A novel mutation in the TMC1 gene causes non-syndromic hearing loss in a Moroccan family.

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Research paper

A novel mutation in the TMC1 gene causes non-syndromic hearing loss in a Moroccan family.

Amina Bakhchaneâ, Hicham Charouteâ, Halima Nahiliâ, Rachida Roky^b, Hassan Roubaâ, Majida Charifâ,c,d, Guy Lenaers^c,d,1, Abdelhamid Barakatâ,1,⁎

aLaboratoire de Génétique Moléculaire Humaine, Département de Recherche Scientiﬁque, Institut Pasteur du Maroc, Casablanca, Morocco

bUniversité Hassan II Ain Chock, Laboratoire de Physiologie et génétique moléculaire, Km 8 Route d'El Jadida, B.P. 5366 Maarif, Casablanca 20100, Morocco

cInstitut des Neurosciences de Montpellier, U1051 de l'INSERM, Université de Montpellier I et II, BP 74103, 34091 Montpellier cedex 05, France

dPREMMi, Université d'Angers, CHU Bât IRIS/IBS, Rue des Capucins, 49933 Angers cedex 9, France

a b s t r a c t a r t i c l e i n f o

Article history:

Received 12 March 2015

Received in revised form 29 June 2015 Accepted 22 July 2015

Available online xxxx

Keywords:

Hearing loss TMC1 Mutation

Whole exome sequencing Morocco

Autosomal recessive non-syndromic hearing loss (ARNSHL) is one of the most common genetic diseases in human and is subject to important genetic heterogeneity, rendering molecular diagnosis difficult. Whole- exome sequencing is thus a powerful strategy for this purpose. After excluding GJB2 mutation and other common mutations associated with hearing loss in Morocco, whole-exome sequencing was performed to study the genetic causes of one sibling with ARSHNL in a consanguineous Moroccan family. Afterfiltering data and Sanger sequencing validation, one novel pathogenic homozygous mutation c.1810CNG (p.Arg604Gly) was identified in TMC1, a gene reported to cause deafness in various populations. Thus, we identified here thefirst mutation in theTMC1gene in the Moroccan population causing non-syndromic hearing loss.

1. Introduction

Hearing loss is one of the most common sensory disorder world- wide, occurring in approximately 0.2% of newborns (Hilgert et al., 2009). The majority of congenital cases are due to genetic factors and inherited across generations. Hereditary hearing loss is highly heterogeneous, with autosomal recessive non-syndromic hearing loss (ARNSHL) being the most frequent accounting for 80% of all cases. To date, a total of 142 loci have been mapped for non syndromic deafness and 90 genes have been identiﬁed, including 31 autosomal dominant, 55 autosomal recessive and 4 X-linked genes (http://hereditaryhearingloss.

org). GJB2 mutations are responsible for almost 50% of all cases with ARNSHL in most populations (Diaz-Horta et al., 2012). Conversely, the

majority of other deafness mutations are private and found in very few families, if not in a single one (Duman and Tekin, 2012). Thus screening of ARNSHL genes by standard molecular procedures is exten- sively long, expensive and time consuming (Diaz-Horta et al., 2012).

Whole-exome sequencing (WES) is lately viewed as a substitute to more conventional procedures for genetic diagnosis, as it authorizes a speciﬁc enrichment and sequencing of all exons of protein-coding genes, including those involved in ARNSHL (Diaz-Horta et al., 2012).

Next to the usual genes related to hearing loss published previously in Morocco, mainly GJB2 (Abidi et al., 2007; Abidi et al., 2008), LRTOMT (Charif et al., 2012) and also MT-RNR1 (Nahili et al., 2010), we here de- scribe theﬁrst mutation in theTMC1gene in the Moroccan population affected by ARNSHL, a gene which was already identiﬁed as causative for DFNA36 and DFNB7/11 deafness presentations (Kurima et al., 2002).

