Congenital severe to profound hearing loss affects 1 in 1000 neonates, of which approximately one-half is of genetic origin.［1］ Seventy percent of the patients with inherited congenital deafness without non-auditory features, are described as nonsyndromic hearing loss, with 75%-80% are inherited in autosomal recessive manner, 20%-25% in dominant and 1%-1.5% in X-linked.［2］ To date, more than 70 non-syndromic loci have been mapped and 40 deafness genes have been identified (http://www.uia.ac.be/dnalab/hhh). Despite this hetero-geneity, up to 50% of prelingual recessive non-syndromic deafness can be attributed to mutations in GJB2, which responsible to autosomal recessive nonsyndromic deafness DFNB1 and dominant deafness DFNA3.［3,4］
GJB2 encodes a connexin protein called connexin 26 (Cx26), a transmembrane protein that oligomerizes with five other connexin molecules to form a connexon (gap junction). It has been hypothesized that gap junctions in cochlea, especially Cx26, provide an intercellular passage by which potassium ions are transported to maintain high levels of the endocochlear potential, which is essential to initiate action potentials in sensory hair cells.［5］ Immunochemical experiments have shown Cx26 expression in the stria vascularis, basement membrane, limbus and the spiral prominence of the cochlea.［6］ Mutant GJB2 results in the defects in Cx26 that may reduce the efficiency of potassium ions circulation and consequently lead to impaired hearing sensitivity. Extensive screening studies for GJB2 mutations have been undertaken within the different population with hearing impairment, which suggest that mutations in this gene account for at least 50% of genetic hearing loss in white population and the features of mutations in this gene accord to ethnic backgrounds.［7-9］
The purpose of this study was to investigate the frequency and the features of GJB2 mutation in the Chinese congenital deafness patients.
This study was part of the one for deafness-gene mutations screening among the patients with congenital hearing loss in Chinese in our institute. One hundred and thirty-one unrelated patients with congenital sensorineural deafness, of whom 69 were autosomal recessive and 27 were dominant and 35 were sporadic, were from the out-patient clinic of the Department of Otolaryngology, Second Xiangya Hospital of Central South University and three hearing rehabilitation schools in Hunan Province of China. We presumed that the pattern of deafness inheritance in these families with at least two deafness patients in one generation or skipped generations was recessive, and at least one patient in every generation was dominant. Clinical evaluation to the patients included personal history and the following examinations: ① otologic examination; ② pure tone audiometry (PTA) including air and bone conduction and/or auditory-evoked brainstem response (ABR); ③ physical and biochemical examinations, including thyroid, liver, kidney and nervous system. There are no evidences of any obvious syndrome and exclusion of other risk factors which could lead to deafness, including chronic middle-ear infection, otologic trauma, ototoxic antibiotics, Meniere disease and so on. Most of the patients showed severe to profound deafness by PTA/ABR. Blood samples from 100 healthy juniors with no hearing loss were collected as control subjects.
Genomic DNA (gDNA) was isolated from the peripheral blood lymphocytes of the subjects by using the Genomic DNA Purification Kit (Promega, USA), and suspended in Tris-EDTA buffer and stored at -4℃ until use. One pair primers of GJB2 gene: forward primer: TCT TTT CCA GAG CAA ACC GC, reverse primer: CTG GGC AAT GCG TTA AAC TGG,［10］ were used to produce a template for sequencing. PCR was performed with PE9600 DNA Amplifier (Perking, USA) and amplification conditions were as follows: reaction volume 50 μl, including 1×PCR buffer, 1.5 mmol/L MgCl2, 1.60 mmol/L dNTPs (Takara, China), direct and reverse primer each 100 ng, Tag enzyme at 0.5 U (Takara, China), gDNA at 100 ng; pre-denaturation at 94℃ for 2 minutes, denaturation at 94℃ for 10 seconds, annealing at 60℃ for 20 seconds, and extension at 72℃ for 45 seconds for a total 32 cycles, followed by a post-PCR extension step at 72℃ for 6 minutes. PCR products with 725 bp that covering the entire coding region of GJB2 were electrophoresed on 6% polyacrylamide gel (29∶1) to verify their quantity. Sequencing was accomplished in both directions by use of the ABI PRISM BigDye Terminator Cycle Sequencing Kit and an ABI 377 DNA Automatic Sequencer (Perking, USA).
