MUTATION IN BRIEF

HUMAN MUTATION Mutation in Brief #322 (2000) Online

© 2000 WILEY-LISS, INC.

Received 20 December 1999; Revised manuscript accepted 10 February 2000.

Identification of Seven Novel SNPs (FiveNucleotide and Two Amino Acid Substitutions) inthe Connexin31 (GJB3) GeneNúria López-Bigas1, Raquel Rabionet1, Elisabeth Martínez1, Isabel Banchs1, Víctor Volpini1,Jeffery M. Vance2, Maria Lourdes Arbonés1, and Xavier Estivill1

1Medical and Molecular Genetics Center-IRO, Hospital Duran i Reynals, L'Hospitalet, Barcelona; 2Department ofMedicine, Duke University Medical Center, Durham, North Carolina, USA.

*Correspondence to Xavier Estivill, Medical and Molecular Genetics Center-IRO, Hospital Duran i Reynals, Avia.Castelldefels Km 2.7, L'Hospitalet de Llobregat, 08907 Barcelona, Catalonia, Spain; E-mail: estivill@iro.es

Contract grant sponsor: Fondo de Investigaciones Sanitarias; Contract grant number: 99/10917

Communicated by Mark H. Paalman

Connexin31 (GJB3) has been associated with hearing impairment and erythrokeratodermiavariabilis. We have analyzed this gene in samples from patients with peripheralneuropathies, deafness and controls and have found several single nucleotide polymorphisms(SNPs). In the noncoding exon 1 of GJB3 two small deletions, 581del2 and 632del4(GenBank accession number AF052692), were found at frequencies of 30% and 14%,respectively. In exon 2 we found two amino acid changes, R32W (1227C-T) and V200I(1731G-A), and three nucleotide variants not affecting the amino acid sequence, 1610G-A,1700C-T and 1931C-T. Most of these changes were found at similar frequencies in patientswith deafness, patients with peripheral neuropathies and control subjects. V200I, 1700C-Tand 1610G-A were found associated in three unrelated patients with deafness and in afourth patient with peripheral neuropathy, but were not detected in control subjects. © 2000Wiley-Liss, Inc.

KEY WORDS: GJB3, Connexin31, Deafness, Peripheral Neuropathy, SNPs

INTRODUCTION

Gap junction channels permit the rapid exchange of ions, secondary messengers and small metabolites betweenneighbouring cells. Until now eleven human connexin genes have been described, several of which have beenassociated with diseases such as Charcot-Marie-Tooth (GJB1) (MIM# 304040), hearing loss (GJB2, GJB3 andGJB6) (MIM# 121011 and 603324), cataract (GJA3 and GJA8) (MIM# 121015 and 600897), erythrokeratodermiavariabilis (GJB3) and mutilating keratoderma (GJB2). The human GJB6 gene has recently been cloned and foundmutated in a family with hearing loss with partial penetrance (Grifa et al., 1999).

Human GJB3 has been described recently (Wenzel et al., 1998) and mutations in this gene have been found inpatients with dominant (Xia et al., 1998) and recessive (Liu et al., 2000) hearing impairment and in patients witherythrokeratodermia variabilis (Richard et al., 1998; Wilgoss et al., 1999). We report here the molecular analysisof GJB3 in 153 patients with deafness and 110 with peripheral neuropathy. Several single nucleotide

2 López-Bigas et al.

polymorphisms (SNPs) in the GJB3 gene were identified.

MATERIAL AND METHODS

For mutation analysis exons 1 and 2 of GJB3 were both PCR amplified in two fragments each. The sequence ofGJB3 was obtained from GenBank (accession number AF052692). The sequences of the primers used were: 5’-ctcgcaggcatcacgcatgc-3’ and 5’-actcctgccctagaccagat-3’ for the first part of exon 1, 5’-gtagtgaggcatccacaaag-3’and 5’-ctgcgagaattagaggaaaaacc-3’ for the second part of exon 1, 5’-acctattcattcatacgatgg-3’ and 5’-gagtgtgcagcaggtagagg-3’ for the first part of exon 2, and 5’-ctacctgttcagcctcatctt-3’ and 5’-cctgcatttcccattggcag-3’for the second part of exon 2. PCR was performed in a 25 µl total volume, containing 100 ng of genomic DNA, 7.5pmols of each primer, 250 µM of each dNTP, 1.5 mM MgCl2. The conditions for the reactions were: 94oC for 5min; 35 cycles of 94oC for 30 sec, 58oC for 30 sec and 72oC for 40 sec, and a final extension of 72oC for 7 min.

