- Email: [email protected]
Absence of KCNQ4 mutation in Bengali families with ADNSHL originated from West Bengal, India
Accepted Manuscript Absence of KCNQ4 mutation in Bengali families with ADNSHL originated from West Bengal, India Bidisha Adhikary, Biswabandhu Bankura, Subhradev Biswas, Silpita Paul, Madhusudan Das PII:
S0165-5876(17)30252-5
DOI:
10.1016/j.ijporl.2017.06.004
Reference:
PEDOT 8567
To appear in:
International Journal of Pediatric Otorhinolaryngology
Received Date: 12 January 2017 Revised Date:
6 June 2017
Accepted Date: 9 June 2017
Please cite this article as: B. Adhikary, B. Bankura, S. Biswas, S. Paul, M. Das, Absence of KCNQ4 mutation in Bengali families with ADNSHL originated from West Bengal, India, International Journal of Pediatric Otorhinolaryngology (2017), doi: 10.1016/j.ijporl.2017.06.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Absence of KCNQ4 mutation in Bengali families with ADNSHL originated from West Bengal, India
Department of Zoology,University of Calcutta,35 Ballygunge Circular Road,Kolkata-700019,
West Bengal, India 2
Department of E.N.T,Institute of Post Graduate Medical Education & Research, 244 A J C
Bose Road,Kolkata-700020,West Bengal, India
Name: Prof. Madhusudan Das
M AN U
* Corresponding author
SC
1
RI PT
BidishaAdhikary1, BiswabandhuBankura1, Subhradev Biswas2, Silpita Paul1, Madhusudan Das1*
Postal address: Department of Zoology,University of Calcutta,35 Ballygunge Circular Road,Kolkata-700019, West Bengal, India
AC C
EP
Telephone: 09831281756
TE D
Email: [email protected]
ACCEPTED MANUSCRIPT
Absence of KCNQ4 mutation in Bengali families with ADNSHL originated from West Bengal, India
Department of Zoology, University of Calcutta, 35 Ballygunge Circular Road,Kolkata-700019,
West Bengal, India 2
Department of E.N.T, Institute of Post Graduate Medical Education & Research, 244 A J C
Bose Road,Kolkata-700020,West Bengal, India
Name: Prof. Madhusudan Das
M AN U
* Corresponding author
SC
1
RI PT
Bidisha Adhikary1, Biswabandhu Bankura1, Subhradev Biswas2, Silpita Paul1, Madhusudan Das1*
Postal address: Department of Zoology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata-700019, West Bengal, India
AC C
EP
Telephone: 09831281756
TE D
Email: [email protected]
1
ACCEPTED MANUSCRIPT
Abstract Objective: Autosomal Dominant Non-Syndromic Hearing Loss (ADNSHL) is extremely
RI PT
heterogeneous in nature. More than 60 loci with 30 different genes have been identified linked to ADNSHL. Mutation in KCNQ4 is considered as one of the most common causative factor responsible for ADNSHL. No study focused on the genetic alteration of KCNQ4 gene among
SC
hearing loss patients in India. The present study for the first time was carried out to determine the mutation spectrum of KCNQ4 gene in ADNSHL patients of West Bengal state, India.
M AN U
Method: Twenty nine individuals from 10 independent ADNSHL family (with two or more generation affected) were studied both clinically and genetically. Most of the patients showed moderate progressive sensorineural hearing loss. Mutation analysis was conducted for KCNQ4
TE D
gene using polymerase chain reaction followed by direct sequencing.
Results: Neither any reported nor a novel pathogenic mutation in KCNQ4was detected in our studied group, in contrast to the findings among East Asians.
EP
Conclusion: The result of the present study suggests that mutations in KCNQ4 gene are
AC C
unlikely to be a major causative factor of ADNSHL in our studied patients from West Bengal, India, pointing to other genes might be responsible for ADNSHL in our studied patients.
