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. Author manuscript; available in PMC: 2013 Mar 14.
Published in final edited form as: Am J Med Genet A. 2008 Apr 1;146(7):934–936. doi: 10.1002/ajmg.a.32207

Infrequency of Two Deletion Mutations at the DFNB1 Locus in Patients and Controls

Hsiao-Yuan Tang 1, Monica J Basehore 2,3, Gregory L Blakey 2,3,4, Sandra Darilek 3, John S Oghalai 1, Benjamin B Roa 2,3, Ping Fang 2,3, Raye Lynn Alford 1,*
PMCID: PMC3597119  NIHMSID: NIHMS447912  PMID: 18324688

To the Editor

Mutations in GJB2, encoding the gap junction beta-2 protein Connexin 26, are associated with autosomal recessive nonsyndromic sensorineural hearing loss (NSHL), autosomal dominant NSHL, and several forms of syndromic sensorineural hearing loss [Denoyelle et al., 1997, 1998; Kelsell et al., 1997; Richard et al., 1998, 2002, 2004; Maestrini et al., 1999; van Geel et al., 2002; van Steensel et al., 2002; Brown et al., 2003]. Mutations in GJB6, encoding the gap junction beta-6 protein Connexin 30, are associated with autosomal recessive NSHL, autosomal dominant NSHL, and hidrotic ectodermal dysplasia [Grifa et al., 1999; Lamartine et al., 2000; Lerer et al., 2001; Pallares-Ruiz et al., 2002; del Castillo et al., 2002].

GJB2 and GJB6 lie adjacent to one another on human chromosome 13q12 and together comprise the autosomal recessive NSHL locus DFNB1 [Van Camp and Smith, 2007]. Autosomal recessive NSHL that maps to DFNB1 is caused by biallelic mutations in GJB2, biallelic mutations in GJB6, or compound heterozygous mutations in GJB2 and GJB6 [Denoyelle et al., 1997; Kelsell et al., 1997; Lerer et al., 2001; Pallares-Ruiz et al., 2002; del Castillo et al., 2002; Del Castillo et al., 2005]. DFNB1 mutations reported to date include more than 80 mutations in GJB2 and two large deletions involving GJB6 [Lerer et al., 2001; Pallares-Ruiz et al., 2002; del Castillo et al., 2002; Del Castillo et al., 2005; Ballana et al., 2007]. In this study, a cohort of hearing impaired patients and a multi-ethnic control group were evaluated for mutations in GJB2 and the two known deletions involving GJB6.

Patients were identified through the Bobby R. Alford Department of Otolaryngology—Head and Neck Surgery, the Department of Molecular and Human Genetics, and the Baylor DNA Diagnostic Laboratory of Baylor College of Medicine (BCM) as previously described [Tang et al., 2005, 2006]. The patient group is comprised of 324 probands undergoing clinical evaluation for hearing loss. The nature, degree, laterality and age of onset of the hearing loss in patients were not always known. Information about the ethnicity of patients was not always provided. Controls were obtained from the Baylor Polymorphism resource (http://www.cardiogene.org) as previously described [Tang et al., 2005, 2006]. The control group used in this study includes 63 Caucasian, 74 Hispanic, 72 Asian, and 71 African American individuals. The hearing status of the controls is not known. This work was approved by the BCM Institutional Review Board.

Patients and controls were evaluated for DNA sequence variations in GJB2 as previously described [Tang et al., 2005, 2006] and for deletions in GJB6 using a triplex PCR test based on the assay of Del Castillo et al. [2005]. The triplex PCR test incorporated the following primers from Del Castillo et al. [2005]: GJB6-1R and BKR-1 to amplify across the breakpoints of the 309 kb deletion; and, Cx30Ex1A and Cx30Ex1B to amplify GJB6 exon 1. To complete the triplex PCR reaction and amplify across the breakpoints of the 232 kb deletion, primers were designed with sequences 5′-TAGATACGCTTAGCTTCTGCAG-3′ (delBK-1A) and 5′-GTCAGTGTGCATGTGAAAA-GATG-3′ (delBK-1B). PCR was conducted with 70 ng genomic DNA, 15 pmol of primers GJB6-1R, BKR-1, delBK-1A and delBK-1B, 10 pmol of primers Cx30Ex1A and Cx30Ex1B, 0.25 mM each dNTP, 1.25 U Taq DNA Polymerase (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and 1 × PCR Buffer as provided by the manufacturer, in a total volume of 20 μl. PCR was conducted as follows: 94°C × 2 min; 40 cycles of 94°C × 30 sec, 58°C × 30 sec, and 70°C × 1 min; and 70°C × 5 min. PCR conditions were modified slightly by the Baylor DNA Diagnostic Laboratory to facilitate incorporation of the assay into the laboratory work flow. PCR reactions were resolved on 1% agarose gels and visualized by ethidium bromide staining and Polaroid photography.

