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Annals of Clinical & Laboratory Science 38:352-360 (2008)
© 2008 Association of Clinical Scientists

Genetic Analysis of Presbycusis by Arrayed Primer Extension

Juan Rodriguez-Paris1, Charles Ballay4,*, Michelle Inserra4,**, Katrina Stidham4, Tahl Colen4, Joseph Roberson4, Phyllis Gardner2 and Iris Schrijver1,3
1 Departments of Pathology, 2 Medicine, and 3 Pediatrics, Stanford University School of Medicine, Stanford, California; 4 California Ear Institute, Palo Alto, San Ramon, and San Jose, California

Address correspondence to Iris Schrijver, M.D., Department of Pathology, L235, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA; tel 650 724 2403; fax 650 724 1567; e-mail ischrijver{at}stanfordmed.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using the Hereditary Hearing Loss arrayed primer extension (APEX) array, which contains 198 mutations across 8 hearing loss-associated genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5, 12S-rRNA, and tRNA Ser), we compared the frequency of sequence variants in 94 individuals with early presbycusis to 50 unaffected controls and aimed to identify possible genetic contributors. This cross-sectional study was performed at Stanford University with presbycusis samples from the California Ear Institute. The patients were between ages 20 and 65 yr, with adult-onset sensorineural hearing loss of unknown etiology, and carried a clinical diagnosis of early presbycusis. Exclusion criteria comprised known causes of hearing loss such as significant noise exposure, trauma, ototoxic medication, neoplasm, and congenital infection or syndrome, as well as congenital or pediatric onset. Sequence changes were identified in 11.7% and 10% of presbycusis and control alleles, respectively. Among the presbycusis group, these solely occurred within the GJB2 and SLC26A4 genes. Homozygous and compound heterozygous pathogenic mutations were exclusively seen in affected individuals. We were unable to detect a statistically significant difference between our control and affected populations regarding the frequency of sequence variants detected with the APEX array. Individuals who carry two mild mutations in the GJB2 gene possibly have an increased risk of developing early presbycusis.

Keywords: presbycusis, genetic mutations, GJB2 gene, microarray analysis of genomic DNA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Age-related hearing loss, or presbycusis, is the most common type of hearing loss in elderly individuals and constitutes an important public health concern in industrialized nations. It occurs in >25% of people aged 65 yr and increases, both in prevalence and severity, with advancing age [1]. This multifactorial disorder is thought to be precipitated by numerous contributing factors including noise, other environmental exposures, trauma, vascular insults, metabolic changes, and genetic susceptibilities [2–7]. It is characterized by a reduction in hearing sensitivity that begins in the high frequencies and progresses to encompass the mid- to low frequencies, concomitant with a reduction of language discrimination in environments with background noise. In many affected individuals, the speed of central processing is reduced and the isolation of sound is hampered, leading to difficulties in understanding speech. As a result of these additive impairments, presbycusis adversely affects communication, safety, and the quality of life [8].

Historically, presbycusis was divided into 4 groups according to the patterns of hearing loss, which correlated with the location of the hearing defect as determined by temporal bone analysis: sensory (outer hair-cell loss); neural (ganglion-cell loss); metabolic (strial atrophy); and cochlear conductive (stiffness of the basilar membrane) [9]. These groups are no longer considered inclusive [10–12] and alternative categories have been proposed, including those encompassing patterns associated with central conditions [13–15]. At present, the most widely accepted otologic etiologies of presbycusis are an atrophy of the stria vascularis or an impairment in the production or availability of energy ("metabolic presbycusis"), but this field of investigation continues to evolve [16–18].

