Part IV: CASE STUDIES: Using Human Genome Epidemiology Information to Improve Health Chapter 24

From Epidemiology to Clinical Practice: The Connexin Connection

Aileen Kenneson, Coleen Boyle

Tables | Figures | References

Individuals who are deaf or hard-of-hearing account for a significant portion of the population. Currently about one sixth of the U.S. population (i.e. about 40 million individuals) have some degree of hearing loss, a half of a million of which fall into the severe-to-profound range.(1) Hearing loss may occur late in life, or may be present at birth, as is the case for one to three in 1000 newborns.(2) Formerly, these children were not diagnosed until they were two to three years of age,(3) resulting in developmental delay, particularly in the arena of language development.(4) However, recent technological advances and increased public health attention have resulted in the development of Early Hearing Detection and Intervention (EHDI) programs in most of the United States. EHDI programs seek the early identification of infants with hearing loss via universal newborn hearing screening programs followed by linkage with appropriate intervention options, of which there are many, resulting in the avoidance of developmental delays.(4-6)

Genetic evaluations are often included as part of the medical care of individuals with hearing loss. Based on family history and physical examination, clinical geneticists categorize hearing loss into syndromic and non-syndromic cases, a distinction that is useful both for appropriate clinical care and estimation of recurrence rates.(7) Recent advances in our understanding of single-gene causes of both syndromic and non-syndromic hearing loss are likely to increase the role of specific genetic tests in the evaluation of individuals with hearing loss.

As genetic research progresses and the demand for use of genetic testing increases, genetic tests often move rapidly from research interest into clinical use, sometimes before the clinical utility of the test is fully defined.(8,9) This is the case for GJB2, a gene recently implicated in up to 50% of cases of non-academic hearing loss in some populations. Thus, GJB2 testing may serve as a model by which we can examine the translation of research advances into clinical and public health use.

Background

Translation of sound waves into integrated neuronal signals in the brain is an amazingly complex process, so it is not surprising that hundreds of genes are involved in the development and operation of this machinery.(10) Variation in any one of these genes can result in hearing loss. The extent of locus heterogeneity of hearing loss is illustrated by the more than 400 hereditary syndromes which claim hearing loss as a component,(11) and which account for about 30% of cases of hearing loss.(12-14) Heterogeneity is also observed in non-syndromic cases, which are typically sensorineural in nature, and for which there is linkage evidence for almost 70 loci, including autosomal recessive variants in 75-80% of non-syndromic cases (designated with the prefix DFNB), autosomal dominant variants in 20-25% (DFNA), and X-linked variants in 1-1.5% (DFN).(15)

The estimated numbers and types of loci for non-syndromic hearing loss based on linkage analysis are: 30 autosomal recessive, 29 autosomal dominant, 8 X-linked, and two mitochondrial.(16) The Gap Junction Beta 2, GJB2, gene was recently identified as the source of both the DFNB1 (autosomal recessive) and the DFNA3 (autosomal dominant) loci.(17-20) The GJB2 gene encodes for connexin 26, a beta class gap junction protein expressed in the cochlea and in the epidermis. Connexin 26 hexamers form channels between cells that, when open, allow cell-to-cell diffusion of small molecules. This function is necessary for the recycling of potassium in the cochlea that is critical for sensorineural hearing function.(21) More than 90 variants of the GJB2 gene have been reported, and many are rare; recessive alleles, dominant alleles, and polymorphisms have all been described.(22)

Epidemiological Findings

Contribution of GJB2 Variants to Hearing Loss

A large number of studies related to the association between hearing loss and GJB2 variants in a broad range of populations have been published in the past few years. In general, the conclusions are limited by factors such as small sample sizes, lack of population-based ascertainment methods and lack of population descriptions. For example, a common source of ascertainment is hearing loss clinics; potential biases in this scenario include self-selection for clinic attendance, and under-ascertainment of mild or unilateral hearing loss. Ascertainment details are often not published, and reports often lack population descriptions including age, sex, and race/ethnicity. Comparison between studies is also limited by the differences in inclusion criteria, which have included all familial cases, recessive cases, sporadic cases, or all cases combined. However, despite the limitations, enough data have been amassed that we can begin to develop a picture of the relationship between GJB2 and hearing loss.

