The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):67
DOI 10.1186/s12881-016-0329-8

REVIEW

Open AccessUtilization of amplicon-based targeted
sequencing panel for the massively parallel
sequencing of sporadic hearing impairment
patients from Saudi Arabia
Ashraf Dallol1,2*, Kamal Daghistani2, Aisha Elaimi1, Wissam A. Al-Wazani1, Afaf Bamanie3, Malek Safiah4,Samira Sagaty5, Layla Taha6, Rawabi Zahed1, Osama Bajouh1,3, Adeel Gulzar Chaudhary2, Mamdooh Abdullah Gari2,
Rola Turki3, Mohammed Hussein Al-Qahtani2 and Adel Mohammed Abuzenadah1,2,6
From 3rd International Genomic Medicine Conference
Jeddah, Saudi Arabia. 30 November - 3 December 2015

Abstract
Background: Hearing Impairment (HI) can have genetic or environmental causes and in some cases, an interplay of
both. Genetic causes are difficult to determine as mutations in more than 90 genes have been shown recently tobe responsible for HI. Providing a genetic diagnostic test for HI is therefore a challenge especially for ethnic groups
where GJB2 mutations are shown to be rare.
Results: Here we show the design and implementation of an amplicon-based targeted sequencing panel thatallows the simultaneous sequencing of 87 HI genes. Mutations identified included known pathogenic mutations
and novel variants with unknown significance. The diagnostic rate of this panel is 28 % when only pathogenic
variants were reported. However, an additional 28 % harbored recurrent combinations of novel or rare single
nucleotide variants in the OTOF or PCDH15 genes. Such combinations were not identified in healthy individuals.
Conclusions: Targeted sequencing approach is a very useful strategy for the identification of mutations affecting
the HI genes because of its relatively fast turn-around time and cost effectiveness compared to whole-exome
sequencing. Further novel or rare variants could be identified by implementing a large-scale screening of HI using
our panel which will eventual lead to a higher diagnostic rate.
Keywords: Hearing impairment, NGS, Targeted sequencing, Saudi Arabia

Background
Hearing loss is a common disease found in many populations worldwide ast is estimated that at least 50 % ofprelingual hearing impairment (HI) is heritable either asa phenotype in many syndromes or in a non-syndromic
fashion [1]. The heritability of HI follows all types of* Correspondence: adallol@kau.edu.sa
1
Center of Innovations in Personalized Medicine, King Abdulaziz University,
P.O. Box 80216, Jeddah 21589, Kingdom of Saudi Arabia
2
Center of Excellence in Genomic Medicine Research, King Abdulaziz
University, Jeddah, Kingdom of Saudi Arabia
Full list of author information is available at the end of the article

Mendelian inheritance and can be passed down to subsequent generations in autosomal/X-linked recessive or
dominant manner. The large number of genes requiredfor hearing causes such complexity in inheritance patterns. Mutations in any one of such genes is sufficient
to cause various degrees of HI. Mutations in GJB2,SLC26A4, MYO15A, OTOF, and CDH23 genes are themost frequent causes of autosomal recessive HI at varying degrees [2]. On average, about 50 % of any givencohort of autosomal recessive HI will have a pathogenic
mutation in the GJB2 gene [3–6]. However, the frequency of HI-causing GJB2 gene mutations is© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):67

Page 2 of 23

population-dependent as it is most predominant HI
gene in Caucasians and rather rare in South East Asians
or Arab populations [7, 8].
The prevalence of the 35delG mutation in GJB2 genein Caucasians has allowed its use as a genetic screening
tool for early detection and diagnosis of HI in newborns.
However, such a test is not applicable in other ethnic
groups due to its rarity. In fact, since over 90 genes havenow been shown to be associated with HI, it is not
feasible to offer classical genetic testing for HI in many
populations. The advent of massively parallel sequencing and whole-exome sequencing (WES) led to a
rapid increase in the rate of identifying the genetic determinants of HI [9]. In order to achieve such aim,
whole-exome sequencing is applied to several members of the affected family in order to identify the genetic cause (s) of HI amongst hundreds of candidate
variants [10, 11]. Furthermore, WES is expensive andtime-consuming and it is not yet amenable for largescale screening of HI that will invariably include sporadic cases. Amplicon-based targeted sequencing is acost-effective tool to precede WES that will allow thefocused sequencing of relevant HI genes and improvethe diagnostic rate of such tests [12, 13].
In this study, we designed and validated a custommade amplicon-based targeted sequencing panel for
HI that allows the simultaneous sequencing of 87
genes shown previously to be associated with various
forms of HI.

