July/August 2003 ⅐ Vol. 5 ⅐ No. 4 article Frequency and distribution of GJB2 (connexin 26) and GJB6 (connexin 30) mutations in a large North American repository of deaf probands Arti Pandya, MD1, Kathleen S. Arnos, PhD2, Xia J. Xia, PhD1, Katherine O. Welch, MS2, Susan H. Blanton, PhD3, Thomas B. Friedman, PhD4, Guillermina Garcia Sanchez, MD5, Xiu Z. Liu MD, PhD6, Robert Morell, PhD4, and Walter E. Nance, MD, PhD1 Purpose: Profound hearing loss occurs with a frequency of 1 in 1000 live births, half of which is genetic in etiology. The past decade has witnessed rapid advances in determining the pathogenesis of both syndromic and nonsyn- dromic deafness. The most significant clinical finding to date has been the discovery that mutations of GJB2 at the DFNB1 locus are the major cause of profound prelingual deafness in many countries.1 More recently, GJB2 mutations have been shown to cause deafness when present with a deletion of the GJB6 gene. We report on the prevalence of GJB2 and GJB6 mutations in a large North American Repository of DNA from deaf probands and document the profound effects of familial ethnicity and parental mating types on the frequency of these mutations in the population. Methods: Deaf probands were ascertained through the Annual Survey of Deaf and Hard of Hearing Children and Youth, conducted at the Research Institute of Gallaudet University. Educational, etiologic, and audiologic information was collected after obtaining informed consent. DNA studies were performed for the GJB2 and GJB6 loci by sequencing and PCR methods. Results: GJB2 mutations accounted for 22.2% of deafness in the overall sample but differed significantly among Asians, African-Americans and Hispanics and for probands from deaf by deaf and deaf by hearing matings, as well as probands from simplex and multiplex sibships of hearing parents. In our sample, the overall incidence of GJB2/GJB6 deafness was 2.57%. Conclusion: GJB2 mutations account for a large proportion of deafness in the US, with certain mutations having a high ethnic predilection. Heterozygotes at the GJB2 locus should be screened for the GJB6 deletion as a cause of deafness. Molecular testing for GJB2 and GJB6 should be offered to all patients with nonsyndromic hearing loss. Genet Med 2003:5(4):295–303. Key Words: GJB2 (connexin 26), GJB6 (connexin 30), genetic hearing loss, national DNA repository, prevalence Approximately 1 in 1000 children is born with a hearing loss Autosomal recessive transmission is found in 77% and auto- severe enough to require special education services and an- somal dominant in 22% of genetic deafness. X-linked and mi- other 1 to 2 in 1000 have a lesser but clinically significant hear- tochondrial forms are much less common in most popula- ing loss. The deafness in at least half of all infants with pro- tions.4 Recently, significant progress has been made in found hearing loss can be attributed to genetic factors,2,3 and it documenting the extreme degree of locus heterogeneity is estimated that more than 400 loci may contribute to syn- through the mapping and cloning of several dozen genes for dromic and/or nonsyndromic deafness. Nonsyndromic forms syndromic and nonsyndromic deafness. As of 2002, nearly 70 account for approximately 67% of genetic deafness, whereas a genes for nonsyndromic hearing loss have been localized and 2 specific syndrome can be identified in about 33% of cases. the protein product has been identified for approximately one- third of these.5 The pace at which additional genes are discov- 1 From the Department of Human Genetics, Medical College of Virginia, Virginia Common- ered is expected to increase in the coming years because of the wealth University, Richmond, Virginia; 2Department of Biology, Gallaudet University, Washington, DC; 3Department of Human Genetics, University of Virginia Section on Genet- availability of cochlea-specific cDNA libraries and completion ics; 4Laboratory of Molecular Genetics, National Institute on Deafness and Other Commu- of the sequencing of the human and mouse genomes.6 nication Disorders, National Institutes of Health, Bethesda, Maryland; 5Instituto de la Co- In addition to confirming locus heterogeneity, molecular 6 municacion Humana, Lomas de Plateros, Mexico; Department of Otolaryngology, studies of hereditary deafness have revealed a more complex University of Miami, Miami, Florida. pattern of inheritance in some cases. Examples of digenic deaf- Arti Pandya, MD, Department of Human Genetics, Medical College of Virginia, Virginia 7–11 Commonwealth University, PO Box 980033, Richmond, VA 23298-0033. ness, deafness secondary to gene-environment interac- 12,13 14 Received: February 6, 2003. tions, or deafness suppressed by specific modifier genes Accepted: April 30, 2003. have been documented. Future studies are likely to uncover DOI: 10.1097/01.GIM.0000078026.01140.68 additional examples of oligogenic transmission where the Genetics IN Medicine 295 Pandya et al. deafness is attributable to the combined effects of genes at sample of nearly 50,000 