Clinical Studies of Families With Hearing Loss Attributable to Mutations in the Connexin 26 Gene (GJB2/DFNB1)
Objective. This retrospective study describes the phenotype associated with the single most common cause of genetic hearing loss. The frequency of childhood deafness is estimated at 1/500. Half of this hearing loss is genetic and ∼80% of genetic hearing loss is nonsyndromic and inherited in an autosomal recessive manner. Approximately 50% of childhood nonsyndromic recessive hearing loss is caused by mutations in the connexin 26 (Cx26) gene (GJB2/DFNB1), making it the most common form of autosomal recessive nonsyndromic hearing loss with a carrier rate estimated to be as high as 2.8%. One mutation, 35delG, accounts for ∼75% to 80% of mutations at this gene.
Methods. Hearing loss was examined in 46 individuals from 24 families who were either homozygous or compound heterozygous for Cx26 mutations. A subset of these individuals were examined for vestibular function, otoacoustic emissions, auditory brainstem response, temporal bone computed tomography, electrocardiography, urinalyses, dysmorphology, and thyroid function.
Results. Although all persons had hearing impairment, no consistent audiologic phenotype was observed. Hearing loss varied from mild-moderate to profound, even within the group of families homozygous for the common mutation 35delG, suggesting that other factors modify the phenotypic effects of mutations in Cx26. Furthermore, the hearing loss was observed to be progressive in a number of cases. No associations with inner ear abnormality, thyroid dysfunction, heart conduction defect, urinalyses, dysmorphic features, or retinal abnormality were noted.
Conclusion. Newborns with confirmed hearing loss should have Cx26 testing. Cx26 testing will help define a group in which ∼60% will have profound or severe-profound hearing loss and require aggressive language intervention (many of these patients will be candidates for cochlear implants).
Approximately 1/500 children are born with a hearing loss of sufficient magnitude that some type of intervention is needed to help with communication.1 Because of the high frequency and clinical impact of congenital hearing impairment, early detection has become an important public health problem. Neonatal screening for early identification of hearing impairment has been recommended by the National Institutes of Health (1993) and the Joint Committee on Infant Hearing Screening (1994). The rationale for early detection lies with the persuasive argument that early intervention influences significantly and positively a child's ability to communicate and learn.2,,3 Additionally, technologic advances using auditory brainstem response (ABR) or otoacoustic emissions (OAE) testing make such newborn screening program economically feasible. Simultaneous with the development of technologies for identifying and quantifying hearing loss, advances in biology are yielding more and better information about the specific causes of hearing loss. The purpose of this report is to present new clinical data on the most common type of childhood recessive nonsyndromic hearing loss and to show how this information impacts on the question of early screening for hearing impairment.
Genetic causes account for at least 50% of all childhood hearing impairment.4 The bulk of these are nonsyndromic and most of these, ∼80%, have an autosomal recessive etiology. The number of different genetic loci involved in nonsyndromic recessive hearing loss (NSRHL) has been estimated at between 20 and 150 (http://dnalab-www.uia.ac.be/dnalab/hhh/).5 The first to be discovered was called DFNB1,6 which was later shown to be attributable to mutations in the connexin 26 (Cx26) gene on chromosome 13.7–9 Mutations in the Cx26 gene are responsible for ∼50% of all the cases of childhood NSRHL,8–10 and one mutation, 35delG, is responsible for greater than half of all the pathologic Cx26 mutations. A second common mutation, 167delT, has been observed to occur solely in the American (presumably Askenazi) Jewish population. The importance of Cx26 is underlined by the fact that mutations in it are responsible for 40% of genetic childhood hearing loss. Here we present an analysis of the clinical symptoms of 46 cases of Cx26-associated hearing loss to define better the prognostic and counseling value of Cx26 testing.
SUBJECTS AND METHODS
The criterion for inclusion in our study required two or more siblings with nonsyndromic sensorineural hearing loss with no history of similar hearing loss in their parents, aunts, or uncles. Blood was drawn, processed, and genotyped as described previously.9DNA from nonsyndromic recessive hearing loss families were analyzed for mutations in Cx26 using heteroduplex analysis, and variant samples were sequenced. Sixty-eight families have been tested. Of the families, 58 were subjects of a previous report9 and were tested completely for mutation in the coding region of the Cx26 gene. However, 10 new families have been screened for only the 35delG and 167delT mutations. Thirty-eight were observed not to have any mutation in Cx26, 6 were observed to have a mutation in only one homolog, and 24 were observed to have two mutations, one each in the paired homologues. Only 46 of 49 persons from the 24 families showing a mutation in each of the Cx26 gene pair are analyzed in this study because we did not have audiograms on 3 individuals.
