Lack of Methylation Changes in GJB2 and RB1 Non-coding Regions of Cochlear Implant Patients with Sensorineural Hearing Loss

Abstract

Objective. Recent advances in epigenetic studies continue to reveal novel mechanisms of gene regulation and control, however little is known on the role of epigenetics in sensorineural hearing loss (SNHL) in humans. We aimed to investigate the methylation patterns of two regions, one in RB1 and another in GJB2 in Filipino patients with SNHL compared to hearing control individuals.Methods. We investigated an RB1 promoter region that was previously identified as differentially methylated in children with SNHL and lead exposure. Additionally, we investigated a sequence in an enhancer-like region within GJB2 that contains four CpGs in close proximity. Bisulfite conversion was performed on salivary DNA samples from 15 children with SNHL and 45 unrelated ethnically-matched individuals. We then performed methylation-specific real-time PCR analysis (qMSP) using TaqMan® probes to determine percentage methylation of the two regions.Results. Using qMSP, both our cases and controls had zero methylation at the targeted GJB2 and RB1 regions. Conclusion. Our study showed no changes in methylation at the selected CpG regions in RB1 and GJB2 in the two comparison groups with or without SNHL. This may be due to a lack of environmental exposures to these target regions. Other epigenetic marks may be present around these regions as well as those of other HL-associated genes.

Full text

Lack of Methylation Changes in GJB2 and RB1
Non-coding Regions of Cochlear Implant Patients
with Sensorineural Hearing Loss
Angelo Augusto M. Sumalde, MD, PhD,1,2,3 Ivana V. Yang, PhD,4 Talitha Karisse L. Yarza, MClinAud,5,6
Celina Ann M. Tobias-Grasso, BSN, AuD,7 Ma. Leah C. Tantoco, MD, MClinAud,3,5,6 Elizabeth Davidson,4
Abner L. Chan, MD,1,3 Mahshid S. Azamian, MD, MPH, CCRP,8 Teresa Luisa G. Cruz, MD, MHPEd,1,3
Seema R. Lalani, MD,8 Maria Rina T. Reyes-Quintos, MD, MClinAud, PhD,3,5,6 Eva Maria Cuongco-de la Paz, MD,9,10
Regie Lyn P. Santos-Cortez, MD, PhD2 and Charloe M. Chiong, MD, PhD1,3,5,6
1College of Medicine, University of the Philippines Manila, Manila, Philippines
2Department of Otolaryngology – Head and Neck Surgery, School of Medicine, University of Colorado Anschutz Medical Campus (CU-AMC), Aurora, Colorado, USA
3Department of Otolaryngology-Head and Neck Surgery, Philippine General Hospital, University of the Philippines Manila, Manila, Philippines
4Department of Medicine, School of Medicine, University of Colorado Anschutz Medical Campus (CU-AMC), Aurora, Colorado, USA
5Philippine National Ear Institute, National Institutes of Health, University of the Philippines Manila, Manila, Philippines
6Newborn Hearing Screening Reference Center, National Institutes of Health, University of the Philippines Manila, Manila, Philippines
7MED-EL, Innsbruck, Austria
8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA
9National Institutes of Health, University of the Philippines Manila, Manila, Philippines
10Philippine Genome Center, UP Diliman Campus, Quezon City, Philippines
ABSTRACT
Objecve. Recent advances in epigenec studies connue to reveal novel mechanisms of gene regulaon and control,
however lile is known on the role of epigenecs in sensorineural hearing loss (SNHL) in humans. We aimed to
invesgate the methylaon paerns of two regions, one in RB1 and another in GJB2 in Filipino paents with SNHL
compared to hearing control individuals.
Methods. We invesgated an RB1 promoter region that was previously idened as dierenally methylated in
children with SNHL and lead exposure. Addionally, we invesgated a sequence in an enhancer-like region within
GJB2 that contains four CpGs in close proximity. Bisulte conversion was performed on salivary DNA samples from
15 children with SNHL and 45 unrelated ethnically-matched individuals. We then performed methylaon-specic
real-me PCR analysis (qMSP) using TaqMan® probes to determine percentage methylaon of the two regions.
Results. Using qMSP, both our cases and controls had zero methylaon at the targeted GJB2 and RB1 regions.
Conclusion. Our study showed no changes in methylaon at the selected CpG regions in RB1 and GJB2 in the two
comparison groups with or without SNHL. This may be due to a lack of environmental exposures to these target
regions. Other epigenec marks may be present around these regions as well as those of other HL-associated genes.
Keywords: GJB2, hearing loss, methylaon, qMSP, RB1,
sensorineural
INTRODUCTION
An estimated 50% of hearing loss (HL) is believed to be
genetic in origin, with 122 non-syndromic hearing loss genes
identied to date and unique population-specic variants
being continually discovered.1 On the other hand, there is
little evidence on the role of epigenetics in HL. Epigenetics
includes numerous biological processes that result in a change
in phenotype without altering the DNA sequence, most
eISSN 2094-9278 (Online)
Published: September 28, 2023
hps://doi.org/10.47895/amp.v57i9.5200
Corresponding author: Regie Lyn P. Santos-Cortez, MD, PhD
Department of Otolaryngology – Head and Neck Surgery
School of Medicine
University of Colorado Anschutz Medical Campus
12700 E. 19th Ave. MS:8606, Aurora, CO 80045 USA
Email: regie.santos-cortez@cuanschutz.edu
ORCiD: hps://orcid.org/0000-0002-9958-2535
VOL. 57 NO. 9 2023116
ORIGINAL ARTICLE