Key words: Hereditary hearing impairment, ADNSHL, KCNQ4, Pathogenic mutation
2
ACCEPTED MANUSCRIPT
1. Introduction Hereditary hearing impairment is a most common neurosensory disorder. In most cases, hearing
RI PT
loss shows genetic heterogeneity and can be inherited in different patterns, autosomal recessive, autosomal dominant, X- linked and mitochondrial mode of inheritance. Among all, Autosomal Dominant Non Syndromic Hearing Loss (ADNSHL) is extremely heterogenic in nature. To date, more than 60 loci with 30 different genes have been identified associated with ADNSHL [1].The
SC
genetic epidemiologies of ARNSHL and ADNSHL are fundamentally different because in
M AN U
ARNSHL mutations in just a few genes (e.g. the DFNB1 locus, SLC26A4 CDH23 or MYO15) account for greater than 50% of hereditary cases. This does not happen with ADNSHL. Till date several genes for example WFS1, KCNQ4, COCH, GJB2, MYO1A and TECTA have found to be associated with this clinical condition [2]. However, due to such genetic heterogeneity, lack of recurrent mutations and availability of limited number of sample, routine genetic testing for
TE D
ADNSHL has lagged [3]. Based on the previous studies on several population, it has been found that among all genes mutation in KCNQ4 gene is one of the most frequent cause of ADNSHL [2]. KCNQ4 is a member of the voltage gated potassium channel family, located on DFNA2
EP
locus [4].Protein codes for KCNQ4, consists of 695 amino acid with six trans-membrane
AC C
domains and a hydrophobic P-loop region, in between the transmembrane domain S5and S6 which forms the K+ selective channel pore. The fourth transmembrane domain contains the voltage sensor, which is responsible for the electrically driven conformational change that leads to channel opening. Functional channels are formed by a tetrameric assembly of KCNQ4 subunits, typically in homotetrameric form [5-7].KCNQ4 is expressed in the outer hair cells. The function of outer hair cells is mainly to tune the cochlea by converting their receptor potential into a mechanical force. Mechanical stimulation of the stereocilia of hair cells leads to an influx
3
ACCEPTED MANUSCRIPT
of K+ ion into it by the large electrical voltage (~150 mV) across apical membranes. The potassium taken up by hair cells leaves these cells via their basolateral membrane. KCNQ4 is important for the basolateral removal of K+ from hair cells. Therefore, mutation in KCNQ4, is
RI PT
expected to disrupt K+ ion removal from outer hair cells leads to slow degeneration of these cells ultimately hearing impairment takes place [8].To date, 20 different pathogenic mutations in KCNQ4 have been reported and they are mostly missense mutation with a dominant negative
SC
mechanism [9].
Although the hereditary hearing impairment has long been recognized in India, only
M AN U
a few studies regarding ADNSHL have been reported from here [10, 11]. Moreover, till date no data is available regarding the frequency of KCNQ4 mutations among ADNSHL patients in India, whereas this gene is the most common cause of nonsyndromic dominant deafness in comparison to other reported genes. Therefore, the objective of the present study was to identify
India.
TE D
the frequency of KCNQ4 mutations among ADNSHL patients in the population of West Bengal,
2. Materials and methods
EP
A total of 29 individuals from 10 independent ADNSHL families (with two or more generation
AC C
affected) were included in this study. The pedigree of 10 families was presented in Supplementary Figure 1. They were from ENT outpatient department of Institute of postgraduate medical education & research (IPGME & R), Kolkata, Ali Yavar Jung National Institute of Speech and Hearing Disability (Eastern Regional Center) and from the Kolkata deaf and dumb school. Age of onset, degree of hearing loss, progression of hearing loss, tinnitus, vertigo, and use of aminoglycosides and other clinical abnormalities of participants were examined by doctors, audiologists and anamnestic evaluation. Audiometric evaluation was done by Brainstem 4
ACCEPTED MANUSCRIPT
Evoked Response Audiometry (BERA). The degree of hearing impairment was based on average threshold calculated over frequencies of 500, 1000 and 2000 Hz in the better ear. All subjects underwent CT scan examination for identification of any inner ear malformation. None of the
RI PT
subjects included in our study had any other associated neurological signs, visual dysfunction and diabetes mellitus. A total of 40 healthy individuals with normal hearing and without any family history of hearing loss from same geographical region were included as control. The
SC
study protocol was approved by the ethics committee of IPGME & R, Kolkata. Written informed
M AN U
consent was obtained from all participants prior to blood sampling.
2.1 Mutation analysis
We sequenced the KCNQ4 gene in 29 individuals from 10 different ADNSHL family and 40 controls. Peripheral blood samples were collected from the members of ADNSHL family and
TE D
control individuals and genomic DNA was extracted from the peripheral blood leucocytes by using QIAamp Blood Kit (QIAGEN, Hilden, Germany). For the analysis of KCNQ4 gene the entire coding exons including exon-intron boundaries and approximately 60 bp of the flanking
EP
intron regions were amplified by polymerase chain reaction (PCR), using specific primers (Supplementary Table 1).
AC C
The PCR was performed in a total of 25 µl reaction contained 40–100 ng of genomic DNA 1.5 mM MgCl2, 100 µM of each dNTP,0.4µM of each primer and 0.5 U of Taq DNA polymerase (Applied Biosystems). Denaturation at 95 °C for 30 s, annealing at 54–62 °C for30 s, and extension at 72 °C for 50 s × 44 cycles were performed. The PCR products were subjected to direct sequencing using a TaqDyeDeoxy Terminator sequencing kit (PE Applied Biosystems, City, USA) with an ABI Prism 377 DNA sequencer (PE Applied Biosystems).