Variation from wild-type sequence was detected by SSCP/heteroduplex analysis, as described elsewhere (Salaand Espinosa-Parrilla, 1999). Direct automatic sequencing of variant fragments was performed with the sameprimers on an automatic genetic analyzer.

RESULTS AND DISCUSSION

Several different SSCP migrating patterns in exon 1 and 2 of GJB3 were observed in patients with peripheralneuropathies or deafness. Sequencing of the abnormal fragments showed two small deletions in the noncodingexon 1 of GJB3, 581del2 and 632del4, two missense changes in exon 2, R32W and V200I, and three nucleotidesubstitutions not affecting the amino acid sequence in exon 2, 1610G-A, 1700C-T and 1931C-T. None of thesechanges have been described previously. In these patients we found also the nucleotide polymorphism N119N,previously reported by Xia et al., (1999). In the analysis of control subjects we found similar frequencies of mostof these changes as in patients with deafness or peripheral neuropathy. Variants V200I, 1700C-T and 1610G-Awere found associated in three unrelated patients with deafness and in a fourth one with peripheral neuropathy, butwere not detected in control subjects (Table 1).

The two SNPs detected in exon 1 of GJB3 are very informative and therefore should be useful for segregationand linkage disequilibrium studies. Although mutations in exon 1 of GJB1 have been reported in families affectedof X-linked Charcot-Marie-Tooth (Ionasescu et al., 1996), the functional significance of changes in connexinnoncoding exons are unclear. To evaluate a potential involvement of these nucleotide changes in disease,segregation analyses were performed in ten families with peripheral neuropathies that were positive for one or bothchanges. Each of these changes were found in both patients and unaffected subjects of these families, and severalaffected subjects were negative for these deletions. These results exclude the implication of these two changes inthe neuropathy in our families, indicating that these variants are polymorphisms.

We included in our study normal and affected subjects of CMT2A families (Ben Othmane et al., 1993) whichare linked to 1p34. We found two individuals with 1931C-T substitution, one subject with 581del2 and one with632del4. These changes do not correlate with disease status, indicating that they are not the cause of CMT2A.

Two amino acid substitutions were identified, R32W and V200I. C→T transversion at nt 1227 results in achange (R32W) from a positive charged amino acid, arginine (CGG) to a noncharged amino acid, tryptophan(TGG), which is a highly conserved residue across connexins. G→A transversion at nt 1731 results in aconservative change (V200I) from valine (GTA) to isoleucine (ATA), located in a nonconserved position acrossconnexin31 of different species. In mouse and rat connexin 31 amino acid 200 is an isoleucine instead of a valine,which seems to indicate that this substitution does not affect functional properties of connexin. V200I was foundassociated in each individual with two nucleotide substitutions, not affecting the amino acid sequence, Y189(1700C-T) and P159 (1610G-A). All four families that were positive for the three associated changes wereascertained from different centers and are unrelated at least in five generations; however it is likely that they derivefrom a common ancestor. The possibility of a gene conversion event leading to these changes, due to homologieswith other connexins, is unlikely due to the many differences at the nucleotide level between β-connexins for theregions bearing these nucleotide changes.

Since mutations in GJB3 have been associated with deafness and erythrokeratodermia variabilis, the new SNPsdescribed here should be useful in segregation and linkage disequilibrium analysis in families affected of these andother disorders.

SNPs in Connexin31 (GJB3) 3

TABLE 1. Nucleotide and amino acid changes in GJB3 (Connexin31) in patients withperipheral neuropathy or deafness and in control subjects

GJB3 Peripheral neuropathy (%) Deafness (%) Controls (%)

581del2 33/110 (30) -* 30/103 (29)

632del4 15/110 (14) -* 14/103 (14)

1931C-T 6/110 (5) 24/153 (16) 6/46 (13)

R32W 2/110 (2) 7/153 (5) 8/46 (18)

V200I 1/110 (1) 3/153 (2) 0/46 (-)

1700C-T 1/110 (1) 3/153 (2) 0/46 (-)

1610G-A 1/110 (1) 3/153 (2) 0/46 (-)

*Exon 1 was not analyzed in deafness patients.

REFERENCES

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