Given the extraordinary genetic heterogeneity of non-syndromic hearing loss, it was believed that no single gene would play a significant role in its etiology. So it was surprising to discover that sequence variations at the GJB2 locus account for up to 50% of cases of non-syndromic prelingual sensorineural hearing loss in some populations. While more than 90 alleles have been described in the literature, three account for the majority of GJB2-related hearing loss in studied populations: 167ΔT, 35ΔG and 235ΔC, the most common variant alleles in the Ashkenazi Jewish population(23,24), populations of northern European descent(25-32), and in the Korean(33) and Japanese(34-36) populations, respectively.

Figure 24-1 summarizes data from several sources and depicts the contribution of the GJB2 variants to hearing loss in several different populations around the world. The available population-specific epidemiological data consistently indicate population differences in two key measures that are important to determine the clinical validity of genetic testing: (1) the percent of cases of non-syndromic hearing loss that is associated with GJB2 variants, and (2) the population frequency of the different GJB2 alleles. For example, non-syndromic sensorineural hearing loss is associated with GJB2 variants in almost 50% of cases in Israel, but only 8% in Korea and 20% in Japan. Likewise, the 35ΔG allele accounts for about 10-20% of cases of non-syndromic hearing loss in persons of northern European descent, but about 30-40% of cases in Mediterranean regions.

Table 24-1 presents the contribution of GJB2 variants to non-syndromic hearing loss in several populations. The table presents both the percent of cases of hearing loss associated with GJB2 variants in general, as well as the percent of cases of hearing loss that are associated with the most common allele in that population. For example, in European and North American whites, 38% of individuals with hearing loss carry one or more GJB2 variant allele, but 34% of individuals with hearing loss carry one or more copies of 35ΔG. Thus, the 35ΔG allele accounts for the majority of variant alleles in this population. In contrast, 50% of Ashkenazi Jewish individuals with hearing loss carry a GJB2 variant allele, but only 31% carry at least one copy of the 167ΔT variant. Thus, there is more allelic heterogeneity in the Ashkenazi Jewish population than there is among European and North American Whites. Table 24-1 also demonstrates that these alleles are not uncommon in the general population. The 235ΔC allele is carried by 1% of individuals in Korea and Japan, and the 35ΔG allele is carried by 1 in 50 Whites. In the Ashkenazi Jewish population, the 167ΔT allele may be carried by up to 8% of the general population.

In general, there is a lack of phenotype data related to the contribution of the less common GJB2 variants to hearing loss. In addition, because of the small numbers of individuals with mild, unilateral, and late-onset hearing loss included in studies, the potential involvement of GJB2 variants with the full spectrum of forms of hearing loss has not been fully assessed, particularly in the case of the less common variants.

GJB2 Variants and Age of Onset

To fully assess the relationship between GJB2 variants and age of onset, genotype data is needed on individuals with congenital (present at birth), non-congenital prelingual, postlingual, and late-onset (after age 30) hearing loss. In the absence of newborn hearing screening, hearing loss is usually not diagnosed until late infancy or early childhood. Thus, in most published studies, it is not possible to distinguish between congenital and non-congenital prelingual hearing loss.
Only one published study has examined the contribution of GJB2 variants to congenital hearing loss. Allele-specific methods were used to determine the prevalence of the 35ΔG and 167ΔT genotypes in 42 infants identified with hearing loss through the Rhode Island universal newborn hearing screen. The study identified three 35ΔG homozygotes, two 35ΔG/167ΔT compound heterozygotes, and one 35ΔG carrier. The two compound heterozygotes were reported as having Ashkenazi Jewish ancestry. The remaining newborns were of mixed European background. Thus, the 35ΔG and 167ΔT genotypes in this newborn population with hearing loss did not differ from other American populations with hearing loss who were ascertained in childhood, and who were of similar race/ethnicity (Table 24-1 ).(37)

More studies of this type, as well as studies including documented non-congenital prelingual hearing loss, are needed to assess the relationship between GJB2 variants and congenital hearing loss. In this regard, it is important to note that there have been case reports of newborns who passed the newborn hearing screen but were diagnosed with GJB2-related hearing loss later in infancy.(38,39) It is not clear whether these cases represent false negative results of the newborn hearing screening programs or are indicative of a non-congenital and/or progressive nature of some GJB2-related cases of hearing loss.