5
ACCEPTED MANUSCRIPT
3. Results A total of 29 individuals from 10 independent ADNSHL families were included in the study. All the patients were from non-consanguineous family. Severity of hearing loss (three frequency
RI PT
average of 500, 1000 and 2000 Hz) varied from mild to profound; 17% of the patients showed mild (30-49dB), 55% had moderate (50-69dB), 21% had severe (70-89dB) and7% had profound(>90dB) hearing loss. The age of onset also varied from congenital to 40. 72% persons
SC
had progressive hearing loss, 17% persons had tinnitus and 21% persons were identified to have vertigo. CT scan examination of all 29 individuals revealed that none of the patients had any
M AN U
inner ear malformation. Table 1 shows the clinical features of all 29 patients from 10 ADNSHL families.
Table 1: Clinical features among 15 family members with history of hearing loss found in this study: Onset (years)
(years)
1
51
2
8
Moderate
AC C
16
12
Age Frequency of Progression Tinnitus Vertigo hearing loss
TE D
Age
EP
Family
Moderate
+
+
-
-
Temporal Bone (CT)
KCNQ4variants
Normal
P291P (hetero), T293T (hetero)
-
-
Normal
P291P (hetero), T293T (hetero)
71
24
Severe
+
-
-
Normal
50
20
Moderate
+
-
-
Normal
45
21
Moderate
+
-
+
Normal
17
14
Moderate
+
+
+
Normal
6
ACCEPTED MANUSCRIPT
3
14
13
Mild
-
-
-
Normal
40
1
Severe
+
-
+
Normal
P291P
(hetero),
T293T (hetero) -
58
16
Moderate
+
-
-
26
15
Mild
+
-
-
6
5
Moderate
-
+
60
16
Moderate
+
-
55
10
Moderate
35
12
Moderate
Normal
RI PT
-
Normal
Normal
SC
-
-
Normal
-
Normal
7
p.Thr293Thr (hetero)
+
+
-
Normal
+
-
-
Normal
6
5
p.Pro291Pro (hetero), p.Thr293Thr (hetero)
Moderate
+
-
-
Normal
Mild
-
-
-
Normal
EP
11
AC C
6
32
p.Pro291Pro (hetero),
TE D
5
Congenital Profound
M AN U
4
9
60
7
Severe
+
-
-
Normal
32
8
Moderate
+
-
+
Normal
7
6
Mild
-
-
-
Normal
35
Unknown
Severe
+
-
-
Normal
9
Congenital Severe
-
-
-
Normal
p.Pro291Pro
7
ACCEPTED MANUSCRIPT
(hetero), p.Thr293Thr (hetero)
10
34
Profound
+
-
+
Congenital Severe
-
-
-
45
10
Moderate
+
+
-
15
11
Moderate
+
-
-
14
10
Mild
-
-
40
12
Moderate
18
6
Moderate
Normal
RI PT
2
Normal
Normal
Normal
SC
9
60
-
M AN U
8
Normal
+
+
+
Normal
+
-
-
Normal
None of the 29 hearing impaired individuals exhibited any pathogenic variants in KCNQ4 gene,
TE D
including most commonp.Trp276Ser or any other disease-causing amino-acid variant. Only two synonymous amino acid changes, first one Proline>Proline at amino acid position 291 and
EP
another Threonine > Threonine at the position 293 were identified in the study (Table 2). It was observed that these two synonymous changes transmitted together in patients and control group
AC C
as they are situated in same linkage disequilibrium in south Asian population according to 1000 genome project. The minor allele frequency ofp.Pro291Pro and p.Thr293Thr among case (10.34%) and control (7.5%) individuals was almost same which indicates that these two synonymous changes are not the causal factor for ADNSHL in our population.