deaf and hard of hearing students who more than one locus and/or specific environmental influences. receive special education services because of their hearing loss. Perhaps the most remarkable and clinically significant dis- In 1997, a brief family history questionnaire was sent to the covery to date has been the finding that mutations involving a parents of about 30,000 of these students. Approximately 6,000 single gene, GJB2 (also known as connexin 26), are the most (20%) returned the questionnaire and gave permission for a common cause of congenital hereditary deafness in many pop- genetic counselor to contact them by phone. The process was ulations.1,15,16 The GJB2 gene encodes connexin 26, a compo- repeated in 2001 for students who were new to the Annual nent of gap junctions. Gap junctions are widely expressed in Survey, resulting in additional responses. Each proband is the cochlea and are thought to participate in the recycling of counted only once. In families with two or more probands, all potassium ions from hair cells to the cochlear endolymph.17 probands are counted. Allowing for multiple probands in a Mutations of GJB2 have been estimated to account for 30% to family adjusts for variation in family size and segregation ratio 40% of all cases of profound, prelingual hereditary deafness in among families. Study participants were also recruited from the United States, with a carrier frequency of 2.5% in a Mid- the student body at Gallaudet University, as well as the genetics western US population.18 Testing in many other populations clinics at the Medical College of Virginia and the Instituto de la has shown that mutations of GJB2 explain 50% to 80% of Comunicacion Humana in Lomas de Plateros, Mexico. nonsyndromic recessive deafness16,19 and 10% to 37% of deaf- Interviews to obtain family and medical history information ness of unknown cause.20,21 One particular mutation, 35delG, from deaf participants or their parents were conducted by ge- accounts for approximately 70% of all recessive mutations of netic counselors by phone (voice or telecommunication device the gene.16 The 167delT mutation has a high prevalence in the for the deaf) or in person. Open-ended interviews were con- Ashkenazi Jewish population,8 with a carrier frequency of ap- ducted to explore relevant medical information and pedigree proximately 3% to 4%. GJB2 screening has become widely data. Systematic data were collected on the age at onset, sever- available, in part because the small size of the single coding ity and progression of the hearing loss, and potentially impor- exon facilitates gene sequencing. PCR-based sequence analysis tant factors such as viral infections, antibiotic administration, has been shown to be an efficient method for identifying surgical procedures, etc. Records were collected to document pathogenic mutations in this gene and is rapidly emerging as the type, dose, and duration of relevant antibiotics use. A preg- the standard of care for the evaluation of newborn infants as nancy and birth history was also obtained. Copies of audio- well as older children and adults with nonsyndromic deafness metric studies were requested on all affected individuals in the of uncertain cause.22,23 Using this and other methods, more family. When there was evidence for progression, serial audio- widespread use of screening on a clinical basis, particularly in grams were requested. A structured systems review was used to newborns and young children who are identified with hearing elicit evidence for syndromic forms of hearing loss such as loss, will doubtless become more common in the future. Waardenburg syndrome, Pendred syndrome, Jervell and Recently, it has been shown that GJB2 mutations, when Lange-Nielseon syndrome, Branchio-Oto-Renal syndrome, present with mutations in other nonallelic, functionally related Alport syndrome, Norrie disease, Usher syndrome, and genes, cause deafness. A 342-kb deletion including GJB6, X-linked congenital fixation of the stapes. When the pedigree which encodes gap junction protein connexin 30 (Cx30), fails data could be extended to relatives living before 1900, an at- to complement in trans with GJB2 mutations to cause tempt was made to link the data to the family history records deafness.11 collected by EA Fay.24 For any deaf or hard of hearing individ- We have established a nationwide DNA Repository of sam- uals identified in the family, information on parents, siblings ples from deaf probands to pursue a sequential screening strat- (number and hearing status of each), spouse and children egy for the identification of new genes for deafness. As a by- (number and hearing status) was obtained. Information about product, this strategy yields estimates of the prevalence of ethnicity was also collected. Blood samples on the proband, known mutations for deafness. Here, we describe the results of parents and siblings were drawn and shipped to the molecular sequential screening of this Repository for mutations in the genetics laboratory at the Medical College of Virginia.