sequencing panel was designed and manufactured by
Life Technologies. Barcoded Ampliseq libraries (2pools) were prepared using the Ampliseq Library kit 2.0
from 10 ng of DNA (concentration determined using
the Qubit™ fluorometer). Templated spheres were prepared using 100 pM of DNA from each library using
the Ion OneTouch 2.0 machine. Template-positive
spheres from the barcoded libraries were multiplexed
and loaded onto Ion chips 316 or 318 version 2.0 andsequencing was performed using the Ion Sequencing
200 v2 kit from Life Technologies. Processing of the
Ion Torrent Personal Genome Machine (PGM) runs
was achieved with the Torrent Suite version 4.4.3. The
Coverage Analysis plugin was used to calculate coverage efficiency and read depth. The Ion Reporter v4.6
was used to identify and annotate variants.

Materials and methods

Results
We have adopted the amplicon-based targeted sequencing approach in order to develop a more efficientmethod for the detection of mutations affecting thedeafness genes. In this approach, sequencing is limited
to the genes known or has been previously demonstrated
to play a role in causing HI. (Table 1) lists the genes included in this panel obtained from the deafness variation
database (http://deafnessvariationdatabase.org).
Eighty-seven genes were selected and custom primers
were designed and manufactured through the ampliseq
portal (http://ampliseq.com). The design resulted in an
excellent coverage of 97.42 % generating 2697 amplicons
with a size range of 125–275 bp in two pools and generating 500.44 kb of DNA sequence. Identified variants
were confirmed by Sanger sequencing.
We have managed to screen 25 individuals affectedwith HI using this approach. Additionally, we havescreened samples from 6 healthy volunteers with no history of HI as a control. Seven patients were found tohave pathogenic (according to clinvar database) mutations affecting OTOF, MYO7A, TMC1 and GJB3 genes(Table 2; Fig. 1). The homozygous c.2239C > A change in
the OTOF gene causes a premature termination of thePatients

Samples from 25 HI patients were collected following
the appropriate local ethical protocols and guidelines
from the Audiology clinic at King Abdulziz University
Hospital in Jeddah, Saudi Arabia or the Audiology department of the King Fahad General Hospital, Jeddah
Institute for Speech and Hearing (JISH) as well as AlAmal secondary school for the hearing impaired, Jeddah,
Saudi Arabia. All samples were selected according to thediagnosis of bilateral severe-to-profound hearing impairment as determined by audiologists in the study team.
The patients ages ranged from 4 to 22 years old. Control
samples were obtained from non-HI patients and aged
18–40 years old. DNA was extracted from peripheral
blood DNA using the standard protocol of the QIAGEN
Blood DNA Extraction kit. The study was approved by
the local ethical committee.
Targeted sequencing of deafness genes

Eighty-seven genes involved in HI were determined using
the Deafness Variation Database (http://deafnessvariation
database.org). The gene names were submitted for primer
design to the http://Ampliseq.com website were a targeted

DNA sequencing using the sanger method

Pathogenic and novel variants were confirmed by custom oligonucleotide primers designed to flank thevariant of interest in order to prepare PCR fragments
for sequencing. PCR was performed at optimal condition for each primer pair and products were purified
using ethanol precipitation. DNA sequencing was performed using the BigDye v. 3.1 and analyzed on the
DNA Analyzer 3500 from Life Technologies. Additionally, TaqMan® SNP genotyping assays, either customdesigned or pre-made, were utilized in the validation
process or screening further cases.