This study examines mutations in the Cx26 gene in relation to the degree, stability, and symmetry of hearing loss. Other parameters investigated include vestibular function (rotary chair), OAE, ABR, thin section temporal bone computed tomography (CT), electrocardiography (EKG), urinalyses, dysmorphlogy examination, and thyroid function studies.
Medical, genetic, and audiologic information pertaining to each patient with NSRHL was requested and reviewed when received. Four families were invited and came to Boys Town National Research Hospital for evaluation. These families were seen by members of our genetics, otolaryngology, and audiology staff. Ophthalmology and radiology consultations also were obtained. All those with hearing loss had 1.5 mm thin section CT of the temporal bone, and one member of each family had perchlorate washouts. These procedures were performed at the University of Nebraska Medical Center through a research agreement with the Department of Radiology. Three other families were seen previously at Boys Town National Research Hospital, but did not necessarily undergo the same testing conducted on the invited families. The tests performed varied among families. Table 1shows the clinical tests performed and the corresponding sample sizes for data collected on patients. Three individuals did not have standard audiograms. One was an adult who declined formal testing, and the other 2 were young children for whom only sound field data were available.
Commonly accepted ranges of hearing thresholds were used as descriptors of the level of hearing impairment in the families, ie, <25 dB pure tone hearing thresholds defined normal hearing; 25 dB to 44 dB, mild hearing loss; 45 dB to 65 dB, moderate hearing loss; >65 dB to 85 dB, severe hearing loss; and >85 dB, profound hearing loss.
The degree of loss was classified according to the better ear. No response was recorded as 130 dB for calculation of modified pure tone average. Asymmetric hearing loss was defined as a 10 dB difference between ears in three frequencies, 15 dB in two frequencies, and 20 dB at one frequency. A modified pure tone average (mPTA) is defined as the hearing loss average of 500, 1000, 2000, and 4000 Hz.
The index of progressive hearing loss in dB/y was calculated from the most widely spaced, most complete audiometric data when typanogram findings were normal. It is defined as the difference in hearing acuity as measured by mPTA for both ears combined.
For the most part, our cases are young, of European ancestry, and equally divided among males and females (Table 2).
The degree of hearing loss was determined on 46 of the subjects with Cx26 mutations. These data are shown in Table 3 in relation to the type of mutation. ABR results were consistent with pure tone thresholds.
Homozygous 35delG Mutations
Thirty-three hearing impaired individuals from 16 families were homozygous for the 35delG mutation. There was considerable variation in the degree of residual hearing loss. Approximately two thirds (20/33) of the 35delG homozygotes had a severe-profound or profound hearing loss.
Homozygous 167delT Mutations
Six hearing-impaired individuals from three Jewish families were homozygous for the 167delT mutation. All these cases had moderate-severe to profound hearing impairment, and the clinical impression was that the hearing impairment was more severe and less variable than that observed for the 35delG homozygotes. Although the trend to greater severity is interesting, the small sample size lessens its significance.
Compound Heterozygotes for Cx26 Mutations
The remaining 5 families with 10 affected individuals were compound heterozygotes with 8 of the individuals from 4 families having a 35delG mutation and one of four other unique mutations, whereas 2 individuals from the fifth family had the 167delT mutation in combination with a 631–632delGT mutation.
Quantitative Assessment of the Hearing Loss Differences Between Genotypes
mPTA was analyzed to determine whether differences existed between the three groups (Table 4). As expected from the distribution of hearing losses (Table 3), individuals with the 167delT mutations showed significantly lower variance, and the mean hearing impairment was 14 dB greater for the 167delT patients than for the 35delG cohort. This difference was statistically significant atP < .05. Although the hearing loss was intermediate for the compound heterozygotes, it was not statistically different from that for the 35delG category. However, this compound heterozygous group was the youngest (Table 2), and 3 showed progressive hearing loss (Table 5). Approximately half of all genotypes showed asymmetric hearing between ears.
Progression of Hearing Loss
One third (10 of 30) of the cases with serial audiograms showed a progressive hearing loss (Tables 5, 6) of at least 1 dB mPTA annual loss. If we changed our criteria to 2 dB mPTA per year, 7 individuals have progressive hearing loss (Table 6).