2. Patients and methods

2.1. Ethics statements and DNA preparation

This study was approved by the ethic committee on research of Pas- teur Institute of Morocco. We collected written informed consents from all patients before including them in the study. One unrelated Moroccan family showing prelingual, profound and bilateral sensorineural hearing loss took part to this study because of its parental consanguinity and the existence of two siblings with hearing loss issues (Fig. 1.A). Further Gene xxx (2015) xxx–xxx

Abbreviations:ARNSHL, autosomal recessive non-syndromic hearing loss; DFNA, deafness, autosomal dominant 36; DFNB, deafness, autosomal recessive; ESP, Exome Sequencing Project; EVS, Exome Variant Server; GJB2, gap junction protein, beta 2, 26 kDa; LRTOMT, leucine rich transmembrane and O-methyltransferase domain con- taining; MT-RNR1, Ribosomal RNA, Mitochondrial, 12S; PolyPhen, Polymorphism Phenotyping; SIFT, Sorting Intolerant From Tolerant; TMC1, transmembrane channel-like 1; WES, Whole-exome sequencing.

⁎ Corresponding author at: Laboratoire de Génétique Moléculaire et Humaine, Département de Recherche Scientiﬁque, Institut Pasteur. 1, Place Louis Pasteur, C.P.

20360 Casablanca, Morocco.

E-mail address:[email protected](A. Barakat).

1equal level contributors.

Contents lists available atScienceDirect

Gene

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / g e n e

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Clinical examination of the subjects disqualiﬁed any symptom or mal- formation that could be suggestive of a syndromic form of hearing loss. Using the phenol chloroform method, Genomic DNA was extracted from the blood from affected and unaffected family members. The patients were previously tested negative for the most common connexin (GJB2, GJB6 and GJB3), mitochondrial (12sRNA) mutations and the 242GNA mutation in LRTOMT gene. We also included in this study 90 healthy Moroccan with normal hearing.

2.2. Exome sequencing

Whole-exome sequencing was performed on patient SF107.3 by Otogenetics Corporation (Norcross, GA, USA). After initial sample qual- ity control, fragmented genomic DNA was ampliﬁed using Illumina li- brary preparation kit (Illumina, Inc., San Diego, CA). The resulting libraries were captured by Agilent Human exome V5 (51 Mb) capture kit, followed by paired end sequencing on a Hiseq2000 platform (Illumina, San Diego, USA), according to the manufacturer's protocol.

We obtained a minimal exome coverage of 53X, which provided sufﬁcient depth to analyze variants.

2.3. Read mapping and variant analysis

Short read alignment and variants calling were performed using the DNAnexus software package. Paired-end short reads were aligned against the human genome reference sequence hg19 (GRCh37). The aligned short reads were analyzed to identify single nucleotide varia- tions (SNVs) and indels (insertions and deletions). Weﬁltered variants based on dbSNP (build 135) and 1000 genome project databases. Vari- ants with a frequency less than 1% were considered as rare. Impacts of each variant on protein structure were predicted by SIFT (Sorting Intol- erant From Tolerant), PolyPhen-2 (Polymorphism Phenotyping) and Proven softwares. The remaining variants were viewed using the DNAnexus Genome Browser.

2.4. Veriﬁcation of variants (Sanger sequencing)

To ascertain the segregation with the disease phenotype in this family, Sanger sequencing was performed to validate the mutation in the

candidate gene. Speciﬁc primers were designed using Primer3 (http://

primer3.ut.ee/) (Table 1).

2.5. Protein structure prediction and analysis of stability

The exact three-dimensional structure ofTMC1is not available;

therefore, homology modeling approach was used to predict a 3D model of the protein. CPHmodels-3.2 Server built a protein 3D model based on the homologous protein structures available in protein data bases (PDB) (Nielsen et al., 2010). The effect of the amino acid substitution onTMC1protein structure was analyzedin silicousing the I-Mutant 2.0 (Capriotti et al., 2005) and SDM (Site Directed Mutator) (Worth et al., 2011) methods. The native and mutant protein structures were vi- sualized using YASARA (Krieger and Vriend, 2014).

3. Results

3.1. Exome sequencing and SNPﬁltering

By whole exome sequencing, we obtained 42 279 068 reads with an average sequence depth of 53×. About 97% of the total short reads were mapped to human reference sequence. A total of 121,839 single nucleotide polymorphisms, 6205 deletions and 5404 insertions were identiﬁed.