8
ACCEPTED MANUSCRIPT
Table 2: Nucleotide variants of KCNQ4 gene in patients and controls. Domain
Category
Reported/ novel
Numbers found in patients (n-29)
Numbers found in controls (n-40)
c.873G>A
Pro291Pro
P-loop region
Synonymous variant
rs:12117176
6
6
c.879G>A
Thr293Thr
P-loop region
Synonymous variant
rs:12143503
6
6
Allele frequency in patients(%)
Allele frequency in control (%)
RI PT
Amino acid change
p-value
10.34%
7.5%
0.78
10.34%
7.5%
0.78
M AN U
SC
Nucleotide Change
4. Discussion
In the present study, we have conducted a comprehensive genetic screening of KCNQ4 in10 Bengali families with ADNSHL to establish the mutation spectrum. The patients belong to mixed ethnic backgrounds, Indo-European and Austro-Asiatic [12]. It is noteworthy that this is
TE D
the first study of screening KCNQ4 gene among ADNSHL patients in Indian population. The majority of the families in our study (7/10) had post lingual progressive hearing loss. Moreover, audiometric evaluation of patients revealed that degree of hearing loss in most of the patients
EP
was moderate (55%). Absence of any inner ear malformation, indicates that hearing loss was not caused by alteration in inner ear structure. From genetic analysis of the KCNQ4 gene, two
AC C
synonymous changes in the p loop region of K+ channel p.Pro291Pro and p.Thr293Thr in the same linkage group were identified. These two variants were also found in control individuals with almost similar frequency. The finding of the current study indicates that KCNQ4 may not play a significant role in ADNSHL in our study. In a previous study Coucke et al., also failed to identify any KCNQ4mutation among large member of patients from an Indonesian family [4]. Contrary to our 9
ACCEPTED MANUSCRIPT
findings, KCNQ4 mutation is one of the common cause of ADNSHL among Dutch [4, 13, 14], American [15-17], Japanese [3, 18] and Chinese [19]. Thus far, 16 missense mutations and 4 deletions in KCNQ4 have been reported. Majority of these are private mutations found in a
RI PT
particular ADNSHL family in particular countries [19]. A missense mutation p.Gly285Ser in KCNQ4, was the first identified mutation associated with dominant deafness in a small French family [8]. Interestingly, Coucke et al., identified another mutation, p.Gly285Cys at the same
SC
amino acid position in an American family [4]. In contrast to recessive deafness, where recurrent mutations are common - such as c.235delC, c.35delG, c.167delT in GJB2 [20, 21], p.His723Arg
M AN U
in SLC26A4 [22], and p.Pro204Leu in CDH23 [23]; these are rare for dominant deafness gene. So far, p.Trp276Ser is the missense mutation of KCNQ4 channel that has been detected in multiple ADNSHL families from different racial origin (Belgiam, Dutch, Japanese). This observation suggests that Trp276 is a mutational hot spot [24].The Trp276 is situated in the pore
TE D
region of KCNQ4 channel and play a crucial role in K+ channel function, presumably holding the pore open at a correct diameter. Therefore mutation at this position alters channel function and leads to hearing loss [25].In addition to p.Trp276Ser, other mutations in p loop region p.Leu274His [13,
EP
14], p.Leu281Ser [15], p.Gly296Ser [26] and p.Trp.275Arg [19] were found to contribute in ADNSHL in different geographic locations around the world. Mutations in KCNQ4 were also
AC C
found in the positions other than the P-loop region in ADNSHL patients. The mutation p.Phe182Leu in third transmembrane domain was reported in a Chinese family [27] while three different missense mutations p.Trp241stop, p.Glu260Lys and p.Asp262Val were found by Hildebrand et al., at fifth transmembrane domain in American family [16]. Coucke et al., also reported p.Gly321Ser mutation in sixth transmembrane domain in a Dutch family [4]. In a recent study with 287 probands from independent Japanese ADNSHL families, Naito et al., found four missense mutations (p.Val230Glu, p.Pro291Ser, p.Pro291Leu and p.Arg291Leu), and concluded 10
ACCEPTED MANUSCRIPT
that KCNQ4 is a common cause of ADNSHL with a frequency of 6.62% in Japanese population [3]. Besides common missense mutations, one recurrent deletion - c.211delC was observed in 13 unrelated Japanese families. The frequency of c.211delC among Japanese was 4.53% and it
RI PT
accounted for 68.4% of all KCNQ4 mutations and was predicted to exert pathogenic effects on hearing physiology [3].
SC
KCNQ4gene has been reported as the major contributor to ADNSHL among many populations in the world including East Asian - Japanese and Chinese population. Although, present study
M AN U
conducted in South Asia, did not identify any pathogenic variants in KCNQ4. Population studies clearly displayed, Japanese can be classified under two main ethnic groups: the Hondo and the Ryukyu [28] and Chinese belong to Han Chinese [29].While, the individuals included in the present study, belong to mixed ethnic background - Indo European and Austro Asiatic [12]. These observations highlight, that distinct ethnicity variation altered KCNQ4 mutations spectrum
TE D
in non-syndromic hearing loss.
It is generally noted that in India, information which is important for proper diagnosis of
EP
autosomal dominant non-syndromic deaf individuals like - detailed health data, pedigree, family history; are often not available to the physicians. Therefore, identification of genes associated
AC C
with hearing loss, and identification of families with autosomal dominant deafness is difficult. The development of proper diagnostic services and improvement in collecting clinical data and pedigree for genetic deafness would prevent the frustration of incomplete etiological diagnosis among dominant deaf individuals in India.
11
ACCEPTED MANUSCRIPT
5. Conclusion In summary, our finding shows that the mutation in KCNQ4 is likely to be rare (<9%) in our studied families from West Bengal. Possibly mutations in other known/unknown gene may be
genes associated with ADNSHL in patients from West Bengal.
SC
Conflict of interest There is no conflict of interest.