Several biological pathways, loci, and genes have recently been associated with presbycusis. Mitochondrial DNA mutations, mitochondrial dysfunction, and mitochondrial haplotypes have all been implicated [19–23]. Mouse models have been used to identify mitochondrial and nuclear genes that promote age-related hearing loss [24]. In humans, genome-wide screening has identified chromosome regions 11p, 11q13.5, and 14q, all of which overlap with genes known to cause congenital hearing loss [25]. Another such screen suggested that genetic variation in the DFNA18 locus on chromosome 3q may contribute to presbycusis in the general population [6]. At the gene level, associations have been identified with ACTG1, NAT2, KCNQ4, GSTM1, GSTT1, and the GRHL2 genes [26–30], and changes in gene expression levels have been related to this type of hearing loss [31]. Despite this recent progress, there is not yet a complete understanding of the relative contribution of each of these genetic factors, and of the role of genes associated with congenital hearing loss in the susceptibility and development of age-related hearing impairment. A suggested genetic evaluation for patients with age-related hearing loss, therefore, is not yet available or appropriate.

The rationale for our study was to determine if patients with presbycusis have a higher mutation frequency in genes that are clearly or potentially associated with congenital sensorineural hearing loss, compared to the general population. We hypothesized that even heterozygous carriers of recessive mutations may have an increased risk of developing presbycusis over time. The arrayed primer extension (APEX) platform was chosen because it has been used reliably by us and others as a relatively comprehensive molecular testing tool for congenital sensorineural hearing loss.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study subjects  The study was conducted at Stanford University with 94 presbycusis patients who were recruited at the California Ear Institute under IRB approval with informed consent, and with 50 unaffected control individuals in the same age range, who were recruited at Stanford under a separate IRB approval process. This cross-sectional study included patients between the ages of 20 and 65 yr at the time of the first available audiogram, with apparently adult-onset sensorineural hearing loss of unknown etiology of any configuration and degree (>10 dB at two or more frequencies). Even though it cannot be entirely excluded that this group may include patients with adult-onset hearing loss of specific etiologies, or individuals with mild hearing loss of earlier onset, all individuals in this study carried a diagnosis of (early) presbycusis.

Ideally, a normal audiogram prior to the onset of hearing loss would be included as evidence of the development of presbycusis, but since patients do not come to clinical attention prior to the onset of complaints, it was not possible to include such information in our study. We included fairly young individuals to enrich our study population for genetic etiologies rather than cumulative environmental factors. All individuals with known or suspected causes of hearing loss were excluded from the study. Such factors included significant exposure to noise, a history of trauma, intra-uterine infection, ototoxic medications, tumor or other known conditions that affect hearing, congenital or pediatric hearing loss, or the manifestation of a recognized syndrome.

Mutation selection  The 198 mutations on the APEX microarray were previously selected from relatively well characterized genes for which mutations and sequence variants have been reported. The genes on this microarray are the nuclear genes GJB2, GJB6, GJB3, GJA1, SLC26A4, and SLC26A5, and mitochondrial genes 12S-rRNA and tRNA Ser. These genes have been associated with mostly non-syndromic, sensorineural hearing loss, although evidence that the GJB3 and GJA1 genes are associated with hearing loss is as yet inconclusive. Mutations were chosen from the literature and from additional web-based sources: (1) the Connexin Deafness home page (http://davinci.crg.es/deafness/), (2) the Mitomap database (www.mitomap.org/), (3) the Hereditary Hearing Loss home page (http://webh01.ua.ac.be/hhh/), and (4) the Human Gene Mutation Database (www.hgmd.cf.ac.uk/) [32]. The mutation list includes single nucleotide changes, which are, from a technical perspective, the most straight forward to detect with the APEX platform. However, it also includes insertions and deletions, including the ~309-kb deletion affecting the GJB6 gene [32].