These variants wereﬁltered to identify pathogenic variants, with the following strategy: we focused on missense, nonsense and frameshift mutations and excluded intronic, 5′UTR, 3′UTR, and synonymous variants. We discarded common variants reported in NHLBI Exome Sequencing Project (ESP), Exome Variant Server (EVS) (Tennessen et al., 2012), and the 1000 Genomes Project, with a frequency greater than 1%. Deleterious SNPs predicted by SIFT, PolyPhen-2 and Proven softwares were retained. According to the pedigree, we hypothesized an autosomal recessive mode of inheritance. Thus, we selected Fig. 1.(A) Pedigree of the SF107 family, presenting the segregation of the p.Arg604Gly mutation. (B) Electrophoregram showing the heterozygous (top) and homozygous (bottom) c.1810CNG mutation.

Table 1

Sequences of the primers used to validate the mutation by Sanger sequencing.

TMC1-Ex20-F TTTAAGAAGTATCTTGGGGAACTG

TMC1-Ex20-R GGATCTCATTTCCACCAACC

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homozygous and compound heterozygous mutations. Based on this strategy, we identiﬁed a single novel deleterious mutation c.1810CNG (p.Arg604Gly) at homozygous state in the exon 20 of theTMC1gene.

This mutation (chr9: 75435804) is localized in a 4 megabases homozygous region located on chromosome 9q21.13. This variant has not yet been reported and is absent from all actual databases. Using Sanger sequencing, we found that both parents were heterozygous, the two affected offspring were homozygous for the mutation, while the healthy siblings were heterozygous or homozygous wild-type (Fig. 1.B). This novel mutation was analyzed on 63 Moroccan probands from recessive multiplex families without genetic diagnosis in order to identify the same mutation in another family, and on 90 Moroccan controls. None of the patients and the controls subjects showed this novel mutation.

3.2. Modeling and stability analysis of mutant structure

Homology model building methods was used to generate three- dimensional structure ofTMC1protein, using CPHmodels-3.2 Server.

Structural analyses were performed for evaluating the impact of the

p.Arg604Gly missense mutation on the protein stability. This mutation is predicted by the I-Mutant 2.0 and SDM (Site Directed Mutator) methods to be highly destabilizing and causing conformational changes on the protein 3D structure, thus possibly leading to protein dysfunction.

Indeed, theTMC1mutation involves the change of Arginine a polar hy- drophilic residue in general found on protein surface, to Glycine a neutral hydrophobic residue, in general found inside the protein structure. In this respect, our prediction study revealed that in the native protein structure, the Arginine-604 is found on polypeptide chain surface (Fig. 3A), whereas in the mutant protein, the Glycine is found in the inte- rior of the folded polypeptide (Fig. 3B). Thus, this mutation may lead to changes inTMC1protein folding, and consequently to its inactivation or degradation.

4. Discussion

Not only whole exome sequencing is an effective way for screening mutations in a huge number of genes, but it is also less expensive if we compare this method to other classical genetic methods, especially

Fig. 2.TMC1gene mutation identiﬁed in family SF107 with autosomal hearing loss. A: Cytogenetic location of TMC1 gene locus on chromosome 9 and mutation position. B: Schematic structure of TMC1 protein. C: Amino acid conservation map across species demonstrating that Arginine at position 604 is well conserved throughout vertebrates.