Only four studies have addressed the possibility of an association between GJB2 variants and postlingual hearing loss. Three of these studies did not detect any GJB2 variants among individuals with postlingual hearing loss, including 11 individuals in Israel (age of onset undefined)(24), 16 in France (onset before age 20)(26) and 39 in Japan (onset between 3 and 30 years).(36) The fourth study, taking place in Austria, found 4 carriers of GJB2 variants among 16 individuals with postlingual (undefined) hearing loss.(40) The genotypes were L90P/I20T (onset in first decade), L90P/35ΔG (onset in first decade), 35ΔG/+ (onset in first decade), and G160S/+ (onset in fourth decade). The L90P allele is of interest in this population because it is seen in 2 of 16 postlingual cases, and 3 of 53 prelingual cases of hearing loss. Thus, this allele may contribute to postlingual, as well as prelingual, hearing loss. The failure to detect GJB2 variants in the other three postlingual studies may be due to a higher prevalence of the L90P allele in the Austrian population, as this allele was detected only rarely in individuals with hearing loss in France (2 of 88)(26) and Italy (3 of 147)31,41, but not at all in Israel (0 of 102)(23,24), Japan (0 of 94)(34-36), Korea (0 of 147)(33), Tunisia (0 of 70)(42) or the United Kingdom (0 of 210).(30)

Another allele, C202F, has also been implicated in postlingual hearing loss, as it was observed to co-segregate with hearing loss (age of onset between 10 and 20 years) over five generations in a French family.(43) This allele was not detected in 95 French control individuals(43), nor has it been reported in other studies that provided sequence data on controls, including 100 Korean newborns(33), 209 Japanese individuals(34-36), and 119 additional French individuals.(27)
No published studies have assessed the possible relationship between GJB2 variants and late-onset (after age 30) hearing loss. Thus, additional population-based studies involving individuals with congenital, non-congenital prelingual, postlingual, and late-onset hearing loss will be needed to fully assess the relationship between GJB2 variants and age of onset.

Factors Influencing Phenotypic Outcome

Many study groups have reported that the degree of hearing loss of individuals with the same GJB2 genotype varies in severity, even within sibships.(23,24,26,31) This suggests that other factors, genetic and/or environmental, may be acting to modify the phenotypic outcome of GJB2 variant alleles. Hearing loss is typically described as 50% genetic and 50% environmental in nature, involving a wide range of both genetic and environmental factors(12); any number of these factors could potentially influence the phenotypic expression of GJB2 variant genotypes.

Influence of Environmental Factors

The possible contribution of environmental factors to GJB2-related hearing loss has not been assessed. Most of the studies pertaining to the contribution of GJB2 variants to hearing loss excluded cases with known risk factors from the genetic analysis. These factors include infections (e.g., meningitis, rubella), low birth weight, ventilator use, ototoxic medications (e.g., aminoglycosides), and hyperbilirubinemia. However, a case report of two individuals with hearing loss originally attributed to rubella infection that were later found to be homozygous for the 167ΔT variant 39 suggests that the presence of known risk factors should not necessarily preclude genetic analysis. Indeed, the proportion of GJB2 cases that have been attributed to other causes has not been elucidated, and the possibility of gene-environment interactions has not been examined. Studies pertaining to the relationship between GJB2 variants, environmental factors, and hearing loss may identify factors that modify the GJB2 phenotype, and may implicate GJB2 variants in the susceptibility to known ototoxic factors. In addition to lending clues about the developmental etiology of hearing function, studies of this nature are important for accurate genetic counseling. For example, in the above case report, the couple would have originally been counseled that the chance of having a child with hearing loss was low due to the environmental nature of their hearing loss. However in retrospect, it actually was 25% due to the recessive nature of their alleles.