OAE data were evaluated in relation to criteria developed for separating ears with normal hearing from those with hearing loss.11,,12 The present data correlated well with behavioral thresholds, ie, OAEs tended to be present where hearing was normal and absent for frequencies in which hearing loss was present.
Audiograms were available on 13 parents. Nine have normal hearing, 2 have normal to mild hearing loss, and 2 have normal to moderate loss. Acoustic immittance and OAE data were available on 6 of the parents and the results of these tests were compatible with the audiogram in all cases. These results are not indicative of any clear-cut effect in the heterozygote.
Vestibular function studies were normal for all but 2 of the 13 patients studied. All these patients were homozygous for the 35delG mutation. One patient had caloric testing indicating 54% unilateral vestibular paresis and was symptomatic with recurrent vertigo and migraine. The second patient had a low frequency phase lead that may be attributable to immaturity because this child was born at 31 weeks' gestation and the testing was conducted at 7 months of age. The vestibular results were considered to be within expectation and do not indicate that the Cx26 mutation has any labyrinthine effect.
No inner ear malformations, including enlarged vestibular aqueducts, were identified in the 19 individuals who underwent CT of the temporal bone.
EKGs were performed in 11 patients from 5 families. The age range of the patients was 9 to 20 years. All EKG studies were normal, indicating that no conduction defect was present in any of the individuals studied.
None of the individuals affected presented with any history of goiter or thyroid problem. Four perchlorate washout tests were performed, and all were normal.
Routine urinalysis was performed on 20 cases. Two abnormal urinalysis results were seen with proteinuria, and both were from the same family.
Ophthalmology examinations were normal for all patients on whom these evaluations were performed.
Two patients were observed with dermatologic problems (1 with atopic dermatitis at age 13 months and 1 with eczema).
GJB2 encodes the gene for connexin 26, one member of a family of gap junction proteins.13 The general function of these proteins is to allow communication between cells allowing transfer of signal molecules, electrolytes, and metabolites. The function of Cx26 in the cochlea is as yet not defined clearly. One hypothesis is that Cx26 is involved in potassium recycling in the cochlea.14
Lack of Associated Symptoms
The results reported in this report confirm the nonsyndromic nature of Cx26 mutations. Only hearing is affected. From the clinician's perspective, this means that ancillary diagnostic tests may not be necessary. Preliminary data suggest that CT of temporal bone is not necessary. Whenever a new hearing-impaired child is seen, pediatricians, otolaryngologists, and geneticists are understandably concerned about the possibilities of Usher, Pendred, Jervall-Lange-Neilsen, and other syndromes. The finding that a Cx26 mutation is responsible for a particular case reduce the extent of additional diagnostic testing resulting in reduction of medical costs.
Data presented here show no evidence of human developmental problems. However, a knockout mutation in mouse of Cx26 is lethal,15suggesting that Cx26 plays some vital role in murine development. CT findings of the inner ear are normal. None of our cases presented with significant anomalies that could be proven to be associated with the Cx26 mutation. Although Cx26 is not implicated directly with conduction defects of the heart, there is established association with connexin 4316 raising the possibility that Cx26 mutations also might affect that organ. However, our data do not support this hypothesis, but our patients are young and may not as yet manifest cardiac problems (Table 2).
Progressive Hearing Loss
With regard to progression of hearing loss, 10/30 (with sufficient audiometric data to determine stability, fluctuation, or progression) were observed to show progression of their hearing impairment (Table 5and 6). In 8 of 10 cases, the four frequency average change was ≥10 dB, and for 4 of these patients, the average change was >20 dB (these changes are based on the mPTA and thus are averaged across four frequencies). A history of progression was not related to gender, nor was it related to the type of mutation present, although the sample size was limited for a comparison between genotypic groups. Clearly, there is some process occurring that leads to a worsening of hearing over time in some individuals with Cx26 mutations. We speculate that a larger number of children will show progressive hearing loss if followed from birth. The nature of the progression, its frequency, and the severity of the hearing loss at birth are issues that could be addressed better if Cx26 genotyping were performed in conjunction with failed newborn hearing screening so that the hearing of Cx26 cases were followed from birth.