The GJB2 genotypes of 135 of the patients and all 280 controls in this study were previously reported [Brown et al., 2003; Tang et al., 2005, 2006] and are summarized in Table I, together with the GJB2 genotypes of the additional 189 patients included in this study. Many patients included in this study were selected because they carried mutations or variants of unknown or controversial clinical significance in GJB2.

TABLE I.

Summary of GJB2 Genotypes

GJB2 genotypea Patients Controls
Wild typeb 225 200
One or more benign polymorphisms 43 62
One or more variants of unknown/controversial clinical significance 16 14d
Single pathogenic mutation 32c 4
Two pathogenic mutations 8 0
Total 324 280
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a

Except for the IVS1 + 1G >A mutation (alternatively described as U43932.1:g.1632G >A or c.-23 + 1G >A) only coding region sequence variations are included.

b

Compared to reference sequence: GenBank Accession Numbers M86849, U43932, and XM_007169.

c

One of these 32 patients carries a heterozygous de novo mutation presumed to be dominant [Brown et al., 2003]. Of the remaining 31 patients, 3 also carry a variant of unknown/controversial clinical significance and 7 also carry one or more benign polymorphisms.

d

Seven of these 14 controls also carry one or more benign polymorphisms.

Among the 648 patient and 560 control chromosomes analyzed in this study, the 232 kb deletion involving GJB6 [Del Castillo et al., 2005] was not found. The 309 kb deletion involving GJB6 [Lerer et al., 2001; Pallares-Ruiz et al., 2002; del Castillo et al., 2002] was observed only once, in a patient of unknown ethnicity who is heterozygous for a p.Gln57X (p.Q57X) mutation in GJB2 (data not shown). In this patient, detection of heterozygous mutations in GJB2 and GJB6 supports an etiologic diagnosis of DFNB1-associated hereditary hearing loss.

These data support and extend the findings of prior studies which have suggested that the 232 and 309 kb deletion mutations in GJB6 may not be common in all populations. It is likely that founder effects, population stratification, and assortative mating at least partially explain the variable frequency of these deletions in different populations [Lerer et al., 2001; Pallares-Ruiz et al., 2002; del Castillo et al., 2002; Fitzgerald et al., 2004; Gualandi et al., 2004; Roux et al., 2004; Dalamon et al., 2005; Del Castillo et al., 2005; Seeman et al., 2005].

It is curious that prior studies have consistently demonstrated larger than expected numbers of heterozygous GJB2 mutation carriers in patient cohorts [Estivill et al., 1998; Green et al., 1999; Roux et al., 2004; Hutchin et al., 2005; Tang et al., 2006; Putcha et al., 2007]. Although some hearing impaired patients would be expected to be GJB2 carriers by chance, the cumulative data support the existence of additional, as yet unknown, mutations at the DFNB1 locus. Alternatively, it is possible that carriers of recessive mutations in GJB2 are at an increased risk for hearing loss from other causes [Estivill et al., 1998; Green et al., 1999; Roux et al., 2004; Tang et al., 2006; Putcha et al., 2007]. The observations of this and prior studies suggest that further investigation of the DFNB1 locus is needed. Such studies may ultimately permit etiologic diagnoses for many patients currently lacking an explanation for their hearing loss.

Acknowledgments

Grant sponsor: Allbritton-Alford Fund; Grant sponsor: Alkek Foundation; Grant sponsor: Brown Foundation.

