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352 Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008

Genetic Analysis of Presbycusis by Arrayed Primer Extension

Juan Rodriguez-Paris,1 Charles Ballay,4* Michelle Inserra,4** Katrina Stidham,4 Tahl Colen,4
Joseph Roberson,4 Phyllis Gardner,2 and Iris Schrijver1,3
Departments of 1Pathology, 2Medicine, and 3Pediatrics, Stanford University School of Medicine, Stanford,
California; 4California Ear Institute, Palo Alto, San Ramon, and San Jose, California

Abstract. 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 insults, metabolic changes, and genetic suscep-

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
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@stanfordmed.org.
* New affiliation: Dept of Otolaryngology, Kaiser Permanente,
Santa Clara, CA.
** New affiliation: Otolaryngology practice, Kerrville, TX.
0091-7370/08/0400-0352. $3.15. © 2008 by the Association of Clinical Scientists, Inc.
tibilities [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
 Genetic analysis of presbycusis by arrayed primer extension 353
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
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 oligo-
nucleotides were designed to enable accurate discrimination
of a nucleotide substitution in the designated position.
354 Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008
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.
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
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
Table 1. Genotypes identified in 50 unaffected controls.
Heterozygous (n) Compound heterozygous (n)
GJB2; 35delG 1 GJB2; V37I / I203T* 1
GJB2; V27I* 1
GJB2; M34T 1
GJB2; V37I 2
SLC26A4; L597S 1
SLC26A4; D724G 1
SLC26A5; IVS2-2A>G** 1
Total 8 1
* Polymorphism; ** Unknown clinical significance
 Genetic analysis of presbycusis by arrayed primer extension 355

Table 2. Genotypes identified in 94 presbycusis patients.

Heterozygous (n) Homozygous (n) Compound heterozygous (n)

GJB2; 35delG 1 GJB2; M34T/M34T 1 GJB2; V27I/V37I/E114G 1
GJB2; V27I* 2 GJB2; V37I/V37I 1 GJB2; V27I*/E114G* 2
GJB2; M34T 1
GJB2; V37I 1
GJB2; I203T* 2
GJB2; N206S 1
SLC26A4; F335L 1
SLC26A4; L597S 3

Total 12 2 3

* Polymorphism

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.
1. The relatively common M34T amino acid
substitution was detected by the APEX array in
both the forward and reverse sequence directions.
Identified mutations. The sequence changes
identified in 50 control individuals (100 separate
alleles) and 94 presbycusis subjects (188 separate
alleles) are summarized in Tables 1 and 2. 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).
No individuals in the control group carried
two pathogenic mutations (Table 1). In the
presbycusis group, however, there were two
individuals homozygous for the mild mutations
M34T and V37I, respectively (Table 2) [34-37].
An audiogram of the patient with the V37I/V37I
genotype demonstrated a characteristic hearing loss
pattern for presbycusis (Fig. 2a). 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. 2b.
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
356 Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008

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.

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.
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
Arrayed primer extension (APEX) technology is
especially well-suited for the molecular diagnosis of
conditions that can result from multiple mutations
 Genetic analysis of presbycusis by arrayed primer extension 357
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
358 Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008
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.
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