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GENETIC TESTING Volume 11, Number 1, 2007 © Mary Ann Liebert, Inc. DOI: 10.1089/gte.2006.9994 Allelic Frequencies of the 35delG Mutation of the GJB2 Gene in Different Brazilian Regions C.A. OLIVEIRA, 1 C.J. PIMPINATI, 1 F. ALEXANDRINO, 1 L.A. MAGNA, 2 A.T. MACIEL-GUERRA, 2 and E.L. SARTORATO 1 ABSTRACT Mutations in the GJB2 gene, which encodes the protein connexin 26, are a major cause of autosomal reces- sive deafness. The most frequent mutation, 35delG, has a carrier frequency as high as 4% in some countries, and this frequency varies in different ethnic groups. Most of the Brazilian population results from intereth- nic crosses of people from three continents (European, African, and Amerindian), and the proportion of each varies according to the geographical region of the country. To verify if the different ethnic composition of Brazilian regions leads to variable 35delG carrier frequencies, we performed the screening of the 35delG mu- tation using DNA from dried-blood filter paper samples obtained from 1,856 newborns from 10 cities in dif- ferent regions. The 35delG mutation was found in 25 individuals (1.35%), indicating an overall carrier fre- quency of 1:74. This frequency was 1:47 in the north, 1:64 in the southeast, 1:85 in the south and 1:124 in the northeast, but these differences were not significant. The overall frequency of the 35delG allele was estimated as 0.0067, and comparison between expected and observed genotype frequencies indicates that the population is in Hardy–Weinberg equilibrium. 1 INTRODUCTION I N DEVELOPED COUNTRIES, approximately 1 in 1,000 children is born with a hearing loss severe enough to require special education services, and about 60% of the cases of isolated deaf- ness have a genetic origin (Morton 1991). In Brazil, there are no official data about the prevalence of hearing impairment, ranging from 2 to 7 per 1,000, depending on the region. Most cases of hearing loss are due to environmental factors, such as congenital infections (mainly rubella), perinatal anoxia, ker- nicterus, and meningitis. However, the proportion of genetic causes tends to increase as a result of improvements in health care (Simões and Maciel-Guerra 1992; Russo 2000). It is estimated that more than 400 loci may contribute to syn- dromic and/or nonsyndromic deafness (Gorlin et al. 1995). Per- haps the most remarkable and clinically significant discovery has been the finding that mutations involving a single gene, GJB2, are the most common cause of congenital hereditary deafness in many populations. The GJB2 gene encodes the con- nexin 26 (Cx26) protein, and one specific mutation, namely 35delG, accounts for approximately 70% of GJB2 mutant alle- les in populations with a European ancestry, with a carrier fre- quency of 2.3–4% (Estivill et al. 1998; Antoniadi et al. 1999; Green et al. 1999; Storm et al. 1999). Brazilian families with nonsyndromic deafness have been an- alyzed to determine the frequency and type of GJB2 gene mu- tation. Mutations in this gene were found in 22% and 27% of the familial and sporadic cases, respectively, and the 35delG mutation accounted for 84.2% and 87.5% of the mutated alle- les, which supports the hypothesis that this mutation is not rare in the Brazilian population (Oliveira et al. 2002; Piatto et al. 2004). In a previous study performed in a city of the southeastern region of Brazil, the 35delG mutation carrier rate was 0.97% in randomly selected neonates (Sartorato et al. 2000). A more recent study revealed 2 heterozygotes among 100 Caucasian 1 Centro de Biologia Molecular e Engenharia Genética/ CBMEG, Laboratório de Genética Humana and 2 Departamento de Genética Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), São Paulo, Brazil. 1 Present address: UNICAMP/CBMEG, Cidade Universitária Zeferino Vaz s/n, Barão Geraldo, Campinas-SP 13083-970, Brazil. 2 Present address: UNICAMP/FCM, Cidade Universitária Zeferino Vaz s/n, Barão Geraldo, Campinas-SP 13083-970, Brazil.