The authors thank Laura Molinari, Susan D. Fernbach, John W. Belmont, and the Baylor Polymorphism Resource (http://www.cardiogene.org) for the control population used in this work; Ignacio del Castillo and Heidi Rehm for positive controls for the deletion assays; Weihong Jin for technical support; and Aletta Moore for critical review of this manuscript. This work was supported by the Allbritton-Alford Fund, the Alkek Foundation, and the Brown Foundation (HYT, RLA).

References

  1. Ballana E, Ventayol M, Rabionet R, Gasparini P, Estivill X. [Accessed August 3 2007];Connexins and Deafness Homepage. 2007 Available at: http://www.crg.es/deafness.
  2. Brown CW, Levy ML, Flaitz CM, Reid BS, Manolidis S, Hebert AA, Bender MM, Heilstedt HA, Plunkett KS, Fang P, Roa BB, Chung P, Tang HY, Richard G, Alford RL. A novel GJB2 (connexin 26) mutation, F142L, in a patient with unusual mucocutaneous findings and deafness. J Invest Dermatol. 2003;121:1221–1223. doi: 10.1046/j.1523-1747.2003.12550_4.x. [DOI] [PubMed] [Google Scholar]
  3. Dalamon V, Beheran A, Diamante F, Pallares N, Diamante V, Elgoyhen AB. Prevalence of GJB2 mutations and the del(GJB6-D13S1830) in Argentinean non-syndromic deaf patients. Hear Res. 2005;207:43–49. doi: 10.1016/j.heares.2005.04.012. [DOI] [PubMed] [Google Scholar]
  4. del Castillo I, Villamar M, Moreno-Pelayo MA, Del Castillo FJ, Alvarez A, Telleria D, Menendez I, Moreno F. A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. N Engl J Med. 2002;346:243–249. doi: 10.1056/NEJMoa012052. [DOI] [PubMed] [Google Scholar]
  5. Del Castillo FJ, Rodriguez-Ballesteros M, Alvarez A, Hutchin T, Leonardi E, de Oliveira CA, Azaiez H, Brownstein Z, Avenarius MR, Marlin S, Pandya A, Shahin H, Siemering KR, Weil D, Wuyts W, Aguirre LA, Martin Y, Moreno-Pelayo MA, Villamar M, Avraham KB, Dahl HH, Kanaan M, Nance WE, Petit C, Smith RJ, Van Camp G, Sartorato EL, Murgia A, Moreno F, del Castillo I. A novel deletion involving the connexin-30 gene, del(GJB6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 nonsyndromic hearing impairment. J Med Genet. 2005;42:588–594. doi: 10.1136/jmg.2004.028324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Denoyelle F, Weil D, Maw MA, Wilcox SA, Lench NJ, Allen-Powell DR, Osborn AH, Dahl HH, Middleton A, Houseman MJ, Dode C, Marlin S, Boulila-ElGaied A, Grati M, Ayadi H, BenArab S, Bitoun P, Lina-Granade G, Godet J, Mustapha M, Loiselet J, El-Zir E, Aubois A, Joannard A, Levilliers J, Garabedian E-N, Mueller RF, McKinlay Gardner RJ, Petit C. Prelingual deafness: High prevalence of a 30delG mutation in the connexin 26 gene. Hum Mol Genet. 1997;6:2173–2177. doi: 10.1093/hmg/6.12.2173. [DOI] [PubMed] [Google Scholar]
  7. Denoyelle F, Lina-Granade G, Plauchu H, Bruzzone R, Chaib H, Levi-Acobas F, Weil D, Petit C. Connexin 26 gene linked to a dominant deafness. Nature. 1998;393:319–320. doi: 10.1038/30639. [DOI] [PubMed] [Google Scholar]
  8. Estivill X, Fortina P, Surrey S, Rabionet R, Melchionda S, D’Agruma L, Mansfield E, Rappaport E, Govea N, Mila M, Zelante L, Gasparini P. Connexin-26 mutations in sporadic and inherited sensorineural deafness. Lancet. 1998;351:394–398. doi: 10.1016/S0140-6736(97)11124-2. [DOI] [PubMed] [Google Scholar]
