Non-syndromic neurosensory autosomal recessive deafness (NSRD) is the most common form of genetic hearing loss. Previous studies defined at least 15 human NSRD loci. Recently we demonstrated that DFNB1, located on the long arm of chromosome 13, accounts for ~80% of cases in the Mediterranean area. Further analysis with additional markers now identifies several recombinants which narrow the candidate region to ~5 cM, encompassed by markers D13S141 and D13S232 and including several ESTs and candidate genes, including the connexin26 (GJB2) gene. Analysis of PCR products from our affected patients' DNA shows two frameshift mutations in the connexin26 gene. Deletion of a G within a stretch of six Gs at position 35 of the GJB2 cDNA (mutation 35delG) leads to premature chain termination and is present in 63% of NSRD chromosomes, demonstrating linkage to chromosome 13. Deletion of a T at position 167 of GJB2 (mutation 167delT), also resulting in premature chain termination, was detected in another patient. Four neutral sequence polymorphisms were also identified. These findings are in agreement with a recent study showing that mutations in the connexin26 gene are associated with genetic forms of deafness in three Pakistani families and that GJB2 is DFNB1. Connexin26 is a member of a large family of proteins involved in formation of gap junctions, which are involved in electrical synapses and the direct transfer of small molecules and ionic currents between neighboring cells. The identification of GJB2 as the DFNB1 gene should provide a better understanding of the biology of normal and abnormal hearing, help form the basis for diagnosis and may facilitate development of strategies for treatment of this common genetic disorder.
Hearing loss affects ~4% of people under 45 years of age and comprises a broad spectrum of clinical presentations (congenital or late onset, conductive or sensorineural and syndromic or non-syndromic) (1 ). Non-syndromic neurosensory autosomal recessive deafness (NSRD) is the most common form of genetic hearing loss, accounting for ~50% of childhood prelingual deafness (1/2500). Previous studies defined at least 15 human NSRD loci (2 ). After the demonstration that a large proportion of NSRD cases in our patient population (3 ) were linked to DFNB1 (4 ), the candidate region (~14 cM) was further analyzed using additional informative microsatellite markers in an attemptto narrow the interval. Genomic mapping data (http://gdbwww.gdb.org) have placed the connexin26 gene within the interval defined by our linkage studies (5 -7 ). Mammalian connexin genes contain the complete coding region in a single coding exon of ~800 nt, greatly facilitating analysis of the entire protein region using a rapid PCR-based approach using genomic DNA (8 ,9 ). An RNA-SSCP protocol was employed to screen for GJB2 mutations (10 ). Here we describe that mutations in this gene are present in 63% of NSRD chromosomes of patients from the Mediterranean area. These findings are in agreement with the recent identification of mutations in the GJB2 gene in Pakistani families affected with congenital hearing loss, although those mutations differ from those found in our families (11 ).
Several recombination events were observed (Fig. 1 ) which further defined the candidate region on chromosome 13 to ~5 cM flanked by D13S141 and D13S232. Efforts were then focused on defining DFNB1 candidate genes mapping to this approximate chromosomal region which were also expressed in human cochlear cells. Previous studies showed that cochlea cells are interconnected via gap junctions, which are formed by connexons containing connexin26 (12 -15 ).
Connexin26 is a member of a large family of proteins involved in formation of gap junctions which allow direct transfer of small molecules and ions between neighboring cells (16 ). Transmission at gap junction synapses is fast and facilitates production of almost instantaneous action potentials. Gap junctions are rare between mammalian neurons, but are common in non-neural cells such as glia, epithelial cells and smooth and cardiac muscle cells (16 ). There is a large family of connexin proteins, each conferring distinct physiological properties on the different gap junction channels (17 ).
At least two other connexin genes have been implicated in human diseases. Point mutations in the connexin32 gene are associated with X-linked Charcot-Marie-Tooth disease (18 ) and mutations in the connexin43 gene are found in patients with heterotaxia and heart malformation (19 ). Our data on connexin26 mutations associated with genetic forms of deafness is in agreement with a recent report describing connexin26 mutations in DFNA3 and DFNB1 patients from Pakistan (11 ), however, in those cases the mutations differ from those we describe in the Mediterranean population. In our study there were three patients heterozygous for mutation 35delG in whom we did not detect a second mutation in the GJB2 coding region. In addition, there were 12 subjects with deafness linked to the DFNB1 locus (3 ) who failed to show GJB2 mutations. These results suggest that mutations may be located in other regions of the connexin gene or that they may occur in neighboring genes mapping to chromosome 13q11-q12 which may also be involved in congenital deafness.
Several findings support the importance of gap junctions and connexin26 in auditory transduction. The auditory organ has gap junctions between the outer hair cells and supporting cells (including melanocytes), providing a morphological basis for the occurrence of intracellular responses to sound in supporting cells and for electric coupling of receptor cells (20 -22 ). In addition, the endothelium of the scala media of the cochlea is involved in production of a receptor response to the auditory stimulus and is separated from the endolymphatic space by tight junctions in the marginal cell layer, which is coupled by gap junctions (23 ). Finally, immunohistochemical and ultrastructural analysis of connexin26 in the rat cochlea showed that gap junctions in both epithelial and connective tissue cells are involved in recycling endolymphatic potassium ions through the sensory cells during the transduction of voltage gating in these channels (13 ,14 ). Therefore, the identification of mutations in GJB2 in patients with autosomal recessive congenital deafness further supports the involvement of gap junctions in the endocochlear potential of audition.
