GJB2 and GJB6 Mutations in Children with Congenital Cytomegalovirus Infection

0031-3998/07/6106-0687 PEDIATRIC RESEARCH Copyright © 2007 International Pediatric Research Foundation, Inc. Vol. 61, No. 6, 2007 Printed in U.S.A. GJB2 and GJB6 Mutations in Children with Congenital Cytomegalovirus Infection SHANNON A. ROSS, ZDENEK NOVAK, REKHA A. KUMBLA, KUI ZHANG, KAREN B. FOWLER, AND SURESH BOPPANA Departments of Pediatrics [S.A.R., Z.N., R.A.K., K.B.F., S.B.], Biostatistics [K.Z.], Epidemiology [K.B.F.], Maternal and Child Health [K.B.F.], and Microbiology [S.B.], The University of Alabama at Birmingham, Birmingham, Alabama 35233 hearing loss in children with symptomatic infection (4). Urinary excretion of CMV has been correlated with hearing outcome, with SNHL and progressive SNHL associated with viral urinary excretion ⬍4 y (5). In addition, an association between increased viral load during early infancy and hearing loss has been documented in asymptomatic children, although not all children with increased virus burden in infancy develop hearing loss (6). Over the past decade, there has been a dramatic increase in our understanding of the importance of hereditary deafness, which is estimated to be responsible for at least 50%– 60% of childhood hearing loss (7) Gap junctions of the inner ear are thought to play a role in recycling potassium in the cochlea, which is important for sensorineural hearing function. Gap junctions are composed of integral membrane proteins, called connexins, that oligomerize to form intercellular channels. Mutations in connexin genes can alter the function of the encoded protein in the inner ear, resulting in inherited SNHL (8) Although it is believed that approximately 100 genes could be responsible for hearing impairment (9), a large proportion of hereditary deafness cases are due to autosomal recessive mutations in one gene, GJB2, which encodes the protein connexin 26. However, there are racial/ethnic differences in the distribution of the various GJB2 alleles (9). More recently, a large deletion, del(GJB6-D13S1830), involving a gene close to GJB2, called GJB6 (connexin 30), has been described as a cause of deafness in the homozygous state and in heterozygosity with GJB2 mutations (10 –12). To determine whether mutations in GJB2 and GJB6 are more frequent in children with CMV-related hearing loss, we compared GJB2 and GJB6 mutations in congenitally infected children with and without SNHL. In addition, because the frequency of connexin mutations in African American populations has not been well studied, we also examined the frequency of these mutations in a predominantly African American infant population born at The University of Alabama (UAB) Hospital. ABSTRACT: Congenital cytomegalovirus (CMV) infection is a leading cause of sensorineural hearing loss (SNHL) in children. Whether connexin mutations are factors in the development of CMV-related hearing loss has not been explored. We examined gap junction protein beta-2 (GJB2) and gap junction protein beta-6 (GJB6) mutations in 149 children with congenital CMV infection and 380 uninfected neonates. Mutations in GJB2 and GJB6 were assessed by nucleotide sequencing and polymerase chain reaction (PCR) methods, respectively. The study population was predominantly African American, and 4.3% of the subjects were carriers of a connexin 26 mutation. The overall frequency of GJB2 mutations was significantly higher in the group of children with CMV infection and hearing loss (21%) compared with those with CMV infection and normal hearing (3%, p ⫽ 0.017) and the group of uninfected newborns (3.9%, p ⫽ 0.016). Eight previously reported mutations (M34T, V27I, R127H, F83L, R143W, V37I, V84L, G160S), and four novel mutations (V167M, G4D, A40T, and R160Q) were detected. None of the study children had the 342-kb deletion (delGJB6D13S1830) in GJB6, which suggests that this mutation does not play a role in hereditary deafness in the African American population. Although GJB2 mutations were detected in children with and without CMV-related hearing loss, those with hearing loss had a higher frequency of GJB2 mutations. (Pediatr Res 61: 687–691, 2007) C MV is a frequent cause of congenital infection and a leading cause of SNHL in children in the United States and northern Europe. Prospective follow-up studies have shown that approximately 5%–15% of children with asymptomatic or subclinical congenital CMV infection and 40%– 75% of those with symptomatic infection develop hearing loss (1–3). However, current knowledge of the pathogenesis of hearing loss in congenitally infected children is limited. The results of a recent study have shown that the presence of petechiae and intrauterine growth retardation at birth in infants with symptomatic congenital CMV infection was predictive of hearing loss. In contrast, the presence of microcephaly and other neurologic abnormalities at birth was not predictive of Received November 2, 2006; accepted January 15, 2007. Presented in part at the 10th International CMV/Betaherpesvirus Workshop, Williamsburg, VA, April 27, 2005. Correspondence: Shannon A. Ross, M.D., The University of Alabama at Birmingham, CHB 304, 1600 7th Avenue South, Birmingham, AL 35233; e-mail: Supported in part by grants from the National Institutes of Health, the National Institute of Child Health and Human Development (P01 HD 10699), the National Institute of Allergy and Infectious Diseases (P01 AI43681, T32 AI052069), The National Institute on Deafness and Other Communication Disorders (R01 DC02139), the General Clinical Research Center (M01 R00032) and the Children’s Center for Research and Innovation. METHODS Study population. Between 1980 and 2003, 780 children were identified by newborn screening for congenital CMV infection at two hospitals in Birmingham, AL, and enrolled in a long-term prospective natural history Abbreviations: CMV, cytomegalovirus; DBS, dried blood spot; GJB2, gap junction protein beta-2; GJB6, gap junction protein beta-6; PBL, peripheral blood lymphocyte; SNHL, sensorineural hearing loss DOI: 10.1203/pdr.0b013e3180536609 687 688 ROSS ET AL. study. Congenital CMV infection was identified by isolation of the virus in urine or saliva within the first 2 wk of life (13,14). Peripheral blood lymphocytes (PBLs) were available from 149 children (19 children with hearing loss and 130 children with normal hearing), and this group constituted the study population. Infants were classified as having symptomatic congenital CMV infection if they had any of the following clinical findings in the newborn period: petechiae, purpura, jaundice with conjugated hyperbilirubinemia (direct bilirubin ⬎2 mg/dL), thrombocytopenia (⬍100,000/mm3), hepatosplenomegaly, microcephaly, seizures, or chorioretinitis (15). Study children were followed in an interdisciplinary clinic and were monitored with audiologic evaluations at the initial clinic visit at 3– 8 wk of age, every 6 mo until 24 y of age, then annually thereafter according to a standard protocol described previ (...truncated)