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Table 1 Genes included in our HI panel
Gene

Coverage (%)

Gene

Coverage (%)

Gene

Coverage (%)

Gene

Coverage (%)

ACTG1

56.62

GIPC3

98.9

OTOA

97.82

TSPEAR

97.54

AIFM1

100

GJB2

100

OTOF

95.41

USH1C

97.21

ALMS1

99.25

GJB3

78.86

OTOG

98.3

USH1G

100

ATP2B2

100

GJB6

100

OTOGL

99.71

USH2A

98.87

CABP2

100

GPR98

99.42

P2RX2

80.21

WFS1

97.63

CACNA1D

100

GPSM2

100

PCDH15

98.27

WHRN

100

CATSPER2

100

GRHL2

100

PNPT1

97.07

MIR-182100

CCDC50100

GRXCR1100

POU3F4100

MIR-183

66

CDH23

99.55

HGF

99.4

POU4F3

100

MIR96

100

CEACAM16

94.39

ILDR1

95.7

PRPS1

100

DFNB59

100

CIB2

95.87

KCNQ1

86.04

PTPRQ

97.5

ESRRB

91.35

CLDN14

88.36

KCNQ4

87.25

RDX

100

TMPRSS3

100

CLPP

99.78

LARS2

98.63

SERPINB6

97.18

CLRN1

100

LHFPL5

100

SIX1

95.77

COCH

100

LOXHD199.72

SLC26A4

100

COL11A2

99.9

LRTOMT100

SLC26A5

100

CRYL1

99.04

MARVELD2

100

SMAD4

100

CRYM

100

MSRB3

100

SMPX

100

DFNA5

100

MYH14

97.53

STRC

99.82

DIABLO

100

MYH9

98.11

TECTA

98.28

DIAPH1

98.49

MYO15

94.22

TJP2

98.17

DSPP

80.44

MYO1A

100

TMC1

96.54

ESPN

73.28

MYO3A

100

TMIE

79.84

EYA1

100

MYO6

100

TPRN

93.57

EYA4

100

MYO7A

98.72

TRIOBP

92.17

protein at p. Glu747Ter. This mutation was identified ina brother and sister suffering from severe to profound
HI. The homozygous IVS5 + 1G > A change in the
MYO7 gene is a mutation affecting the 5′ splice site andwas found in a brother and sister with severe to profound HI in our screen. The p. Arg830His in MYO7A
gene is a very rare SNP (single nucleotide polymorphisms) with a reported minor allele frequency of
0.0001556 and classified as likely pathogenic by the
ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/).

This variant was identified in two of our patients withsevere to profound HI, albeit one of the patient harboredthe mutation in a heterozygous state thus could not
sufficiently account for the disease. The c.100C > T, p.
Arg34Ter variant in the TMC1 gene is a pathologicalchange according to ClinVar database. The c.100C > T,
p. Arg34Ter homozygous change in the TMC1 genecauses the premature termination of the protein andfound only in one patient. The rs74315318 SNP isc.547G > A change causing a p. Glu183Lys alteration in

Table 2 Pathogenic mutations identified in our cohort
Positiongenotype

genetranscript

codingproteindiagnosis

chr2:26700593

A/A

OTOF

NM_194248

c.2239G > T

p. Glu747Ter

Severe to profoundchr1:35250910

G/A

GJB3

NM_001005752

c.547G > A

p. Glu183Lys

Severe to profoundchr11:76890902

A/A

MYO7A

NM_000260

c.2489G > A

p. Arg830His

Severe to profoundchr11:76867138

A/A

MYO7A

NM_000260

c.470 + 1G > A

IVS5 + 1G > A

Severe to profoundchr9:75309494

T/T

TMC1

NM_138691

c.100C > T

p. Arg34Ter

Severe to profoundThe Author(s) BMC Medical Genetics 2016, 17(Suppl 1):67

Page 4 of 23

Fig. 1 Sanger sequencing validation of the identified pathogenic variants (a) and the Integrated Genome Viewer view of the Ion Torrent BAM file
generated by the targeted sequencing using our custom-made HI panel demonstrating the detection of the p. Glu747Ter/c.2239C > A mutationin the OTOF gene (b)the GJB3 gene. This variant is reported to be pathological in an autosomal dominant fashion in the ClinVar database.
Although we could not identify additional known
pathogenic mutations in our cohort, we have identified 7
patients with a possible genetic aberration underlying
their HI. In these patients, two novel or rare SNVs wereidentified affecting the same gene (Table 3). In particular,
in the OTOF and PCDH15 genes. The rare damaging
SNPs [minor allele frequency (MAF) of 0.001 and 0.006,
respectively] identified in combination in the PCDH15
gene were recurrent in 3 unrelated patients. Furthermore, we have designed custom-made TaqMan® SNP
genotyping assays targeting such mutations in order totest whether the variants identified in this screen recurin other unrelated patients. Interestingly, the combination identified by the panel was not found in 80 additional HI patients or non-HI controls.