In conclusion, we previously demonstrated that a large proportion of families with NSRD showed linkage to the DFNB1 locus. We have now reduced the candidate region to 5 cM and investigated candidate genes within this region, including the GBJ2 gene, which is expressed in the cochlea and may be involved in auditory transduction. Mutation analysis of GJB2 demonstrated two frameshift mutations in affected patients with linkage to DFNB1. The 35delG mutation is the most common mutation, accounting for >63% of the DFNB1 alleles in our patient population. The link between connexin26 and deafness should facilitate our understanding of the biology of normal and abnormal hearing and may provide new ways for prevention and investigation of rational treatment strategies for this common genetic disorder. Until recently deafness was thought to be a highly complex problem because of genetic heterogeneity. This complexity has now been reduced since: (i) NSRD represents the majority (0.70-0.75) of genetic deafness cases; (ii) a large proportion (0.80) of NSRD cases in Mediterraneans is associated with DFNB1; (iii) we have now described a very common frameshift mutation in NSRD patients linked to DFNB1. Therefore, in terms of prevention and counselling, the complexity of genetic deafness has become simple, at least in the Mediterranean population. The new knowledge about the pathogenesis of congenital deafness should also lead to the development of new therapeutic strategies for this genetic disease.
Thirty five patients with NSRD linked to DFNB1 as previously described (3 ) were studied. Diagnosis of NSRD was established according to accepted clinical criteria, including autosomal recessive inheritance.
Nine different microsatellite markers (D13S175, D13S1316, D13S141, D13S1236, D13S143, D13S1275, D13S115, D13S232 and D13S292) from the DFNB1 region were utilized to narrow the candidate gene region in families previously identified to have undergone a recombination event within this region. PCR reactions and conditions were as previously described (3 ).
For the purpose of transcription from genomic PCR fragments, the T7 bacteriophage RNA polymerase promoter sequence was incorporated into one PCR primer of each primer pair. PCR was performed on 500 ng genomic DNA as previously described for 30 cycles with each cycle consisting of denaturation, annealing and extension at 94, 55 and 72oC respectively. Four PCR primer pairs were designed from the mRNA sequence of connexin26 (HUMGAPJUNC, accession no. M86849) to cover 798 bp of coding region plus ~20 bp of 5'-UTR and 3'-UTR. Primers were: F1, 5'-CAT TCG TCT TTT CCA GAG CA-3' (-38 to -19), and R4, 5'-CAC GTG CAT GGC CAC TAG-3' (+285 to +268), (367 bp PCR product); F4, 5'-CGT GTG CTA CGA TCA CTA C-3' (+186 to +204), and R5, 5-AGC CTT CGA TGC GGA CCT T-3' (+391 to +373), (226 bp PCR product); F5, 5'-ACC GGA GAC ATG AGA AGA AG-3' (+290 to +309), and R8, 5'-TTC CAG ACA CTG CAA TCA TG-3' (+602 to +583), (333 bp PCR product); F7, 5'-TAT GTC ATG TAC GAC GGC T-3' (+463 to +481), and R9,5'-TCT AAC AAC TGG GCA ATG C-3' (+702 to +684), (260 bp PCR product). Transcription was with 10 U T7 RNA polymerase in a final volume of 10 [mu]l containing 10 mM DTT, 40 mM Tris, pH 7.5, 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 5 nmol each ribonucleoside triphosphate, 10 U RNasin and 2 [mu]l [35S]UTP (>1000 Ci/mmol). An aliquot of 2 [mu]l of transcribed RNA was mixed with 48 [mu]l 95% (v/v) formamide, 20 mM EDTA, 0.05% (w/v) bromophenol blue and 0.05% (w/v) xylene cyanol. The mixture was then heated at 95oC for 6 min and then chilled on ice for 10 min. An aliquot (4.5 [mu]l) was then loaded onto a 6.5% (w/v) non-denaturing polyacrylamide gel and electrophoresis was performed at 30 W constant power for 13 h. After electrophoresis the gel was dried and subjected to autoradiography for 12 h.
PCR products which after in vitro transcription showed altered mobility compared with normal controls after RNA-SSCP analysis were excised from the gel and DNA sequence determined on an automated sequencer (ABI 373A; Applied Biosystems-Perkin Elmer, Foster City, CA) according to the manufacturers' protocols.
We are indebted to the families for their cooperation and the Studio Logopedico Elvira Signori. The authors thank P.Stanziale for his helpful advice on microsatellite analysis and Dr M.Keller and C.Ponte in the Molecular Biology Core at the du Pont Hospital for Children for automated DNA sequence analysis. This work was supported in part by a grant from Telethon (E.549) and the Italian Ministry of Health (P.G. and L.Z.), the `Fundación Ramón Areces' (X.E., N.G. and M.M.), the Servei Catalá de la Salut (X.E.), NIH-SBIR grant no. 1R43NS/MH34589 (E.M.), the du Pont Hospital for Children, the Nemours Foundation (S.S.) and the Department of Pathology, The Children's Hospital of Philadelphia (P.F.).
*To whom correspondence should be addressed at: The Children's Hospital of Philadelphia, Abramson Pediatric Research Center 310-C, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104, USA. Tel: 215 590 3318; Fax: 215 590 3660; Email: fortina@mail.med.upenn.edu
Human Molecular Genetics
Pages
Introduction
Results
Discussion
Materials And Methods
Patient data
Microsatellite analysis
RNA-SSCP
Sequence analysis
Acknowledgements
References
REFERENCES
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