  9. Fitzgerald T, Duva S, Ostrer H, Pass K, Oddoux C, Ruben R, Caggana M. The frequency of GJB2 and GJB6 mutations in the New York State newborn population: Feasibility of genetic screening for hearing defects. Clin Genet. 2004;65:338–342. doi: 10.1111/j.1399-0004.2004.00233.x. [DOI] [PubMed] [Google Scholar]
  10. Green GE, Scott DA, McDonald JM, Woodworth GG, Sheffield VC, Smith RJ. Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness. JAMA. 1999;281:2211–2216. doi: 10.1001/jama.281.23.2211. [DOI] [PubMed] [Google Scholar]
  11. Grifa A, Wagner CA, D’Ambrosio L, Melchionda S, Bernardi F, Lopez-Bigas N, Rabionet R, Arbones M, Monica MD, Estivill X, Zelante L, Lang F, Gasparini P. Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus. Nat Genet. 1999;23:16–18. doi: 10.1038/12612. [DOI] [PubMed] [Google Scholar]
  12. Gualandi E, Ravani A, Berto A, Burdo S, Trevisi P, Ferlini A, Martini A, Calzolari E. Occurrence of del(GIB6-D13S1830) mutation in Italian non-syndromic hearing loss patients carrying a single GJB2 mutated allele. Acta Otolaryngol Suppl. 2004;552:29–34. doi: 10.1080/03655230410017166. [DOI] [PubMed] [Google Scholar]
  13. Hutchin T, Coy NN, Conlon H, Telford E, Bromelow K, Blaydon D, Taylor G, Coghill E, Brown S, Trembath R, Liu XZ, Bitner-Glindzicz M, Mueller R. Assessment of the genetic causes of recessive childhood non-syndromic deafness in the UK—Implications for genetic testing. Clin Genet. 2005;68:506–512. doi: 10.1111/j.1399-0004.2005.00539.x. [DOI] [PubMed] [Google Scholar]
  14. Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, Mueller RF, Leigh IM. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature. 1997;387:80–83. doi: 10.1038/387080a0. [DOI] [PubMed] [Google Scholar]
  15. Lamartine J, Munhoz EG, Kibar Z, Lanneluc I, Callouet E, Laoudj D, Lemaitre G, Hand C, Hayflick SJ, Zonana J, Antonarakis S, Radhakrishna U, Kelsell DP, Christianson AL, Pitaval A, Der Kaloustian V, Fraser C, Blanchet-Bardon C, Rouleau GA, Waksman G. Mutations in GJB6 cause hidrotic ectodermal dysplasia. Nat Genet. 2000;26:142–144. doi: 10.1038/79851. [DOI] [PubMed] [Google Scholar]
  16. Lerer I, Sagi M, Ben-Neriah Z, Wang T, Levi H, Abeliovich D. A deletion mutation in GJB6 cooperating with a GJB2 mutation in trans in non-syndromic deafness: A novel founder mutation in Ashkenazi Jews. Hum Mutat. 2001;18:460. doi: 10.1002/humu.1222. [DOI] [PubMed] [Google Scholar]
  17. Maestrini E, Korge BP, Ocana-Sierra J, Calzolari E, Cambiaghi S, Scudder PM, Hovnanian A, Monaco AP, Munro CS. A missense mutation in connexin26, D66H, causes mutilating keratoderma with sensorineural deafness (Vohwinkel’s syndrome) in three unrelated families. Hum Mol Genet. 1999;8:1237–1243. doi: 10.1093/hmg/8.7.1237. [DOI] [PubMed] [Google Scholar]
  18. Pallares-Ruiz N, Blanchet P, Mondain M, Claustres M, Roux AF. A large deletion including most of GJB6 in recessive non syndromic deafness: A digenic effect? Eur J Hum Genet. 2002;10:72–76. doi: 10.1038/sj.ejhg.5200762. [DOI] [PubMed] [Google Scholar]
  19. Putcha GV, Bejjani BA, Bleoo S, Booker JK, Carey JC, Carson N, Das S, Dempsey MA, Gastier-Foster JM, Greinwald JH, Jr, Hoffmann ML, Jeng LJ, Kenna MA, Khababa I, Lilley M, Mao R, Muralidharan K, Otani IM, Rehm HL, Schaefer F, Seltzer WK, Spector EB, Springer MA, Weck KE, Wenstrup RJ, Withrow S, Wu BL, Zariwala MA, Schrijver I. A multicenter study of the frequency and distribution of GJB2 and GJB6 mutations in a large North American cohort. Genet Med. 2007;9:413–426. doi: 10.1097/gim.0b013e3180a03276. [DOI] [PubMed] [Google Scholar]