Mutations in patients with autosomal recessive and sporadic congenital deafness
Nine types of the mutations in GJB2 were detected in this group ( Table ). They consisted of five types of polymorphism, and four types of deafness-causing mutation with 35delG and 235delC frameshift mutation, and 442G→A (Val 148 Ile) missense mutation and 465T→A nonsense mutation. Homozygous 35delG was found in 1 sporadic patient (1/35) ( Fig. 1 ), homozygous 235delC in 1 sporadic (1/35) and 8 recessive patients (8/69) ( Fig. 2 ), heterozygous 235delC in 2 recessive patients (2/69), homozygous 442G→A in 1 recessive patient ( Fig. 3 ), 465T→A, a novel mutation of GJB2, in a proband whose sibling had the same change and their parents were consanguineous married ( Fig. 4 ), respectively. The 235delC mutation was the most common in the recessive patients (10/69, 14.5%). The homozygous (10/69, 14.5%) and the heterozygous (2/69, 2.9%) mutation of GJB2 in the recessive patients (12/69, 17.4%) and homozygous mutations in the sporadic patient (2/35, 5.7%) all had congenital severe to profound sensorineural hearing loss.
Mutations in patients with autosomal dominant congenital deafness
511G→A (Ala 171 Thr) missense mutation and 299-300delAT frameshift mutation ( Fig. 5 ) were found to be segregated to deafness in two dominant families (2/27, 7.4%) ( Table ). The members with 511G→A mutation in the family showed variable audiological manifestations from mild to severe congenital sensorineural deafness. Hearing of the individuals with 299-300delAT mutation in another family were mild to moderate congenital hearing impaired and still further progressed losing in their early childhood.
Mutations in the control subjects
Among the 100 control subjects, six types of mutation—5 types of polymorphism and 1 type of heterozygous deletion (235delC) mutation were found. 109G→A (Val 37 Ile) was the most frequent (15/100, 15%) and 79G→A (Val 27 Ile) was the second common (8/100, 8%) polymorphism in the controls. Three subjects had other 3 types of polymorphism: 341A→G, 506A→G, 608T→C. Twenty-six subjects (26/100, 26%) had polymorphism in GJB2. All the polymorphisms were not associated with deafness.
Six Cx26 monomers assemble to form a hexameric connexon, which localises to the cell membrane. Oligomerisation with a connexon from an adjacent cell results in channel formation, facilitating intercellular transport of small metabolites and ions. Cx26, like other connexins, has a common sequence of structure motifs:［6］ the N-terminal domain (NT), four transmembrane domains (M1, M2, M3, M4), two extracellular domains (E1, E2), the cytoplasmic linking domain (CL) and the C-terminal domain (CT). In the cochlea, Cx26 is expressed in the supporting cells and fibrocytes of the cochlear duct, where it may be involved in the recycling of potassium ion from hair cells back to the endolymph. GJB2 mutation and disruption of Cx26 gap junctions presumably impairs recycling of potassium ion thereby causing hearing impairment.
Currently, more than 80 different recessive mutations have been found in GJB2 gene, but 35delG is an especially hot-spot in the white population and 235delC in East Asian and 167delT in the Ashkenazi Jewish.［11-13］ The most common deafness mutation found in the autosomal recessive and sporadic Chinese patients in this study was 235delC frameshift mutation which resulted in premature termination at codon 81, and the mutant Cx26 protein was 145 amino acids shorter than that of the wild type. The frequency of this mutant allele accounted for approximately 11.6% (16/138 alleles) in recessive patients and 2.9% (2/70 alleles) in sporadic and the carrier frequency in control population was estimated at 0.5% (1/200 alleles), which was similar to the results reported by other scholars.［14］ This type of mutation is also the most common mutation in Japanese and Korean population with inherited deafness. These results might support the opinion that 235delC could be a mutation hot spot in GJB2 in Mongoloid ethnic group.