commonly due to histone acetylation or DNA methylation.2
DNA methylation in promoter regions typically acts to
repress gene transcription.3 For HL, variants in the de novo
methyltransferase DNMT3A aected regulation of genes
involved in otic placode development, suggesting a role for
DNA methylation in inner ear development.4
Despite advancements in investigating DNA methyla-
tion, studies on the epigenetics of HL in humans remain
limited. is is mostly due to the diculty of obtaining
adequate human inner ear tissue for epigenetic study. Studies
on mouse inner ear tissues are more numerous2,3,5, for
example, in a mouse model of the aging cochlea, connexin 26
expression decreased with concomitant hypermethylation of
the promoter region of its encoding gene, GJB26. Although
mouse models could potentially lead to epigenetic targets
in humans, a study that applied the human methylation
proling platform Illumina Innium MethylationEPIC
to mouse samples showed only 1.6% of probes to be
conserved7, indicating low correspondence between human
and mouse methylomes. Previously in Chinese children
with sensorineural hearing loss (SNHL), exposure to lead
and cadmium was associated with dierentially-methylated
regions in the RB1 promoter.8 Currently, this is the only
study that utilized human subjects for an epigenetic study
on SNHL, with blood utilized as a surrogate tissue for the
inner ear. Recent evidence suggests that salivary DNA is a
better surrogate than blood for brain and neural tissue for
methylation studies9,10 and possibly the neuroepithelium
of the inner ear, which would allow direct investigation of
DNA methylation in humans without performing invasive
sampling. In this study, we aimed to investigate DNA
methylation patterns in non-coding regions of GJB2 and RB1
using salivary DNA of Filipino patients with SNHL.
MATERIALS AND METHODS
Ethical approval was obtained from the University
of the Philippines Manila Research Ethics Board and the
institutional review board of the Baylor College of Medicine
and aliated hospitals. Informed consent was obtained from
all study participants.
Salivary DNA samples from 15 Filipino patients who
underwent cochlear implantation (CI) for SNHL (ages 3.5-
21 years, 67% female; Table 1) and 45 unrelated Filipinos
were utilized in this study. CI patient DNA was previously
used in investigations on damaging variants in hearing loss,
otitis media, and temporal bone anomalies.11–15 Control DNA
samples were previously isolated from saliva of a cohort of
Filipino-descent individuals who did not have hearing loss
and were recruited for a study on speech delay.16 Bisulte
Table 1. Clinical Descripons and Hearing Loss (HL) Genes with Variants in Cochlear Implant Paents11–15
Paent No. Prenatal History Other Childhood Disease Temporal Bone Findings HL gene with variant
1Maternal joint pain
during pregnancy
Unremarkable Superior semicircular canal
dehiscence and os media, le
KCNQ4
2Unremarkable Unremarkable EVA, bilateral SLC26A4
3Maternal fever at
6 mos AOG
Microscopic hematuria; primary complex Normal CDH23
4Unremarkable Delayed motor development; white maer
disease by MRI
Normal WFS1
5Unremarkable Primary Koch infecon Normal MYO15A
6Maternal diabetes
at 6 mos AOG
Unremarkable Malformed cochlea, bilateral;
absent cochlear and inferior
vesbular nerves, right
SLC9A3R1
7Unremarkable Unremarkable EVA, bilateral COL4A3
8Unremarkable Unremarkable EVA, bilateral SLC26A4
9Unremarkable Global developmental delay; le foot inversion Normal MYH14
10 Unremarkable Maternal urinary tract infecon and eclampsia
during pregnancy
EVA, le IST1a
11 Unremarkable Global developmental delay Normal SLC12A2b
12 Unremarkable Sepsis at 19 days of age and was prescribed
various anbiocs, one of which was Amikacin
Normal MYO7Ab
13 Unremarkable Turbinate hypertrophy secondary to papillary
allergic rhinis with nodule in nasopharynx
Normal CLDN9b
14 Unremarkable Unremarkable Normal GREB1Lb; CBLN3a
15 Unremarkable Unremarkable EVA; Os media, le GDPD5a
a variant is found in candidate gene15; b variant is novel and found in a known HL gene15
Abbreviaons: AOG, age of gestaon; EVA, enlarged vesbular aqueduct
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GJB2 and RB1 Methylaon and Hearing Loss

