Volume 17 Supplement 1

Proceedings of the 3rd International Genomic Medicine Conference: medical genetics

Open Access

Utilization of amplicon-based targeted sequencing panel for the massively parallel sequencing of sporadic hearing impairment patients from Saudi Arabia

  • Ashraf Dallol1, 2Email author,
  • 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
BMC Medical GeneticsBMC series – open, inclusive and trusted201617(Suppl 1):67

https://doi.org/10.1186/s12881-016-0329-8

Published: 10 October 2016

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 to be 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 that allows 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 % of prelingual hearing impairment (HI) is heritable either as a phenotype in many syndromes or in a non-syndromic fashion [1]. The heritability of HI follows all types of Mendelian inheritance and can be passed down to subsequent generations in autosomal/X-linked recessive or dominant manner. The large number of genes required for 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 the most frequent causes of autosomal recessive HI at varying degrees [2]. On average, about 50 % of any given cohort of autosomal recessive HI will have a pathogenic mutation in the GJB2 gene [36]. However, the frequency of HI-causing GJB2 gene mutations is 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 gene in 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 have now 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 and time-consuming and it is not yet amenable for large-scale screening of HI that will invariably include sporadic cases. Amplicon-based targeted sequencing is a cost-effective tool to precede WES that will allow the focused sequencing of relevant HI genes and improve the diagnostic rate of such tests [12, 13].

In this study, we designed and validated a custom-made 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.

Materials and methods

Patients

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 Al-Amal secondary school for the hearing impaired, Jeddah, Saudi Arabia. All samples were selected according to the diagnosis 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://deafnessvariationdatabase.org). The gene names were submitted for primer design to the http://Ampliseq.com website were a targeted sequencing panel was designed and manufactured by Life Technologies. Barcoded Ampliseq libraries (2 pools) 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 and sequencing 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.

DNA sequencing using the sanger method

Pathogenic and novel variants were confirmed by custom oligonucleotide primers designed to flank the variant 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 custom-designed or pre-made, were utilized in the validation process or screening further cases.

Results

We have adopted the amplicon-based targeted sequencing approach in order to develop a more efficient method for the detection of mutations affecting the deafness 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).
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-182

100

CCDC50

100

GRXCR1

100

POU3F4

100

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

LOXHD1

99.72

SLC26A4

100

  

COL11A2

99.9

LRTOMT

100

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

  

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 affected with HI using this approach. Additionally, we have screened samples from 6 healthy volunteers with no history of HI as a control. Seven patients were found to have 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 the protein at p. Glu747Ter. This mutation was identified in a 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 and was 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 with severe to profound HI, albeit one of the patient harbored the 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 pathological change according to ClinVar database. The c.100C > T, p. Arg34Ter homozygous change in the TMC1 gene causes the premature termination of the protein and found only in one patient. The rs74315318 SNP is c.547G > A change causing a p. Glu183Lys alteration in the GJB3 gene. This variant is reported to be pathological in an autosomal dominant fashion in the ClinVar database.
Table 2

Pathogenic mutations identified in our cohort

Position

genotype

gene

transcript

coding

protein

diagnosis

chr2:26700593

A/A

OTOF

NM_194248

c.2239G > T

p. Glu747Ter

Severe to profound

chr1:35250910

G/A

GJB3

NM_001005752

c.547G > A

p. Glu183Lys

Severe to profound

chr11:76890902

A/A

MYO7A

NM_000260

c.2489G > A

p. Arg830His

Severe to profound

chr11:76867138

A/A

MYO7A

NM_000260

c.470 + 1G > A

IVS5 + 1G > A

Severe to profound

chr9:75309494

T/T

TMC1

NM_138691

c.100C > T

p. Arg34Ter

Severe to profound

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 mutation in the OTOF gene (b)

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 were identified 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 to test whether the variants identified in this screen recur in other unrelated patients. Interestingly, the combination identified by the panel was not found in 80 additional HI patients or non-HI controls.
Table 3

Novel or rare variants identified in our screen

Case

Position

ref

genotype

MAF

gene

protein

coding

SIFT (score)

polyphen

dbsnp

transcript

147

chr2:26689695

G

G/C

NA

OTOF

p. Pro1463Ala

c.4387C > G

Damaging (0)

Probably damaging (0.965)

Novel

NM_194248.2

chr2:26750782

G

G/A

0.008

OTOF

p. Arg49Trp

c.145C > T

Damaging (0)

Probably damaging (1)

rs61746568

 

144

chr2:26695500

A

A/C

0.009

OTOF

p. Cys1251Gly

c.3751 T > G

Tolerated (0.46)

Benign (0)

rs41288773

NM_194248.2

chr2:26750782

G

G/A

0.008

OTOF

p. Arg49Trp

c.145C > T

Damaging (0)