  20. Richard G, White TW, Smith LE, Bailey RA, Compton JG, Paul DL, Bale SJ. Functional defects of Cx26 resulting from a heterozygous missense mutation in a family with dominant deaf-mutism and palmoplantar keratoderma. Hum Genet. 1998;103:393–399. doi: 10.1007/s004390050839. [DOI] [PubMed] [Google Scholar]
  21. Richard G, Rouan F, Willoughby CE, Brown N, Chung P, Ryynanen M, Jabs EW, Bale SJ, DiGiovanna JJ, Uitto J, Russell L. Missense mutations in GJB2 encoding connexin-26 cause the ectodermal dysplasia keratitis-ichthyosis-deafness syndrome. Am J Hum Genet. 2002;70:1341–1348. doi: 10.1086/339986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Richard G, Brown N, Ishida-Yamamoto A, Krol A. Expanding the phenotypic spectrum of Cx26 disorders: Bart-Pumphrey syndrome is caused by a novel missense mutation in G JB2. J Invest Dermatol. 2004;123:856–863. doi: 10.1111/j.0022-202X.2004.23470.x. [DOI] [PubMed] [Google Scholar]
  23. Roux AF, Pallares-Ruiz N, Vielle A, Faugere V, Templin C, Leprevost D, Artieres F, Lina G, Molinari N, Blanchet P, Mondain M, Claustres M. Molecular epidemiology of DFNB1 deafness in France. BMC Med Genet. 2004;5:5. doi: 10.1186/1471-2350-5-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Seeman P, Bendova O, Raskova D, Malikova M, Groh D, Kabelka Z. Double heterozygosity with mutations involving both the GJB2 and GJB6 genes is a possible, but very rare, cause of congenital deafness in the Czech population. Ann Hum Genet. 2005;69:9–14. doi: 10.1046/j.1529-8817.2003.00120.x. [DOI] [PubMed] [Google Scholar]
  25. Tang HY, Xia A, Oghalai JS, Pereira FA, Alford RL. High frequency of the IVS2-2A>G DNA sequence variation in SLC26A5, encoding the cochlear motor protein prestin, precludes its involvement in hereditary hearing loss. BMC Med Genet. 2005;6:30. doi: 10.1186/1471-2350-6-30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tang HY, Fang P, Ward PA, Schmitt E, Darilek S, Manolidis S, Oghalai JS, Roa BB, Alford RL. DNA sequence analysis of GJB2, encoding connexin 26: Observations from a population of hearing impaired cases and variable carrier rates, complex genotypes, and ethnic stratification of alleles among controls. Am J Med Genet Part A. 2006;140A:2401–2415. doi: 10.1002/ajmg.a.31525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van Camp G, Smith R. [Accessed August 7, 2007];Hereditary Hearing Loss Homepage. 2007 Available at: http://webhost.ua.ac.be/hhh/
  28. van Geel M, van Steensel MA, Kuster W, Hennies HC, Happle R, Steijlen PM, Konig A. HID and KID syndromes are associated with the same connexin 26 mutation. Br J Dermatol. 2002;146:938–942. doi: 10.1046/j.1365-2133.2002.04893.x. [DOI] [PubMed] [Google Scholar]
  29. van Steensel MA, van Geel M, Nahuys M, Smitt JH, Steijlen PM. A novel connexin 26 mutation in a patient diagnosed with keratitis-ichthyosis-deafness syndrome. J Invest Dermatol. 2002;118:724–727. doi: 10.1046/j.1523-1747.2002.01735.x. [DOI] [PubMed] [Google Scholar]

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