers C, Buys CHCM. 1999. A loss-of-function mutationin the endothelin-converting enzyme 1 (ECE 1) associated with Hirsch-sprung disease, cardiac defects, and autonomic dysfunction. Am J HumGenet 64:304–308.

Hoth CF, Milunsky A, Lipsky N, Sheffer R, Clarren SK, Baldwin CT. 1993.Mutations in the paired domain of the human PAX3 gene cause Klein-

Waardenburg syndrome (WS-III) as well as Waardenburg syndrometype I (WS-I). Am J Hum Genet 52:455–462.

Kusafuka T, Wang Y, Puri P. 1996. Novel mutations of the endothelin Breceptor gene in isolated patients with Hirschsprung’s disease. HumMol Genet 5:347–349.

Kusafuka T, Wang Y, Puri P. 1997. Mutation analysis of the RET, theendothelin-B receptor, and the endothelin-3 genes in sporadic cases ofHirschsprung’s disease. J Pediatr Surg 32:501–504.

Pingault V, Bondurand N, Kuhlbrodt K, Goerich DE, Prehu M-O, Puliti A,Herbarth B, Hermans-Borgmeyer I, Legius E, Matthijs G, Amiel J,Lyonnet S, Ceccherini I, Romeo G, Smith JC, Read AP, Wegner M,Goossens M. 1998. Sox10 mutations in patients with Waardenburg-Hirschsprung disease. Nat Genet 18:171–173.

Puffenberger EG, Hosoda K, Washington SS, Nakao K, deWit D, Yanag-isawa M, Chakravarti A. 1994. A missense mutation of the endothe-lin-B receptor gene in multigenic Hirschsprung’s disease. Cell 79:1257–1266.

Read AP, Newton VE. 1997. Waardenburg syndrome. J Med Gen 34:656–665.

Sakamoto A, Yanagisawa M, Sawamura T, Enoki T, Ohtani T, Sakurai T,Nakao K, Toyo-oka T, Masaki T. 1993. Distinct subdomains of thehuman endothelin receptors determine their selectivity to endothelin-A-selective antagonist and endothelin-B-selective agonists. J BiolChem 268:8547–8553.

Sakurai T, Yanagisawa M, Masakai T. 1992. Molecular characterization ofendothelin receptors. Trends Pharmacol Sci 13:103–108.

Shah KN, Dalal SJ, Desai MP, Sheth PN, Joshi NC, Ambani LM. 1981.White forelock, pigmentary disorder of the irides and long segmentHirschsprung disease: possible variant of Waardenburg syndrome. JPediatr 99:432–435.

Tassabehji M, Newton VE, Read AP. 1994. Waardenburg syndrome type 2caused by mutations in the human microphthalmia (MITF) gene. NatGenet 8:251–255.

Tassabehji M, Read AP, Newton VE, Harris R, Balling R, Gruss P, Stra-chan T. 1992. Waardenburg’s syndrome patients have mutations in thehuman homologue of the Pax-3 paired box gene. Nature 355:635–636.

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