Probably damaging (1)

rs61746568

 

151

chr2:26696374

C

C/T

0.006

OTOF

p. Arg1157Gln

c.3470G > A

Tolerated (1)

Probably damaging (1)

rs56054534

NM_194248.2

chr2:26781384

C

C/T

0

OTOF

p. Arg19Gln

c.56G > A

Damaging (0.006)

Probably damaging (1)

rs200316189

 

197

chr10:55779975

C

C/A

0.001

PCDH15

p. Ala915Ser

c.2743G > T

Damaging (0.05)

Probably damaging (1)

rs139175351

NM_001142763.1

chr10:55782743

A

A/G

0.006

PCDH15

p. Ile817Thr

c.2450 T > C

Damaging (0.023)

Probably damaging (0.998)

rs61731363

 

350

chr10:55779975

C

C/A

0.001

PCDH15

p. Ala915Ser

c.2743G > T

Damaging (0.05)

Probably damaging (1)

rs139175351

NM_001142763.1

chr10:55782743

A

A/G

0.006

PCDH15

p. Ile817Thr

c.2450 T > C

Damaging (0.023)

Probably damaging (0.998)

rs61731363

 

888

chr10:55779975

C

C/A

0.001

PCDH15

p. Ala915Ser

c.2743G > T

Damaging (0.05)

Probably damaging (1)

rs139175351

NM_001142763.1

chr10:55782743

A

A/G

0.006

PCDH15

p. Ile817Thr

c.2450 T > C

Damaging (0.023)

Probably damaging (0.998)

rs61731363

 

873

chr3:121712010

A

A/G

NA

ILDR1

p. Val529Ala

c.1586 T > C

Tolerated (1)

Benign (0.39)

Novel

NM_001199799.1

chr7:129414567

C

C/T

NA

MIR96

    

rs370173345

NR_029512.1

Discussion and conclusions

We have screened 25 HI patients using a custom-made targeted sequencing panel to simultaneously screen 87 genes known to play a role in HI. Known pathogenic mutations were identified in 7 patients which gives a diagnostic rate of 28 %. The rs74315318 SNP is a c.547G > A change causing a p. Glu183Lys alteration in the GJB3 protein. This variant is reported to be pathological in the ClinVar database [14] according to a published report of its identification in Chinese families with autosomal dominant hearing loss [15]. We have also 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 brother and 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 gene causes the premature termination of the protein and has been previously reported in more than 23 probands with sensorineural hearing loss [1719]. We have identified this variant in the homozygous state in one patient with profound HI. The homozygous IVS5 + 1G > A change in the MYO7A gene is a mutation affecting the 5′ splice site and was found in a brother and sister with severe to profound HI in our screen. This mutation was reported only once previously in a Tunisian family enrolled in a study 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 in this study a C > T change in the sequence of the MIR-96 microRNA which has been recently shown to play a role 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 shown to cause autosomal dominant hearing loss in Italian families by altering pre-miRNA processing [23]. This variant 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 frequency of 0.0006688 and found predominantly in the south Asian population [21].

An interesting observation in this cohort is the co-existence 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 gene variant, p. Pro1463Ala, co-existed with another rare OTOF variant, p. Arg49Trp, (MAF = 0.008) in one patient. Another patient harbored the latter variant with the rare p. Cys1251Gly variant (MAF = 0.009) affecting the same gene. A third unrelated patient harbored the rare 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 or novel variants may reflect a new state of pathogenicity of single 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 whole-exome sequencing which renders possible the screening of large cohorts of sporadic HI patients. Additionally, targeted sequencing combined with next generation sequencing can be utilized as an aid for genetic counseling of families affected with hearing impairment. Furthermore, such test can be applied as a premarital screening tool at a national level in order to significantly reduce the 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

Declarations

Acknowledgements

The authors acknowledge the excellent technical assistance of Najla Filimban, Maram Amin, Fatima Gazzaz and Shireen Turkistani.

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 are available online at http://bmcmedgenet.biomedcentral.com/articles/supplements/volume-17-supplement-1.

Funding

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

Availability of data and materials

Not applicable.

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 and administration of this study. AMA provided logistical support and critical discussion of this manuscript. AD analyzed the data and wrote the manuscript. All read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

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.

Open AccessThis 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, and reproduction 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.

Authors’ Affiliations

(1)
Center of Innovations in Personalized Medicine, King Abdulaziz University
(2)
Center of Excellence in Genomic Medicine Research, King Abdulaziz University
(3)
King Abdulaziz University Hospital, King Abdulaziz University
(4)
Jeddah Institute for Speech and Hearing
(5)
King Fahad General Hospital
(6)
Faculty of Applied Medical Sciences, King Abdulaziz University

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