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GENETIC TESTINGVolume 11, Number 1, 2007© Mary Ann Liebert, Inc.DOI: 10.1089/gte.2006.9994

Allelic Frequencies of the 35delG Mutation of the GJB2Gene in Different Brazilian Regions

C.A. OLIVEIRA,1 C.J. PIMPINATI,1 F. ALEXANDRINO,1 L.A. MAGNA,2

A.T. MACIEL-GUERRA,2 and E.L. SARTORATO1

ABSTRACT

Mutations in the GJB2 gene, which encodes the protein connexin 26, are a major cause of autosomal reces-sive deafness. The most frequent mutation, 35delG, has a carrier frequency as high as 4% in some countries,and this frequency varies in different ethnic groups. Most of the Brazilian population results from intereth-nic crosses of people from three continents (European, African, and Amerindian), and the proportion of eachvaries according to the geographical region of the country. To verify if the different ethnic composition ofBrazilian regions leads to variable 35delG carrier frequencies, we performed the screening of the 35delG mu-tation using DNA from dried-blood filter paper samples obtained from 1,856 newborns from 10 cities in dif-ferent regions. The 35delG mutation was found in 25 individuals (1.35%), indicating an overall carrier fre-quency of 1:74. This frequency was 1:47 in the north, 1:64 in the southeast, 1:85 in the south and 1:124 in thenortheast, but these differences were not significant. The overall frequency of the 35delG allele was estimatedas 0.0067, and comparison between expected and observed genotype frequencies indicates that the populationis in Hardy–Weinberg equilibrium.

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INTRODUCTION

IN DEVELOPED COUNTRIES, approximately 1 in 1,000 childrenis born with a hearing loss severe enough to require special

education services, and about 60% of the cases of isolated deaf-ness have a genetic origin (Morton 1991). In Brazil, there areno official data about the prevalence of hearing impairment,ranging from 2 to 7 per 1,000, depending on the region. Mostcases of hearing loss are due to environmental factors, such ascongenital infections (mainly rubella), perinatal anoxia, ker-nicterus, and meningitis. However, the proportion of geneticcauses tends to increase as a result of improvements in healthcare (Simões and Maciel-Guerra 1992; Russo 2000).

It is estimated that more than 400 loci may contribute to syn-dromic and/or nonsyndromic deafness (Gorlin et al. 1995). Per-haps the most remarkable and clinically significant discoveryhas been the finding that mutations involving a single gene,GJB2, are the most common cause of congenital hereditary

deafness in many populations. The GJB2 gene encodes the con-nexin 26 (Cx26) protein, and one specific mutation, namely35delG, accounts for approximately 70% of GJB2 mutant alle-les in populations with a European ancestry, with a carrier fre-quency of 2.3–4% (Estivill et al. 1998; Antoniadi et al. 1999;Green et al. 1999; Storm et al. 1999).

Brazilian families with nonsyndromic deafness have been an-alyzed to determine the frequency and type of GJB2 gene mu-tation. Mutations in this gene were found in 22% and 27% ofthe familial and sporadic cases, respectively, and the 35delGmutation accounted for 84.2% and 87.5% of the mutated alle-les, which supports the hypothesis that this mutation is not rarein the Brazilian population (Oliveira et al. 2002; Piatto et al.2004).

In a previous study performed in a city of the southeasternregion of Brazil, the 35delG mutation carrier rate was 0.97%in randomly selected neonates (Sartorato et al. 2000). A morerecent study revealed 2 heterozygotes among 100 Caucasian

1Centro de Biologia Molecular e Engenharia Genética/ CBMEG, Laboratório de Genética Humana and 2Departamento de Genética Médica,Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), São Paulo, Brazil.

1Present address: UNICAMP/CBMEG, Cidade Universitária Zeferino Vaz s/n, Barão Geraldo, Campinas-SP 13083-970, Brazil.2Present address: UNICAMP/FCM, Cidade Universitária Zeferino Vaz s/n, Barão Geraldo, Campinas-SP 13083-970, Brazil.

Brazilians (giving a carrier frequency of 2%) and 1 heterozy-gote among 100 Brazilians of African ancestry (1%); no 35delGcarriers were found among 107 Asian Brazilians (Oliveira etal. 2004).

Brazil, the largest country in South America, was colonizedby Portugal, but there were five centuries of interethnic crossesof people from three continents (European, African, andAmerindian), and the proportion of each varied according tothe region. For example, European ancestry is more prominentin the southern and southeastern regions and African and Na-tive American in the northern and northeastern regions. Severalstudies have been performed during recent decades in an at-tempt to characterize the genetic background of the Brazilianpopulation (Alves-Silva et al. 2000; Carvalho-Silva et al. 2001).To verify whether the different ethnic compositions found indifferent Brazilian regions leads to variable 35delG carrier fre-

quencies, we screened for the 35delG mutation in different re-gions of the country.

MATERIALS AND METHODS

Samples

A total of 1,856 random samples of newborns from 10 Brazil-ian cities from four geographic regions were obtained for theanalysis of the 35delG mutation (Fig. 1). The states of westernBrazil did not participate in the study. The protocol was ap-proved by our local Ethics Committee, and written informedconsent was obtained from the parents of all participants. A de-tailed history was obtained for each subject.