Contribution of GJB2 Variants to Syndromic Hearing Loss

The currently published GJB2 studies have generally excluded cases of syndromic hearing loss from analysis, thus precluding the analysis of possible gene-gene interactions in the phenotypic expression of these syndromes. One study in the United Kingdom included DNA analysis of seven families with syndromic hearing loss. The DNA analysis looked only for the 35ΔG allele, which was not detected in any of these families.(44) The small number of participants and the allele-specific DNA analysis limit any conclusions about the role of GJB2 variants in syndromic hearing loss.

Over 400 different recognizable syndromes have hearing loss as a component, varying in degree of loss, age of onset, and penetrance.(11) The aforementioned variation in degree of hearing loss in siblings with identical GJB2 genotypes indicates the importance of genetic and/or environmental backgrounds in the expression of GJB2-related hearing loss. Hence, it is also possible that variants in genes such as GJB2 influence the penetrance and expressivity of hearing loss associated with syndromes. This possibility remains to be explored.

Laboratory Detection Techniques

The known genetic variants in the GJB2 gene are amenable to detection by standard molecular genetic laboratory techniques. The majority of GJB2 variants fall in the 680-basepair coding region in exon 2, and the rest fall in the 3’ untranslated region in exon 1. Detection methods include allele-specific PCR-based methods, scanning technologies such as SSCP, and sequencing. As some common alleles account for the majority of variants in some populations, allele-specific methods are often used, either alone or in conjunction with sequencing methods. A recent survey of laboratory practices pertaining to clinical use of GJB2 testing indicated that U.S. laboratories vary in their chosen methodology. Most of the laboratories used sequencing, either alone or as a follow-up to allele-specific methods. Of the laboratories that employed sequencing, most analysed exon 2 only, while a few sequenced both exons 1 and 2.(45) Most of the published studies that have utilized sequencing methods have included analysis of exon 2 only. Therefore, information is lacking to accurately determine the relative clinical validity and utility of these two methods.

Potential Contribution Of Genetic Information To Improved Health Outcomes

The American College of Medical Genetics recommends the provision of genetic services to individuals with hearing loss “to establish the etiology whenever possible”(46), and GJB2 testing may be one potential option in this process. Several potential clinical uses of GJB2 testing in children with hearing loss have been proposed, including (1) ruling-out risk of syndromic complications, (2) predicting moderate-to-profound hearing loss requiring aggressive language intervention, (3) indicating potential candidacy for cochlear implants use, and (4) allowing genetic counseling regarding recurrence rates.(37,47,48) However, there is little evidence in support of some of these proposed uses, and there are many factors to be weighed in the decision to include GJB2 testing.

Much of the information regarding a child’s phenotype can be obtained through physical examination by audiologists, otolaryngologists and clinical geneticists, so a child’s course of intervention may or may not be significantly altered by the knowledge of GJB2 genotype. It has been argued a GJB2 diagnosis may reduce the burden of additional tests that are traditionally used to rule-out syndromic complications (e.g., ophthalmologic, cardiac, vestibular evaluations).(48) This may be particularly relevant in the case of infants with hearing loss, as the medical tests used to distinguish syndromic and non-syndromic cases may not have as much predictive power during infancy as they do later in childhood. However, the potential role of GJB2 variants in the penetrance and expressivity of hearing loss in syndromes has not been assessed, so while the presence of GJB2 variants in a newborn with hearing loss will most likely be associated with non-academic hearing loss, more data need to be collected to determine the sensitivity and specificity of this use of GJB2 testing. Likewise, the use of GJB2 testing in the prediction of the success of various intervention options has not been assessed.

Determination of a genetic etiology also allows for the provision of recurrence information for the family. However, organizations of professional geneticists, including the American Society of Human Genetics and the American College of Medical Genetics, generally discourage the genetic testing of minors in the absence of direct intervention benefits for the child.(49) Given the Deaf community’s concerns about genetic testing(50), this point is particularly germane in regards to hearing loss because a child may prefer not to know this information as an adult.