APEX arrays and analysis  The APEX microarrays (Asper Biotech) were used as previously described [32]. In brief, wild-type consensus gene sequences for both the sense and antisense directions were used as templates for oligonucleotide primer design (www.ncbi.nlm.nih.gov/Genbank/). These oligonucleotides were designed to enable accurate discrimination of a nucleotide substitution in the designated position. Genomic DNA was then amplified, purified, fragmented, and hybridized to the microarray in an isothermic APEX reaction. Following a washing step, duplicate fluorescent signals for both complementary DNA strands were visualized with a Genorama Quattroimager (Asper Biotech), indicating which nucleotide was present in each individual position under investigation. Thus, every mutation site corresponded with 4 data points for final interpretation. This reduced the potential for false positive signals and allowed a clear distinction between homozygous and heterozygous calls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
APEX array  The 198 mutations on the Hereditary Hearing Loss APEX array were selected to create an assay that would be more comprehensive than the current approach to genetic testing for seemingly non-syndromic hearing loss, and yet still practical in terms of the number and interpretation of included mutations. Thus this single assay can provide a molecular diagnosis for individuals with sensorineural hearing loss, can help focus subsequent testing in those individuals with a single identified mutation, and can offer carrier detection for hearing loss of recessive inheritance. With autosomal recessive inheritance patterns, carriers are expected to have a single (heterozygous) mutation, whereas affected individuals are expected to carry two. When the hearing loss is caused by two mutations on the array, then either compound heterozygosity at two separate mutation sites, or homozygosity at a single mutation site will be observed. When the sensorineural hearing loss is dominant, only a single (dominant) mutation is expected.

In addition to well-characterized pathogenic mutations in the connexin, pendrin, prestin, and mitochondrial genes, a few mutations of uncertain clinical significance were included in this initial version of the array. In the SLC26A5 gene, the functional effects of the IVS2-2A>G splicing variant are not fully understood [33]. Examples in the GJB2 gene include V27I and E114G, which, when present separately, are considered to be innocent polymorphisms, but when present together on a single allele (in Cis) appear to be additive, amounting to the effect of a mild mutation [34]. The I203T variant is now classified as a polymorphism (the connexin-deafness homepage: http://davinci.crg.es/deafness/). Sequence changes M34T and V37I appear to have mild effects, although knowledge regarding the implication of these changes continues to increase [35–38]. Finally, the evidence that the GJB3 and GJA1 genes are associated with hearing loss has not yet been definitively demonstrated, but these genes were initially included on this first version of the microarray because sequence variants have been reported and inclusion of these changes on our array allowed routine investigation of their frequencies. A representative result of the APEX array from a presbycusis subject is presented in Fig. 1Go. The relatively common M34T amino acid substitution was detected by the APEX array in both the forward and reverse sequence directions.


Figure 1
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  		Fig. 1. APEX detection of M34T (101T>C) in the GJB2 gene. Row 1. Wild-type genotype. In the sense direction (S) the wild-type T allele is present, and in the antisense (AS) direction the complementary A allele is identified. Row 2. Heterozygous for M34T. S: the wild-type T allele and mutant C allele are both identified. AS: the wild-type A allele and the mutant G allele are present. Row 3. Wild-type genotype.

 
Identified mutations  The sequence changes identified in 50 control individuals (100 separate alleles) and 94 presbycusis subjects (188 separate alleles) are summarized in Tables 1Go and 2Go. All sequence changes were tabulated, including those classified as polymorphisms (the connexin-deafness homepage: http://davinci.crg.es/deafness/). In the control group, 18% (9/50) carried sequence changes vs 18.1% (17/94) of the presbycusis subjects. The allele frequency of sequence changes was 10% (10/100) in the control group and 11.7% (22/188) in the affected individuals. Polymorphic alleles (including the IVS2-2A>G change in the SLC26A5 gene) were seen in 3% (3/100) of control alleles and 4.3% (8/188) of patient alleles. Thus, pathogenic alleles were identified in 7% (7/100) of the control alleles and 7.5% (14/188) of all alleles in the presbycusis group. This takes into account that the individual with three sequence variants most likely carried the V37I mutation on one allele and the V27I and E114G variants on the other, since these are frequently observed in Cis. We also assumed that the individuals who are compound heterozygous for V27I and E114G carried these variants on opposite alleles, in which case they should be considered innocent polymorphisms. If this were not the case, the pathogenic allele frequency would amount to 8.5% (16/188).