M AN U
Acknowledgement
RI PT
responsible for the phenotype. Therefore, further study is required to screen full spectrum of
The authors would like to thank the participants and their families who gave their consent and collaborated in this study. Funding
TE D
This research was supported by grant from the Department of Biotechnology, Govt. of West Bengal, India [Sanction No-106-BT (Estt.)/RD-20/11].
EP
References
[1]http://hereditaryhearingloss.org
AC C
[2] N. Hilgert, R.J. Smith, G. Van Camp, Forty-six genes causing nonsyndromichearing impairment: which ones should be analyzed in DNA diagnostics?,MutatRes. 681 (2009) 189– 196. [3] T .Naito, S.Y. Nishio, Y. Iwasa, T. Yano, K. Kumakawa,S. Abe et al., Comprehensive genetic screening of KCNQ4 in a large autosomal dominant nonsyndromic hearing loss cohort: genotype-phenotype correlations and a founder mutation, PLoS One. 8 (2013) e63231. [4] P.J. Coucke, P. Van Hauwe, P.M. Kelley, H. Kunst, I. Schatteman, D. Van Velzen et al., Mutations in the KCNQ4 gene are responsible for autosomal dominant deafness in four DFNA2 families, Hum Mol Genet. 8(1999) 1321–1328. 12
ACCEPTED MANUSCRIPT
[5] H.S. Wang, Z. Pan, W. Shi, B.S.Brown, R.S.Wymore, I.S. Cohen, J.E. Dixon and D.McKinnon,KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the Mchannel, Science 282(1998) 1890–1893.
RI PT
[6] B.C.Schroeder, C.Kubisch, V. Stein and T.J. Jentsch, Moderate loss of function of cyclicAMP-modulated KCNQ2/KCNQ3 K+ channels causes epilepsy, Nature 396(1998) 687–690. [7]W.P. Yang, P.C. Levesque, W.A. Little, M.L.Conder, P.Ramakrishnan, M.G.Neubauer and M.A. Blanar, Functional expression of two KvLQT1-related potassium channels responsible for inherited idiopathic epilepsy, J. Biol. Chem. 273(1998) 19419–19423.
M AN U
SC
[8]C. Kubisch, B.C. Schroeder, T. Friedrich, B. Lutjohann, A. El-Amraoui, et al., KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness, Cell 96 (1999) 437–446. [9] L.M. Dominguez, K.M. Dodson, Geneticsof hearing loss: focus on DFNA2, The Application of Clinical Genetics 5(2012)97–104.
TE D
[10] A.Pavithra , J.Chandru, J.M.Jeffrey, N.P.Karthikeyen, C.R.Srisailapathy, Rare compound heterozygosity involving dominant and recessive mutations of GJB2 gene in an assortative mating hearing impaired Indian family, Eur Arch Otorhinolaryngol.(2016) 274(1)119-125. [11]A.Pavithra, M.Selvakumari, V.Nityaa, N.Sharanya, R.Ramakrishnan, M.Narasimhan, C.R.Sr isailapathy, Autosomal dominant hearing loss resulting from p.R75Q mutation in the GJB2 gene: nonsyndromic presentation in a South Indian family, Ann Hum Genet. 79(1) (2015) 76-82.
AC C
EP
[12] A.Basu , N.Mukherjee, S.Roy, S.Sengupta, S.Banerjee, M.Chakraborty, B.Dey, M.Roy, B.Roy, N.P.Bhattacharyya, S.Roychoudhury, P.P.Majumder, Ethnic India: a genomic view, with special reference to peopling and structure, Genome Res. 13(10) (2003) 2277-2290. [13] P. Van Hauwe, P. J. Coucke, R. J. Ensink, P. Huygen, C.W.Cremers, G. V. Camp, Mutations in the KCNQ4 K+ channel gene, responsible for autosomal dominant hearing loss, cluster in the channel pore region, Am. J. Med. Genet. 93 (2000) 184-187. [14] A.M. de Heer , M. Schraders, J. Oostrik, L. Hoefsloot, P.L. Huygen, C.W. Cremers, Audioprofile-directed successful mutation analysis in a DFNA2/KCNQ4 (p.Leu274His) family,AnnOtolrhinolaryngol. 120(4)(2011)243-248.
13
ACCEPTED MANUSCRIPT
[15] Z.Talebizadeh , P.M.Kelley, J.W.Askew, K.W.Beisel, S.D.Smith, Novel mutation in the KCNQ4 gene in a large kindred with dominant progressive hearing loss, Hum Mutat. 14(6)(1999) 493-501.
RI PT
[16]M.S.Hildebrand, D.Tack, S.J.McMordie, A.DeLuca, I.A.Hur, et al., Audioprofile-directed screening identifies novel mutations in KCNQ4causing hearing loss at the DFNA2 locus, Genet Med. 10(2008) 797–804.