Another factor to be considered in the decision to use GJB2 testing is the lack of epidemiological data pertaining to the less common variants. This paucity of information must be considered when counseling families about GJB2 test options and results. Consider, for example, the M34T allele. In 1997, the M34T variant was found to co-segregate with 3 generations of hearing loss in one family in an apparently dominant fashion.(20) This was the first evidence implicating GJB2 in non-syndromic hearing loss. But several years later, a second variant was characterized in this family, found in trans with the M34T allele in the individuals with hearing loss.(51) This suggests that the M34T allele is recessive in nature. Since then, several groups have documented the failure of M34T to co-segregate with hearing loss in several families, raising the possibility that M34T may be a benign polymorphism.(52-55) More recent evidence that supports the recessive allele model includes the observation of M34T compound heterozygotes and homozygotes among individuals with hearing loss but not among control populations.(51-60) This example cautions researchers and clinicians to interpret the role of less common, and hence less-well-characterized, variants with care, particularly in regards to family genetic counseling issues.

These issues will continue to affect an increasing number of families as an increasing number of states are screening newborns for audiologic function so that infants with hearing loss are identified and referred for intervention services very early in life. The role that GJB2 testing will play in conjunction with EHDI programs is still in the process of being defined.

Conclusions

Several areas of current research are aimed at the definition of the clinical utility of GJB2 genetic testing. One such area of research focuses on the potential role of GJB2 genotyping in the prediction of success of various intervention options, such as cochlear implants. A second area of research involves the contribution of GJB2 variants to hearing loss in diverse populations. Given the inter-population variability of the prevalence of GJB2 variants and their apparent contribution to hearing loss, it is would be helpful to define these measures in all potential target populations. In the United States, for example, there is a lack of data pertaining to non-White American populations. Epidemiological data specific to these populations are necessary to provide population-specific determinations of clinical validity and utility.

While GJB2 variants have been shown to be associated with a large fraction of cases of non-syndromic moderate-to-profound prelingual hearing loss, the potential contribution of GJB2 variants to mild, unilateral, late-onset, syndromic, or environmentally-acquired cases of hearing loss has not yet been determined. Research into potential associations such as these may help to unravel the interactions between genetic and environmental influences in the phenotypic expression of hearing function and hearing loss.

The emergence of EHDI programs presents an excellent opportunity for population-based ascertainment of cases of congenital hearing loss. Similar population-based strategies are needed for complete ascertainment of cases of hearing loss arising sometime after the newborn period. Inclusion of all cases of hearing loss, regardless of etiology (syndromic versus non-syndromic, other known genetic factors), presence of known risk factors, degree of hearing loss, and age at onset are required to fully assess the contribution of the GJB2 gene to the spectrum of hearing loss phenotypes. In addition, as greater than 5% of the general population have a hearing loss of some kind(61), the ascertainment of control populations should also be carefully considered in this type of analysis, and should include individuals known not to have a hearing loss. Confounding variables including age, sex, race/ethnicity, and presence of known risk factors are important considerations in case-control analyses of GJB2 variants and hearing loss.

A final note about the process of defining the potential role of genetic testing in medical practice relates to the consideration of the viewpoints of all stakeholders. The case of genetic testing pertaining to hearing loss has raised some important issues in this regard. The goal of the medical community to eliminate disease and disability is at odds with the viewpoint of the Deaf community that hearing loss is not a disability. This viewpoint challenges society to reconsider the definitions of disease and disability. The Deaf community has also expressed concerns that genetic testing will do more harm than good and will devalue individuals with hearing loss.(50) The issues raised by the Deaf community provide a unique opportunity by challenging scientists and society to find culturally-sensitive methods for genetic research and testing that are acceptable to all cultural groups.

Tables

• Table 24-1

Figures

• Figure 24-1

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Address correspondence to Dr. Khoury at
Office of Genomics and Disease Prevention
Centers for Disease Control and Prevention
6 Executive Park, Mail Stop E-82
Atlanta, Georgia 30329