conversion of DNA samples was performed as described in
the EpiTect Bisulte Kit (Qiagen).
Two CpG-rich regions were investigated. The
RB1 promoter sequence contains ve CpG sites (hg19
chr13:48877688, chr13:48877674, chr13:48877677,
chr13:48877679, chr13:48877684).17 Additionally, we
investigated a region located in an enhancer-like region
(EH38E1658502) between exons 1 and 2 of the SNHL-
associated gene GJB2. is region contains four CpGs in
close proximity (hg19 chr13:20766381, chr13:20766387,
chr13:20766397, chr13:20766399) as shown in iMETHYL
and the ENCODE project.18,19 For the GJB2 region, primers
for methylation-specic qPCR (qMSP) were designed using
MethPrimer software.20 qMSP using TaqMan® probes was
then performed using the EpiTect Methylight PCR kit
(Qiagen) using gene-specic primer and probe sets (Table 2).
Standardization was done using the EpiTect PCR
Control DNA Set (Qiagen) which contains 100% methylated
and 100% unmethylated control DNA. Additionally,
75%, 50%, and 25% methylated controls were prepared.
Percentage methylation was calculated using the following
formula:
where Cmeth is percentage methylation, CTFAM is threshold
cycle of the methylated reporter (FAM channel), and CTVIC
is the threshold cycle of the unmethylated reporter (VIC
channel).21
RESULTS
Bisulte-converted DNA samples were of high
concentration and purity based on nanodrop measurements,
260/280 ratio and 260/230 ratio. PCR using RB1 and GJB2
primers produced appropriately-sized bands.
For both RB1 and GJB2, the 100% methylated and
100% unmethylated BSC standards produced only FAM
and VIC signals, respectively, while the 75%, 50%, and
25% methylated standards produced both FAM and VIC
signals appropriately, indicating that the system was able
to distinguish the methylation levels of both target regions.
qMSP of bisulte-converted salivary DNA revealed that for
both CI patients and ethnically-matched controls, only VIC
signals were produced. is indicates zero methylation at the
targeted RB1 promoter and GJB2 enhancer-like regions for
both cases and controls.
DISCUSSION
qMSP of bisulte-converted salivary DNA of Filipino
CI patients and non-hearing-impaired controls revealed
0% methylation at a region in the RB1 promoter previously
described as being dierentially methylated in SNHL
patients exposed to lead and cadmium8 and a target sequence
in an enhancer-like region in the SNHL-associated gene
GJB2. is nding may be due to the lack of sucient
levels of environmental exposures causing dierences in the
target regions. Population-specic dierences may also be
present, as the cohort from the study by Xu et al.8 were of
Chinese descent.
Alternatively, there may be other genetic factors that
could have explained this nding. It must be noted that
the DNA samples from our CI patients were submitted
for exome sequencing,11–15 and in six out of fteen patients,
known variants in HL genes were not identied. Further
exome analysis of these patients, however, revealed potential
candidate genes for HL and temporal bone anomalies in three
patients as well as novel variants in known HL genes in four
patients (patients 10-15; Table 1).15 In the other 10 patients,
rare damaging variants were identied in non-GJB2 HL
genes (patients 1-9; Table 1). For this study, we hypothesized
that dierentially-methylated regions (DMRs) in non-coding
sequences of GJB2 or other known HL-associated genes may
be present in our SNHL patients. In this study, GJB2 was
selected for methylation studies due to it being the most
common HL-associated gene in many world populations22,
however we found zero methylation at the selected enhancer-
like region of GJB2. In addition to the CpG sites we tested,
several enhancer-like regions were located between the rst
and second exons of GJB2. Changes in non-coding regions in
the form of epigenetic marks may be present in these GJB2
regions, or in non-coding regions of other genes for HL.
qMSP has been criticized as being more challenging
in terms of designing primers specic for methylated and
nonmethylated regions23, however, improvements in the
design of protocols and kits which utilize only one primer
set alongside methylation-specic TaqMan® probes as in
the case of this study has allowed easier, high-throughput,
and sensitive evaluation of percentage methylation of target
sequences21. However, we were somewhat limited as to which
region as well as sequence length to investigate due to the use
of TaqMan® probes. A more comprehensive investigation into
these CpG-rich regions, for example using pyrosequencing
or amplicon sequencing following bisulte conversion to
Table 2. Primer and Probe Sets for RB1 and GJB2 Target Regions
Forward primer Reverse primer Methylated probe Unmethylated probe
RB1 5’-GTTTAAGGAG
GGAGAGTGG-3’
5’- AAATAACTATAAA
CCTCATCCCTATCC-3’
FAM5’-AACACGTCCGAACCG
CGCCGAATAC3’-TAMRA
VIC5’-AACACATCCAAACC
ACACCAAATAC3’-TAMRA
GJB2 5’-GGAATTGATTT
TTATTTTTTGGAG-3’
5’- AAAAAAACCACTA
AAATCTTAACCC-3’
FAM-5’CCGAAATCGACT
AACTCCGCGTTA3’-TAMRA
VIC-5’CCAAAATCAACTAA
CTCCACATTA3’-TAMRA
Cmeth = 100/[1+2(CTFAM – CTVIC)]%
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GJB2 and RB1 Methylaon and Hearing Loss