Discussion and conclusions
We have screened 25 HI patients using a custom-madetargeted sequencing panel to simultaneously screen 87
genes known to play a role in HI. Known pathogenic
mutations were identified in 7 patients which gives adiagnostic rate of 28 %. The rs74315318 SNP is ac.547G > A change causing a p. Glu183Lys alteration in
the GJB3 protein. This variant is reported to bepathological in the ClinVar database [14] according to a
published report of its identification in Chinese families
with autosomal dominant hearing loss [15]. We havealso identified a homozygous c.2239C > A change in the
OTOF gene causing premature termination of protein at
p. Glu747Ter. This mutation was identified in a brotherand sister suffering from severe to profound HI. Although not reported in the dbSNP, this variant was previously identified in a Libyan family [16]. The c.100C >T, p. Arg34Ter homozygous change in the TMC1 genecauses the premature termination of the protein and has
been previously reported in more than 23 probands withsensorineural hearing loss [17–19]. We have identified
this variant in the homozygous state in one patient withprofound HI. The homozygous IVS5 + 1G > A change in
the MYO7A gene is a mutation affecting the 5′ splicesite and was found in a brother and sister with severe toprofound HI in our screen. This mutation was reported
only once previously in a Tunisian family enrolled in astudy of Usher 1B syndrome [20]. The p. Arg830His in
the MYO7A gene is a very rare SNP with a minor allele
frequency of 0.0001556 as reported by the exome aggregation consortium (ExAC) data set [21]. This variant
was identified in two of our patients with profound HI.
Genetic factors underlying HI patients from Saudi
Arabia are not fully elucidated and therefore, other mutations may play a role. For example, we have identified

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):67

Page 5 of 23

Table 3 Novel or rare variants identified in our screen
Case Position

ref genotype MAF

gene

proteindbsnp

transcript147

chr2:26689695

G

G/C

NA

OTOF

p. Pro1463Ala c.4387C > G Damaging(0)

Probablydamaging(0.965)

Novel

NM_194248.2

chr2:26750782

G

G/A

0.008 OTOF

p. Arg49Trp

Probablydamaging (1)rs61746568

chr2:26695500

A

A/C

0.009 OTOF

p. Cys1251Gly c.3751 T > G Tolerated(0.46)

Benign (0)rs41288773

chr2:26750782

G

G/A

0.008 OTOF

p. Arg49Trp

Damaging(0)

Probablydamaging (1)rs61746568

chr2:26696374

C

C/T

0.006 OTOF

p. Arg1157Gln c.3470G > A Tolerated(1)

Probablydamaging (1)rs56054534

chr2:26781384

C

C/T

0

p. Arg19Gln

chr10:55779975 C

C/A

chr10:55782743 A

144

151

197

350

888

873

OTOF

codingc.145C > T

c.145C > T

SIFT (score) polyphen

Damaging(0)

NM_194248.2

NM_194248.2

c.56G > A

Damaging(0.006)

Probablydamaging (1)rs200316189

0.001 PCDH15 p. Ala915Ser

c.2743G > T

Damaging(0.05)

Probablydamaging (1)rs139175351 NM_001142763.1

A/G

0.006 PCDH15 p. Ile817Thr

c.2450 T > C Damaging(0.023)

Probablydamaging(0.998)

rs61731363

chr10:55779975 C

C/A

0.001 PCDH15 p. Ala915Ser

c.2743G > T

Probablydamaging (1)rs139175351 NM_001142763.1

chr10:55782743 A

A/G

0.006 PCDH15 p. Ile817Thr

c.2450 T > C Damaging(0.023)

Probablydamaging(0.998)

rs61731363

chr10:55779975 C

C/A

0.001 PCDH15 p. Ala915Ser

c.2743G > T

Probablydamaging (1)rs139175351 NM_001142763.1

chr10:55782743 A

A/G

0.006 PCDH15 p. Ile817Thr

c.2450 T > C Damaging(0.023)

Probablydamaging(0.998)

rs61731363

chr3:121712010 A

A/G

NA

ILDR1

c.1586 T > C Tolerated(1)

Benign (0.39)