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  		Table 1. Genotypes identified in 50 unaffected controls.
 

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  		Table 2. Genotypes identified in 94 presbycusis patients.
 
No individuals in the control group carried two pathogenic mutations (Table 1Go). In the presbycusis group, however, there were two individuals homozygous for the mild mutations M34T and V37I, respectively (Table 2Go) [34–37]. An audiogram of the patient with the V37I/V37I genotype demonstrated a characteristic hearing loss pattern for presbycusis (Fig. 2aGo). In addition, we found three compound heterozygous individuals. Interestingly, the first individual carried three sequence changes, which most likely occurred in the configuration with V37I on one allele and V27I with E114G on the other, since the latter two variants can be observed together [39]. The audiogram of this individual is shown in Fig. 2bGo. The second and third individual carried V27I and E114G. Without subcloning the alleles or testing parent samples, it is not possible to determine the allele configuration. Even though it is plausible that these variants occurred in Cis and the subjects should be classified as functional heterozygotes, we chose the conservative approach of assuming that these variants resided on opposite alleles, and were polymorphisms without a pathogenic effect. Thus, 3.2% (3/94) presbycusis subjects carried two functional GJB2 mutations.


Figure 2
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  		Fig. 2. Sloping high frequency hearing loss in two subjects with sequence changes in the GJB2 gene. (a) Audiogram of a patient with the V37I/V37I genotype. (b) Audiogram of a patient with the V27I/V37I/E114G genotype. The sloping high frequency hearing loss pattern observed in both individuals is characteristic for presbycusis.

 
Of the 8 genes represented on the APEX array, sequence changes were only found in three genes: (1) the GJB2 gene, which encodes the connexin 26 protein and is frequently tested in patients with non-syndromic sensorineural hearing loss, (2) the SLC26A4 gene that encodes the pendred protein, and (3) the SLC26A5 gene that encodes prestin.

Statistical analysis to determine the difference between the frequency of sequence changes in control individuals and presbycusis subjects was calculated using Fisher’s exact test. The percentages in the affected vs unaffected group were very close and did not reach significance (p = 1.000). The odds-ratio of developing presbycusis if one carries a non-polymorphism sequence variant present in the APEX array is 1.069 with a 95% confidence interval of 0.417 to 2.74. Three of 94 presbycusis subjects (3.2%) carried two GJB2 mutations, compared to none of the controls. This difference between the affected and control groups was not significant (p = 0.5515, odds-ratio = 3.863, 95% confidence interval 0.196 to 76.35).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Arrayed primer extension (APEX) technology is especially well-suited for the molecular diagnosis of conditions that can result from multiple mutations located in multiple genes, because it can evaluate all spotted mutations within a single test. APEX microarrays have been developed by us for cystic fibrosis [40], Ashkenazi Jewish mutations [41], and sensorineural hearing loss [32], and by others for conditions such as Usher syndrome [42], and retinal phenotypes associated with the ABCA4 gene [43].

The Hereditary Hearing Loss microarray for sensorineural hearing loss encompasses mutations from six nuclear and two mitochondrial genes, and includes frequently affected genes underlying genetic sensorineural hearing loss. It does not include the Usher genes, for which a separate APEX array can now be used [42]. In this study, we applied our array to a study of mutation frequencies in individuals with presbycusis.

Presbycusis is commonly manifested in the aging population and is characterized by bilateral, sensorineural, high-frequency and often progressive hearing loss. Presbycusis is multifactorial; the genetic contributors to presbycusis are not yet clear, although recent associations with susceptibility regions have been identified through genome-wide linkage analysis [6,25]. Targeted studies of associations with specific candidate genes yielded a positive association with a SNP in the ACTG1 gene [26] and the NAT2 gene [27,29], with a 13 kb region in the KCNQ4 gene [28], with deletion polymorphisms in GSTM1 and GSTT1 [29], and with SNPs in the GRHL2 gene [30].