investigate the entire stretch between exons 1 and 2 of GJB2,
may yield DMRs associated with HL.
Other limitations of this study must be considered.
First, we did not have environmental data such as chemical
exposure from our cases and controls. Second, we only
investigated for methylation patterns in this study, and no
other means of epigenetic control. However, exome data
from our CI cohort were negative for variants in microRNA
genes previously associated with HL in Spanish families.24
ird, the relatively small sample size of our cohort may not
have been enough to detect any dierences in methylation
patterns between cases and controls. Finally, due to the
aforementioned diculty of obtaining human inner ear
tissue, we utilized salivary DNA as a surrogate for inner ear
neuroepithelium.
CONCLUSION
In summary, we showed that in Filipino CI patients
and hearing controls, there were no changes in terms of
methylation in a sequence previously described as a DMR
in hearing-impaired children exposed to lead and cadmium,
as well as a CpG-rich sequence in an enhancer-like
region of GJB2. Further study into these regions as well as
noncoding regions in other HL-associated genes may yield
DMRs associated with HL, preferably using genome-wide
methylation arrays.
Acknowledgments
We thank the patients and individuals who provided
DNA samples. We also thank C Garcia, M Pedro, T
Bootpetch, S Chanthaponh, D Frank and H Jenkins for
general support.
Statement of Authorship
All authors certied fulllment of ICMJE authorship
criteria.
Author Disclosure
All authors declared no conicts of interest.
Funding Source
A.M.S. was funded by the Philippine Council for Health
Research and Development of the Department of Science
and Technology (PCHRD-DOST) under the Research
Enrichment (Sandwich) Grant of the Accelerated Science
and Technology Human Resource Development Program.
Recruitment, DNA collection from the Filipino patients, and
exome sequencing were funded by grants PCHRD-DOST
FP150010 and UP Manila-NIH 2008–005 (to C.M.C.).
Studies on the genetics and epigenomics of hearing loss
are being funded by the US National Institutes of Health –
National Institute on Deafness and Other Communication
Disorders through grant R01 DC019642 (to R.S.C. and
I.V.Y.).
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