SC
[17] J.Arnett, S.B.Emery, T.B.Kim, A.K.Boerst,K.Lee, et al., Autosomal dominant progressive sensorineural hearing loss due to a novel mutation in the KCNQ4 gene, Arch Otol Head Neck Surg. 137(2011) 54–59.
M AN U
[18] J. Akita, S. Abe, H. Shinkawa, W.J. Kimberling, S. Usami, Clinical and genetic features of nonsyndromic autosomal dominant sensorineural hearing loss: KCNQ4 is a gene responsible in Japanese, J Hum Genet. 46(2001) 355–361. [19] H. Wang, Y. Zhao, Y. Yi, Y. Gao, Q. Liu, D. Wang, et al., Targeted highthroughput sequencing identifies pathogenic mutations in KCNQ4 in two large Chinesefamilies with autosomal dominant hearing loss.PLoS One (2014) 9(8) e103133.
TE D
[20]L.VanLaer, P.Coucke, R.F.Mueller, G. Caethoven, K.Flothmann, et al., Acommon founder for the 35delG GJB2 gene mutation in connexin 26 hearing impairment, J Med Genet. 38(2001)515–518.
EP
[21] D. Yan, H.J. Park, X.M. Ouyang, A. Pandya, K. Doi, et al., Evidence of a founder effect for the 235delC mutation of GJB2 (connexin 26) in east Asians, Hum Genet. 114(2003) 44–50.
AC C
[22] H.J. Park, S. Shaukat, X.Z. Liu, S.H. Hahn, S. Naz, et al., Origins and frequencies of SLC26A4 (PDS) mutations in east and south Asians: global implications for the epidemiology of deafness, J Med Genet. 40(2003) 242–248. [23] M. Miyagawa, S.Y. Nishio, S. Usami, Prevalence and clinical features of hearing losspatients with CDH23 mutation: a large cohort study, Plos one (2012) 7(8) e40366. [24] G. Van Camp, P.J. Coucke, J. Akita, E. Fransen, S. Abe, et al., A mutational hot spot in the KCNQ4 gene responsible for autosomal dominant hearing impairment, Hum Mutat. 20(2002)15–19.
14
ACCEPTED MANUSCRIPT
[25] D.A. Doyle, J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, et al., The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity, Science 280 (1998) 69– 77.
RI PT
[26] A. Mencia, D. Gonzalez-Nieto, S. Modamio-Hoybjor, A. Etxeberria, G. Aranguez, et al., A novel KCNQ4 pore-region mutation (p.G296S) causes deafness by impairing cell-surface channel expression, Hum Genet. 123 (2008) 41–53.
SC
[27] C.C. Su, J.J. Yang, J.C. Shieh, M.C. Su, S.Y.Li, Identification of novel mutations in the KCNQ4 gene of patients with nonsyndromic deafness from Taiwan, AudiolNeurotol. 12(2007)20
M AN U
[28]Y.YamaguchiKabata, K.Nakazono, A.Takahashi, S.Saito, N.Hosono, M.Kubo, Y.Nakamura,N. Kamatani, Japanese population structure, based on SNP genotypes from 7003 individuals compared to other ethnic groups: effects on population-based association studies,Am J Hum Genet.83(4)(2008)44556.
AC C
EP
TE D
[29] J.Chen, H.Zheng,J.X. Bei, L.Sun, W.H.Jia,T. Li , F.Zhang, M.Seielstad, Y.X.Zeng, X.Zhang, J. Liu , Genetic structure of the Han Chinese population revealed by genome-wide SNP variation, Am J Hum Genet.85(6)(2009)775-85.