35delG was only found in one sporadic patients in our group which indicated that this type of mutation might be rare in Chinese patients, which was according to the idea that 35delG could be exceptionally low in Chinese deafness population.［14］ 442G→A (Val 148 Ile) missense mutation, which was at the M3 domain of Cx26, might cause recessive deafness by disrupting the synthesis, assembly and structure of Cx26 gap junction.
465T→A nonsense mutation at the M3 domain of Cx26, which truncated 112 amino acids from the E2 to the CT domain, was a novel mutation found by our study. The mutant protein of 465T→A Cx26, as that of 235delC, eliminating the E2-M4-CT domain, caused Cx26 gap junction functional defect and could not form functional channel. All 465T→A and 235delC homozygous individuals had congenital serve to profound deafness, whereas the heterozygotes 465T→A had normal hearing. Two 235delC heterozygotes deafness patients in this group might have another compound-heterozygous mutation in the untranslated regions in GJB2 or mutations in other deafness-related genes. This phenomenon has discovered among other ethnic patients.［15］
511G→A missense mutation and 299-300delAT frameshift mutation were associated with autosomal dominant congenital deafness in our study. 511G→A related to dominant deafness had been reported by Hamelmann et al.［16］ But the frameshift mutation of 299-300delAT was previously reported in only autosomal recessive deafness patients.［17］ Heterozygous mutation of 299-300delAT was found to be segregated with hearing loss in an autosomal dominant deafness family in the study which indicated that this type of mutation was also contributed to autosomal dominant deafness. The mutant protein of 299-300delAT terminated at the codon 113 and only had 112 amino acids, which was 226 amino acids shorter than that of the wild type, and the former 99 amino acids were just the same as the wild one. This change would disrupt the function of Cx26. The individuals with the mutation of 299-300delAT in the family showed congenital mild to moderate impairment and still further progressed losing their hearing in their early childhood. It suggested that the heterozygous mutant protein of 299-300delAT might continue destroying the function of Cx26 gap junction during a short period after birthing.［14,17-19］
GJB2-related hearing loss is prelingual in onset and generally nonprogressive. GJB2 mutation resultant hearing loss usually involves all frequencies and symmetrically at double ears. But progressive and asymmetrical hearing loss has been noted in some cases.［20］ A phenomenon that deafness was more severe in the recessive individuals than in the dominant patients was observed in this deaf group. But the genotype/phenotype correlation and a “typical” audiogram associated with GJB2-related deafness has not happened, making it important to maintain a high index of suspicion for GJB2-related deafness in all individuals with congenital hearing loss of uncertain etiology.
In addition to these deafness-causing mutations, five types of polymorphism were found in the patients and the controls. 109G→A (Val 37 Ile) was the most frequent (15%) and 79G→A (Val 27 Ile) was the second (8%). Also Val 37 Ile was thought to be pathogenic variants leading to a mild phenotype by some scholars.［11］ We still confirm the fact that Val 37 Ile is a polymorphism but not a deafness mutation because of its high rate in the control subjects in our study.
This study shows that there are GJB2 mutations in 17.4% (12/69) of the autosomal recessive and 7.4% (2/27) of dominant patients, and 5.7% (2/35) of the sporadic congenital deafness, respectively. The total mutation rate of GJB2 in Chinese patients with congenital sensorineural deafness is 12.2% (16/131). The 235delC is the most frequent deafness mutation in the recessive and sporadic congenital deafness patients (10/104, 9.6%). The allelic frequency of 235delC was much high in the recessive and sporadic deafness group (20/208, 9.6%) than that in the control subjects (1/200, 0.5%). 465T→A nonsense mutation associated with autosomal recessive deafness is a novel mutation found by this screening. Our study also reveals that 511G→A and 299-300delAT mutations contribute to autosomal dominant hearing loss. The study further supports the view that the common types of mutation in GJB2 based on different ethnic background and that the mutation prevalence in the East Asian deafness population is lower than that in the white population.
Acknowledgements: We thank National Laboratory of Medical Genetics of China, Central South University, Changsha, China for the assistance to this study.
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