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for extremely heterogeneous diseases such as ARNSHL. In this work, WES disclosed in a Moroccan family a novel missense mutation c.1810CNG (p.Arg604Gly) in exon 20 inTMC1gene (Fig. 2.A) which is responsible for the DFNA36 and DFNB7/11 form of deafness (Kurima et al., 2002). This mutation is predicted to be deleterious as stated by Polyphen-2 (Adzhubei et al., 2010), MutationTaster (Schwarz et al., 2010) and SIFT (http://sift.jcvi.org/).TMC1gene encodes a transmembrane channel-like protein 1, which includes six transmembrane domains (Fig. 2.B). This protein does not show similarity to any other known protein and is expressed specifically in hair cells of the co- chlea and in the neurosensory epithelium of the vestibular organ, and mandatory for their normal functions (Kurima et al., 2002). The identified missense mutation c.1810CNG (p.Arg604Gly) can cause structural changes inTMC1structure affecting its function, and eventually leading to the phenotype observed in the affected offspring. The pathogenic effect of the p.Arg604Gly mutation on the three-dimensional structure is suggested by the prediction programs as reported in the graphical model (Fig. 3) In addition, this amino-acid position is well-conserved among species (Fig. 2.C). To date, a total of 52 different mutations have been detected inTMC1gene (Table 2). In North Africa,TMC1mutations were identified in Algeria, Morocco (the current study) and Tunisia. In Tunisia, a novel mutation (c.2260 + 2 T) was recently described by whole exome sequencing in intron 23 ofTMC1(Riahi et al., 2014), while another mutation inTMC1exon 7 c.100CNT (p.R34X) was very frequently found (5.55%) in Tunisian patients with ARSHNL, suggesting a founder effect (Ben Said et al., 2010). This mutation c.100CNT was also present in a family from Algeria (Ammar-Khodja et al., 2015).

In conclusion, using whole exome sequencing, we identiﬁed theﬁrst TMC1 mutation, c.1810CNG (p.Arg604Gly) in the Moroccan population.

The identiﬁcation of supplementary disease-causing mutations in this gene should conﬁrm further the essential role of theTMC1protein in au- ditory function.

Disclosure statements

The authors declare not having any conﬂict of interest.

Fig. 3.Three-dimensional structural modeling. (A) Segments of TMC1 protein highlighting position and orientation of the native amino acid Arginine-604. (B) Same structural prediction as in (A) but with the Glycine-604 substitution showing a reverse orientation of the amino-acid residue.

Table 2

Overview of all TMC1 mutations so far identiﬁed.

Origin Mutation DNA Protein Exon(E)/Intron(I) Type of variant Domain Reference

China c.589GNA p.G197R E11 Missense IC1 Gao et al. (2013)

China c.1171CNT p.Q391X E15 Nonsense EC2 Gao et al. (2013)

China c.1247 TNG p.L416R Missense EC2 Chen et al. (2015)

China c.1312GNA p.A438T Missense EC2 Chen et al. (2015)

China c.236 + 1GNC – Splicing site – Yang et al. (2013)

China c.1107CNA p.N369K Missense TM3 Yang et al. (2013)

China c.1209GNC p.W403C Missense EC2 Yang et al. (2013)

China c.1253 TNA p.M418K E16 Missense EC2 Zhao et al. (2014)

India c.1960ANG p.M654V E20 Missense TM5 Kurima et al. (2002)

India c.237-6 TNG – Intron 7 Splice site regulation – Ganapathy et al. (2014)

India c.453 + 2 TNC – Intron 9 Splice site regulation – Ganapathy et al. (2014)

India c.628_630del p.I210del Exon 11 Deletion TM1 Ganapathy et al. (2014)

India c.800GNA p.G267E Exon 13 Missense EC1 Ganapathy et al. (2014)

India c.1566 + 1GNA – Intron 17 Splice site regulation – Ganapathy et al. (2014)

Iran c.776 + 1GNA – E7 Splice site mutation – Hilgert et al. (2008)

Greece c.2350CNT p.R604X E20 Nonsense EC3 Hilgert et al. (2008)

Morocco c.1810CNG p.Arg604Gly E20 Missense IC3 The present study

Pakistan c. IVS10-8 TNA – I10 Splice site mutation – Kurima et al. (2002)

Pakistan c. IVS13 + 1GNA – I13 Splice site mutation – Kurima et al. (2002)

Santos et al. (2005)

Pakistan c.884 + 1GNA – E13 Splice site mutation – Kurima et al. (2002)

Pakistan c.830ANG p.Y277C E13 Missense TM2 Santos et al. (2005)

Pakistan c.IVS5 + 1GNT Splice site I5 Splice site mutation – Kitajiri et al. (2007b)