Variability in Severity of Hearing Loss
We observed an unexpected wide range of hearing loss from mild-moderate to profound hearing loss, which needs some discussion. One hypothesis we considered was that the phenotype might be related to the specific mutation. However, the data do not support that hypothesis totally. A wide range of severity was observed across the homozygous 35delG genotypic category, but hearing impairment was more constant and severe for 167delT homozygotes. It may be that these differences are mutation-specific. For example, 167delT theoretically would produce a protein larger than that produced by the 35delG mutation, and it is possible that the 167delT protein actually may be functional enough to incorporate itself into the cell membrane and thereby to interfere with intercellular transport in a more drastic manner.
On the other hand, it may be that the differences between 35delG and 167delT are attributable to genetic background differences between Jewish and non-Jewish populations originating in Europe. If so, we might expect to observe more severe hearing loss for 35delG in the Ashkenazi Jewish population. It also is possible that there are nongenetic modifying factors that have yet to be identified.
The variability of hearing impairment for the 35delG homozygotes is perplexing. Most autosomal recessive disorders have fairly consistent phenotypes, especially within sibships, which is not observed for Cx26–35delG. This suggests the possibility of other factors modulating the expression of the mutant gene. One intriguing possibility is that there may be a second connexin gene that shares partial functional redundancy with Cx26. It is conceivable that a second connexin protein can act as a substitute under certain conditions. This would be consistent with the observation of some redundancy for other connexins. Perhaps there are modifying genes in other locations or environmental influences that activate or inactivate the promoter/enhancer regions. If Cx26 is involved in inner ear ion homeostasis, some of these patients are able to function with little loss of hearing, suggesting alternative or compensating homeostatic mechanisms. The loss of Cx26 may affect adversely the development of the auditory system, resulting in variability or asymmetry.
There may be environmental influences, such as noise, that are additive or synergistic with the defects caused by Cx26 mutation, thus increasing hearing loss. As our knowledge of chemicals and other factors that stabilize neural and sensory elements increases, it is conceivable that we may intervene successfully and arrest progression of sensory and neural hearing loss.
The profoundly deaf patients and some of the patients with severe-profound hearing loss will require aggressive language therapy if amplification is not successful. Many of these patients will be candidates for cochlear implantation, which works best when implanted by 24 months of age. Some clinicians are performing cochlear implants as early as 18 months on a clinical trial basis. In our sample, 17/49 have a profound hearing loss and are candidates for a cochlear implant. An additional 13/49 have severe-profound hearing loss, some of whom (approximately one third) will become candidates for cochlear implant. All will need language strategies implemented as early as possible.
Establishing progression on review of multiple audiograms over time is difficult and scientifically hazardous. The progressive nature of the hearing loss in one third of the Cx26 cases (for whom sufficient data exist) is notable and raises several important issues. This means that a child with a moderate loss may progress into the profound range; therapies are very different between these two magnitudes of hearing loss.
Families having a single child with moderate-severe hearing loss or worse will benefit from Cx26 analysis. The genetic nature of a sporadic case often is unappreciated. Testing for Cx26 can quickly identify a significant proportion as being recessive, when positive, genetic, audiologic, and developmental language counseling can be offered so that parents can make informed decisions.
The feasibility and benefit of screening for Cx26 mutation is quickly going to become an important public health issue. The importance of early detection of hearing impairment is well established. Cx26 mutation testing can not pick up all hearing-impaired infants, and it would be unreasonable to expect such testing to replace existing hearing screening programs. Whether ABR- or OAE-based infant hearing screening programs should include Cx26 mutation diagnosis is another matter. The use of Cx26 testing in conjunction with failed infant audiologic testing will help define a group in which ∼40% may not respond to early hearing aid use and will require total language intervention. Many of those children will be candidates for cochlear implantation. Future studies should be prospective, including surveys of newborns who fail diagnostic hearing testing. This will help determine the percentage of children with early progressive hearing loss and the number not responding to traditional hearing amplification at early critical stages of language development.
This work was supported by National Institutes of Health NIDCD Grants P01 DC01813-05 and R01 DC02942-02.
- Received October 23, 1998.
- Accepted November 23, 1998.
Reprint requests to (W.J.K.) Boys Town Research Hospital, Omaha, NE 68131.
- ABR =
- auditory brainstem response •
- OAE =
- otoacoustic emission •
- NSRHL =
- nonsyndromic recessive hearing loss •
- EKG =
- electrocardiography •
- CT =
- computed tomography •
- PTA =
- pure tone average
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- Copyright © 1999 American Academy of Pediatrics