15
S0165-5876(17)30252-5
DOI:
10.1016/j.ijporl.2017.06.004
Reference:
PEDOT 8567
To appear in:
International Journal of Pediatric Otorhinolaryngology
Received Date: 12 January 2017 Revised Date:
6 June 2017
Accepted Date: 9 June 2017
Please cite this article as: B. Adhikary, B. Bankura, S. Biswas, S. Paul, M. Das, Absence of KCNQ4 mutation in Bengali families with ADNSHL originated from West Bengal, India, International Journal of Pediatric Otorhinolaryngology (2017), doi: 10.1016/j.ijporl.2017.06.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Absence of KCNQ4 mutation in Bengali families with ADNSHL originated from West Bengal, India
Department of Zoology,University of Calcutta,35 Ballygunge Circular Road,Kolkata-700019,
West Bengal, India 2
Department of E.N.T,Institute of Post Graduate Medical Education & Research, 244 A J C
Bose Road,Kolkata-700020,West Bengal, India
Name: Prof. Madhusudan Das
M AN U
* Corresponding author
SC
1
RI PT
BidishaAdhikary1, BiswabandhuBankura1, Subhradev Biswas2, Silpita Paul1, Madhusudan Das1*
Postal address: Department of Zoology,University of Calcutta,35 Ballygunge Circular Road,Kolkata-700019, West Bengal, India
AC C
EP
Telephone: 09831281756
TE D
Email: [email protected]
ACCEPTED MANUSCRIPT
Absence of KCNQ4 mutation in Bengali families with ADNSHL originated from West Bengal, India
Department of Zoology, University of Calcutta, 35 Ballygunge Circular Road,Kolkata-700019,
West Bengal, India 2
Department of E.N.T, Institute of Post Graduate Medical Education & Research, 244 A J C
Bose Road,Kolkata-700020,West Bengal, India
Name: Prof. Madhusudan Das
M AN U
* Corresponding author
SC
1
RI PT
Bidisha Adhikary1, Biswabandhu Bankura1, Subhradev Biswas2, Silpita Paul1, Madhusudan Das1*
Postal address: Department of Zoology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata-700019, West Bengal, India
AC C
EP
Telephone: 09831281756
TE D
Email: [email protected]
1
ACCEPTED MANUSCRIPT
Abstract Objective: Autosomal Dominant Non-Syndromic Hearing Loss (ADNSHL) is extremely
RI PT
heterogeneous in nature. More than 60 loci with 30 different genes have been identified linked to ADNSHL. Mutation in KCNQ4 is considered as one of the most common causative factor responsible for ADNSHL. No study focused on the genetic alteration of KCNQ4 gene among
SC
hearing loss patients in India. The present study for the first time was carried out to determine the mutation spectrum of KCNQ4 gene in ADNSHL patients of West Bengal state, India.
M AN U
Method: Twenty nine individuals from 10 independent ADNSHL family (with two or more generation affected) were studied both clinically and genetically. Most of the patients showed moderate progressive sensorineural hearing loss. Mutation analysis was conducted for KCNQ4
TE D
gene using polymerase chain reaction followed by direct sequencing.
Results: Neither any reported nor a novel pathogenic mutation in KCNQ4was detected in our studied group, in contrast to the findings among East Asians.
EP
Conclusion: The result of the present study suggests that mutations in KCNQ4 gene are
AC C
unlikely to be a major causative factor of ADNSHL in our studied patients from West Bengal, India, pointing to other genes might be responsible for ADNSHL in our studied patients.
Key words: Hereditary hearing impairment, ADNSHL, KCNQ4, Pathogenic mutation
2
ACCEPTED MANUSCRIPT
1. Introduction Hereditary hearing impairment is a most common neurosensory disorder. In most cases, hearing
RI PT
loss shows genetic heterogeneity and can be inherited in different patterns, autosomal recessive, autosomal dominant, X- linked and mitochondrial mode of inheritance. Among all, Autosomal Dominant Non Syndromic Hearing Loss (ADNSHL) is extremely heterogenic in nature. To date, more than 60 loci with 30 different genes have been identified associated with ADNSHL [1].The
SC
genetic epidemiologies of ARNSHL and ADNSHL are fundamentally different because in
M AN U
ARNSHL mutations in just a few genes (e.g. the DFNB1 locus, SLC26A4 CDH23 or MYO15) account for greater than 50% of hereditary cases. This does not happen with ADNSHL. Till date several genes for example WFS1, KCNQ4, COCH, GJB2, MYO1A and TECTA have found to be associated with this clinical condition [2]. However, due to such genetic heterogeneity, lack of recurrent mutations and availability of limited number of sample, routine genetic testing for
TE D
ADNSHL has lagged [3]. Based on the previous studies on several population, it has been found that among all genes mutation in KCNQ4 gene is one of the most frequent cause of ADNSHL [2]. KCNQ4 is a member of the voltage gated potassium channel family, located on DFNA2
EP
locus [4].Protein codes for KCNQ4, consists of 695 amino acid with six trans-membrane
AC C
domains and a hydrophobic P-loop region, in between the transmembrane domain S5and S6 which forms the K+ selective channel pore. The fourth transmembrane domain contains the voltage sensor, which is responsible for the electrically driven conformational change that leads to channel opening. Functional channels are formed by a tetrameric assembly of KCNQ4 subunits, typically in homotetrameric form [5-7].KCNQ4 is expressed in the outer hair cells. The function of outer hair cells is mainly to tune the cochlea by converting their receptor potential into a mechanical force. Mechanical stimulation of the stereocilia of hair cells leads to an influx
3
ACCEPTED MANUSCRIPT
of K+ ion into it by the large electrical voltage (~150 mV) across apical membranes. The potassium taken up by hair cells leaves these cells via their basolateral membrane. KCNQ4 is important for the basolateral removal of K+ from hair cells. Therefore, mutation in KCNQ4, is
RI PT
expected to disrupt K+ ion removal from outer hair cells leads to slow degeneration of these cells ultimately hearing impairment takes place [8].To date, 20 different pathogenic mutations in KCNQ4 have been reported and they are mostly missense mutation with a dominant negative
SC
mechanism [9].