Pakistan c.1534CNT p.R512X E17 Nonsense IC3 Kurima et al. (2002)

Pakistan c.536-8 TNA – E11 Splice site mutation – Santos et al. (2005)

Pakistan c.2004 TNG p.S668R E21 Missense EC3 Santos et al. (2005)

Pakistan c.2035GNA p.E679K E21 Missense EC3 Santos et al. (2005)

Pakistan c.1541CNT p.P514L E17 Missense IC3 Kitajiri et al. (2007b)

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Acknowledgments

Authors are indebted to the families that contributed to this study, and to an INSERM-CNRST collaboration program favoring exchanges between our laboratories.

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Table 2(continued)

Origin Mutation DNA Protein Exon(E)/Intron(I) Type of variant Domain Reference

Pakistan c.1543 TNC p.C515R E17 Missense IC3 Kitajiri et al. (2007b)

Pakistan c.IVS3_IVS5 – E5 Deletion – Kurima et al. (2002)

Pakistan c.362 + 18ANG p.Glu122Tyrfs*10 I8 Splice site mutation – Shaﬁque et al. (2014)

Poland – p.S320R E8 Missense IC2 Hassan et al. (2015)

Sudan c.IVS19 + 5GNA – I19 Splice site mutation – Meyer et al. (2005)

The Netherlands c.1763 + 3ANG p.W588WfsX81 I19 Splice site mutation – de Heer et al. (2011)

Tunisia c.2260 + 2 TNA – I23 Splice site mutation – Riahi et al. (2014)

Tunisia c.1764GNA p.W588X E19 Nonsense IC3 Tlili et al. (2008)

Turkey c.776 ANG p.Y259C E13 Missense EC1 Kalay et al. (2005)

Turkey c.821CNT p.P274L E13 Missense TM2 Kalay et al. (2005)

Turkey c.1083_1087delCAGAT p.R362PfsX6 E15 Deletion IC2 Kalay et al. (2005)

Turkey c.767delT p.F255FfsX14 E13 Deletion EC1 Hilgert et al. (2008)

Turkey c.1166GNA p.R389Q E15 Missense EC2 Hilgert et al. (2008)

Turkey c.1330GNA p.G444R E16 Missense TM4 Sirmaci et al. (2009)

Turkey c.IVS6 + 2 TNA Splice site I6 Splice site mutation – Sirmaci et al. (2009)

Turkey c.1685_2280 del – E19–24 Deletion – Sirmaci et al. (2009)

Turkey, India c.1333CNT p.G445R E16 Missense TM4 Sirmaci et al. (2009)

Ganapathy et al. (2014)

North America, China c.1714 GNA p.D572N E19 Missense IC3 Makishima et al. (2004)

Kitajiri et al. (2007a) Wei et al. (2014)

Iran, China c.150delT p.N50KfsX25 Frame-shift indel IC1 Yang et al. (2010)

Yang et al. (2013)

Pakistan, India c.1114GNA p.V372M E15 Missense TM3 Ganapathy et al. (2014).

Santos et al. (2005)

Pakistan, India c.295_296 delA – E8 Deletion IC1 Kurima et al. (2002)

Pakistan, Tunisia, Algeria, Lebanon, Jordan,Turkey, India

c.100 CNT p.R34X E7 Nonsense IC1 Kurima et al. (2002)

Hilgert et al. (2008) Sirmaci et al. (2009) Kitajiri et al. (2007a) Tlili et al. (2008) Ammar-Khodja et al. (2015) Ganapathy et al. (2014)

Sudan, Tunisia, Lebanon, Jordan c.1165CNT p.R389X E15 Nonsense EC2 Meyer et al. (2005)

Hilgert et al. (2008) Tlili et al. (2008)

Turkey, Pakistan, China c.1334 GNA p.R455H E16 Missense TM4 Kalay et al. (2005)

Santos et al. (2005) Yang et al. (2013)

Turkey, Iran c.2030 TNC p.I667T E21 Missense EC3 Sirmaci et al. (2009)

Davoudi-Dehaghani et al. (2014)

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