Molecular analysis

For rapid and cost-effective screening of the 35delG muta-tion, we eluted dried blood spots from 1-mm paper disks andperformed allele refractory mutation system (ARMS-PCR) us-ing primers described elsewhere (Lucotte et al. 2001), inclu-sive of a primer set of the X-Y homologous gene amelogeninas the internal reaction control (Antoniadi et al. 1999).

RESULTS

Our results regarding the 35delG mutation are shown inTable 1. The frequency of this mutation did not differ signifi-cantly differ among the samples from the four Brazilian regions(�2

(3) � 1.42; p � 0.7019); thus, all of the samples could begathered. There were 25 35delG heterozygous subjects amongthe 1,856 neonates, giving an overall carrier frequency of1.35%. The frequency of the 35delG allele was estimated as0.0067, and the frequency of 35delG/35delG deaf individualsas 0.000045 (1:22,300). Comparison between expected and ob-served genotype frequencies indicates that the population is inHardy–Weinberg equilibrium.

OLIVEIRA ET AL.2

TABLE 1. 35delG MUTATION CARRIER FREQUENCY IN BRAZILIAN REGIONS

HeterozygousRegion Frequency Town (state) individuals/n Frequency q q2

North 1/47 Belém (PA) 2/95 1/47 0.0105 0.000110(2/190) (1�9,100)

Southeast 1/64 Sorocaba (SP) 0/135 0/135S.J. Rio Preto (SP) 5/223 1/45E.S. do Pinhal (SP) 3/72 1/24Jundiaí (SP) 3/200 1/100 0.0078 0.000061Patos de Minas 2/200 1/100 (13/1,660) (1�16,400)

South 1/85 Porto Alegre (RS) 2/300 1/150 0.0059 0.000035Joinville (SC) 2/183 1/91 (8/1,366) (1�28,500)Curitiba (PR) 4/200 1/50

Northeast 1/124 Fortaleza (CE) 2/248 1/124 0.0040 0.000017(2/496) (1�59,000)

Total 25/1,856 1/74 0.0067 0.000045(25/3,712) (1�22,300)

q � frequency of the 35delG allele; q2 � expected frequency of 35 delG/35delG deaf individuals.

FIG. 1. Geographic location of Brazilian regions and cities(*) selected for study.

DISCUSSION

Mutations in the GJB2 gene account for up to 50% of prelin-gual recessive nonsyndromic deafness in populations with Eu-ropean ancestry. The 35delG mutation is the most common mu-tation of GJB2 within this ethnic group (Denoyelle et al. 1997).The method of 35delG detection is simple, reliable, and lowcost and is especially adapted to the study of large numbers ofsamples. Our results demonstrate a fairly high carrier frequencyof the 35delG mutation in Brazil, 1/74. This is lower than thatfound in Europe, probably due to the contribution of theAmerindian and Afro-Brazilian ancestral populations.

The highest carrier frequencies were expected in the south-ern and southeastern regions, which stand out with a great ma-jority of European immigrants, and not, as observed, in thenorthern region. This may be due to the fact that the samplefrom the north came from a private service, where the atten-dance is mainly of high-income families, whose ethnicity maydiffer from the population of this region as a whole. Becauseno significant differences were found between Brazilian regionsregarding the frequency of the 35delG mutation, the generalfrequency may be used, in practice, in the whole country.

According to our results, the incidence of hearing loss dueto 35delG homozygosity may be 1/22,300. Compared to otherdisorders that are currently screened country-wide through pub-licly supported programs [phenylketonuria (1/12,000), congen-ital hypothyroidism (1/2,500), cystic fibrosis (1/2,000) and he-moglobinopathies (1/1,000) (Ministério da Saúde)], the 35delGmutation should be considered for inclusion in newborn massscreening programs in Brazil. Furthermore, the genetic testwould be useful, when combined with other diagnostic meth-ods, for early diagnosis with the objective of early intervention.

ACKNOWLEDGMENTS

We would like to thank Vania Belintani Piatto fromFAMERP, Marília Fontenele e Silva from UFC, and FabianeZibermann (Maternidade Darcy Vargas) for their help and sup-port in this work.

This research was supported by grants from Fundação deAmparo à Pesquisa do Estado de São Paulo (FAPESP), Coor-denação de Aperfeiçoamento de Pessoal de Nível Superior(CAPES), and II Prêmio Mostra Puc-Rio/BR.

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Address reprint requests to:Dra. Edi Lúcia Sartorato

Laboratório de Genética HumanaUNICAMP/CBMEG

Cidade Universitária Zeferino Vaz s/nBarão Geraldo, Campinas, SP 13083-970, Brazil

E-mail: [email protected]

35delG MUTATION ALLELE FREQUENCIES IN GJB2 3