Novel

chr7:129414567 C

C/T

NA

MIR96

p. Val529Alain this study a C > T change in the sequence of the MIR96 microRNA which has been recently shown to play arole in hearing [22]. This SNP is extremely rare and according to the ExAC browser [21] carries a minor allele
frequency of 0.0001428. This mutation is also conserved
and lies close to the +57 T > C mutation recently shownto cause autosomal dominant hearing loss in Italianfamilies by altering pre-miRNA processing [23]. Thisvariant was identified in a patient with severe to profound hearing loss. In addition, we have identified a patient that harbored a rare rs369424114 variant in the
MYO7A gene. This variant has a minor allele frequencyof 0.0006688 and found predominantly in the south
Asian population [21].
An interesting observation in this cohort is the coexistence of two damaging variants of PCDH15 gene in
3 unrelated patients. The reported MAF for the p.
Ala915Ser and the p. Ile817Thr variants is 0.001 and
0.006, respectively. Additionally, a novel OTOF genevariant, p. Pro1463Ala, co-existed with another rare
OTOF variant, p. Arg49Trp, (MAF = 0.008) in one

Damaging(0.05)

Damaging(0.05)

NM_001199799.1

rs370173345 NR_029512.1

patient. Another patient harbored the latter variant withthe rare p. Cys1251Gly variant (MAF = 0.009) affecting
the same gene. A third unrelated patient harbored therare p. Arg1157Gln variant in the OTOF gene (MAF =0.001) as well as another rare variant, p. Arg19Gln, affecting the same gene. The co-existence of such rare ornovel variants may reflect a new state of pathogenicity ofsingle nucleotide variants affecting the OTOF and
PCDH15 genes in our cohort.
In conclusion, our study shows that targeted sequencing approach is a very useful strategy for the identification of mutations affecting the HI genes. However,further work is necessary to identify and characterize
novel variants in each ethnic group. Targeted sequencing
is relatively fast and cost effective compared to wholeexome sequencing which renders possible the screeningof large cohorts of sporadic HI patients. Additionally,targeted sequencing combined with next generation sequencing can be utilized as an aid for genetic counselingof families affected with hearing impairment. Furthermore, such test can be applied as a premarital screening

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):67

tool at a national level in order to significantly reducethe burden of HI on the society.
Abbreviations
ExAC: Exome aggregation consortium; HI: Hearing impairment; MAF: Minor
allele frequency; MPS: Massively parallel sequencing; NSHL: Non-syndromic
hearing loss; PGM: Personal genome machine; SNPs: Single nucleotide
polymorphisms; WES: Whole-exome sequencing
Acknowledgements
The authors acknowledge the excellent technical assistance of Najla
Filimban, Maram Amin, Fatima Gazzaz and Shireen Turkistani.

Page 6 of 23

6.

7.

8.

9.

Declaration
This article has been published as part of BMC Medical Genetics Volume 17
Supplement 1, 2016: Proceedings of the 3rd International Genomic Medicine
Conference: medical genetics. The full contents of the supplement areavailable online at http://bmcmedgenet.biomedcentral.com/articles/
supplements/volume-17-supplement-1.

10.

Funding
Funding by the King Abdulaziz City for Science and Technology (KACST) is
acknowledged through project no. ARP-30-304. Publication fees for thisarticle are paid by the Center of Excellence in Genomic Medicine Research(CEGMR), King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.

12.

11.

13.
Availability of data and materials
Not applicable.
14.
Authors’ contributions
KD, AB, MS, SS, RT, OB provided samples and clinical diagnosis. AE, WA, LT,
RZ provided technical support. AGC, MAG, MHA aided in the design andadministration of this study. AMA provided logistical support and critical
discussion of this manuscript. AD analyzed the data and wrote themanuscript. All read and approved the final manuscript.

15.

16.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.

17.

Ethics approval and consent to participate
This study was approved by the Research Committee of the Biomedical Ethics
Unit, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia.

18.

Author details
1
Center of Innovations in Personalized Medicine, King Abdulaziz University,
P.O. Box 80216, Jeddah 21589, Kingdom of Saudi Arabia. 2Center of
Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah,
Kingdom of Saudi Arabia. 3King Abdulaziz University Hospital, King Abdulaziz
University, Jeddah, Kingdom of Saudi Arabia. 4Jeddah Institute for Speechand Hearing, Jeddah, Kingdom of Saudi Arabia. 5King Fahad General
Hospital, Jeddah, Kingdom of Saudi Arabia. 6Faculty of Applied Medical
Sciences, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.

19.

20.

Published: 10 October 201621.
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