It was suggested that unaffected carriers of the common 35delG mutation in the GJB2 gene may have an increased risk of presbycusis, because of indirect evidence of cochlear outer hair cell changes. Individuals without the mutation demonstrated distortion product oto-acoustic emissions that were larger than those of 35delG heterozygotes at all frequencies, and although the number of responses decreased with higher frequencies in both groups, 50–70% of the carriers had no responses between 6000 and 10000 Hz, compared to 30–60% of non-carriers. Thus, the hearing of carriers of the 35delG mutation seemed impaired at the very high frequencies of 8000–10000 Hz, which could remain undetected because these frequencies are not routinely assessed by audiometry or auditory brainstem evoked potentials [44]. In support of this notion, it had been suggested that potassium channel pathology in general might contribute to the development of presbycusis, and specifically through an as yet unidentified SNP in the KCNQ4 voltage-gated potassium channel gene [28]. Van Eyken et al [45], however, demonstrated that there was no increased susceptibility for the development of age-related hearing loss in carriers of the 35delG mutation in the GJB2 gene. Our findings confirm these results for the 35delG mutation, and for the 97 other mutations in the connexin 26 potassium channel gene (GJB2), present on the APEX array.

In a recently published candidate susceptibility gene study that identified a highly significant association of presbycusis with the GRHL2 gene [30], a total of 70 candidate genes were investigated, including GJB2, GJB3, GJB6, SLC26A4, and SLC26A5, mutations, which are represented on the Hereditary Hearing Loss APEX array. Our results are congruent with the findings of this large association study. We did not identify any mutations in genes other than GJB2 and SLC26A4 in our presbycusis population, most likely because of the low general frequency of such changes.

Our study shows that the sensorineural APEX array is not helpful in the genetic work-up of individuals with presbycusis, and that the carrier frequencies of unaffected and affected individuals are not significantly different. It is interesting that the group of individuals clinically diagnosed with early presbycusis includes individuals with more than one mutation in the GJB2 gene, whereas our control group did not. The homozygous samples included the V37I/V37I and the M34T/M34T genotypes. Both these mutations are considered to be mild, but have not previously been associated with age-related or progressive hearing loss [34–37]. It seems plausible that the negative effects on distortion product oto-acoustic emissions observed in 35delG mutation carriers are more pronounced in individuals who carry two mild mutations. The oto-acoustic changes in carriers were observed at all frequencies but were more pronounced in the higher frequencies, which are affected first in age-related hearing loss [44]. The compound heterozygous V37I/V27I/E114G sample would likely fall in the same category. The two patients with V27I and E114G would be considered carriers if these variants occurred on the same allele, and unaffected if they occurred on opposite alleles, and should not be considered part of the compound heterozygous group. For all these individuals it is possible that the hearing loss was pre-existing but undiagnosed. Alternatively, together with accumulating environmental contributors, age-related hearing loss may be precipitated sooner in the presence of a mild GJB2 defect in potassium recycling.

In conclusion, molecular diagnostic genetic testing in adults with age-related hearing loss is very limited at this time, since genetic contributors to presbycusis are just in the process of being discovered. Although GJB2 and most other genes on the APEX array were logical candidate genes for age-related hearing impairment, our findings, together with those in the recent literature [30], indicate that the mutations on the APEX array do not substantially contribute to age-related hearing loss. An exception to this, however, may be the homozygosity or compound heterozygosity for mild GJB2 mutations, in which case the associated hearing loss may, at least potentially, not reach a manifesting threshold early in life. As such, this study raises an interesting genotype-phenotype possibility for GJB2 gene mutations. Considering the small number of homozygous and compound heterozygous individuals, larger studies of presbycusis subjects with documented normal hearing in childhood will be necessary to confirm these findings.


   Footnotes
 
* New affiliation: Dept of Otolaryngology, Kaiser Permanente, Santa Clara, CA. Back

** New affiliation: Otolaryngology practice, Kerrville, TX. Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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