Although the hereditary hearing impairment has long been recognized in India, only
M AN U
a few studies regarding ADNSHL have been reported from here [10, 11]. Moreover, till date no data is available regarding the frequency of KCNQ4 mutations among ADNSHL patients in India, whereas this gene is the most common cause of nonsyndromic dominant deafness in comparison to other reported genes. Therefore, the objective of the present study was to identify
India.
TE D
the frequency of KCNQ4 mutations among ADNSHL patients in the population of West Bengal,
2. Materials and methods
EP
A total of 29 individuals from 10 independent ADNSHL families (with two or more generation
AC C
affected) were included in this study. The pedigree of 10 families was presented in Supplementary Figure 1. They were from ENT outpatient department of Institute of postgraduate medical education & research (IPGME & R), Kolkata, Ali Yavar Jung National Institute of Speech and Hearing Disability (Eastern Regional Center) and from the Kolkata deaf and dumb school. Age of onset, degree of hearing loss, progression of hearing loss, tinnitus, vertigo, and use of aminoglycosides and other clinical abnormalities of participants were examined by doctors, audiologists and anamnestic evaluation. Audiometric evaluation was done by Brainstem 4
ACCEPTED MANUSCRIPT
Evoked Response Audiometry (BERA). The degree of hearing impairment was based on average threshold calculated over frequencies of 500, 1000 and 2000 Hz in the better ear. All subjects underwent CT scan examination for identification of any inner ear malformation. None of the
RI PT
subjects included in our study had any other associated neurological signs, visual dysfunction and diabetes mellitus. A total of 40 healthy individuals with normal hearing and without any family history of hearing loss from same geographical region were included as control. The
SC
study protocol was approved by the ethics committee of IPGME & R, Kolkata. Written informed
M AN U
consent was obtained from all participants prior to blood sampling.
2.1 Mutation analysis
We sequenced the KCNQ4 gene in 29 individuals from 10 different ADNSHL family and 40 controls. Peripheral blood samples were collected from the members of ADNSHL family and
TE D
control individuals and genomic DNA was extracted from the peripheral blood leucocytes by using QIAamp Blood Kit (QIAGEN, Hilden, Germany). For the analysis of KCNQ4 gene the entire coding exons including exon-intron boundaries and approximately 60 bp of the flanking
EP
intron regions were amplified by polymerase chain reaction (PCR), using specific primers (Supplementary Table 1).
AC C
The PCR was performed in a total of 25 µl reaction contained 40–100 ng of genomic DNA 1.5 mM MgCl2, 100 µM of each dNTP,0.4µM of each primer and 0.5 U of Taq DNA polymerase (Applied Biosystems). Denaturation at 95 °C for 30 s, annealing at 54–62 °C for30 s, and extension at 72 °C for 50 s × 44 cycles were performed. The PCR products were subjected to direct sequencing using a TaqDyeDeoxy Terminator sequencing kit (PE Applied Biosystems, City, USA) with an ABI Prism 377 DNA sequencer (PE Applied Biosystems).
5
ACCEPTED MANUSCRIPT
3. Results A total of 29 individuals from 10 independent ADNSHL families were included in the study. All the patients were from non-consanguineous family. Severity of hearing loss (three frequency
RI PT
average of 500, 1000 and 2000 Hz) varied from mild to profound; 17% of the patients showed mild (30-49dB), 55% had moderate (50-69dB), 21% had severe (70-89dB) and7% had profound(>90dB) hearing loss. The age of onset also varied from congenital to 40. 72% persons
SC
had progressive hearing loss, 17% persons had tinnitus and 21% persons were identified to have vertigo. CT scan examination of all 29 individuals revealed that none of the patients had any
M AN U
inner ear malformation. Table 1 shows the clinical features of all 29 patients from 10 ADNSHL families.
Table 1: Clinical features among 15 family members with history of hearing loss found in this study: Onset (years)
(years)
1
51
2
8
Moderate
AC C
16
12
Age Frequency of Progression Tinnitus Vertigo hearing loss
TE D
Age
EP
Family
Moderate
+
+
-
-
Temporal Bone (CT)
KCNQ4variants
Normal
P291P (hetero), T293T (hetero)
-
-
Normal
P291P (hetero), T293T (hetero)
71
24
Severe
+
-
-
Normal
50
20
Moderate
+
-
-
Normal
45
21
Moderate
+
-
+
Normal
17
14
Moderate
+
+
+
Normal
6
ACCEPTED MANUSCRIPT
3
14
13
Mild
-
-
-
Normal
40
1
Severe
+
-
+
Normal
P291P
(hetero),
T293T (hetero) -
58
16
Moderate
+
-
-
26
15
Mild
+
-
-
6
5
Moderate
-
+
60
16
Moderate
+
-
55
10
Moderate
35
12
Moderate
Normal
RI PT
-
Normal
Normal
SC
-
-
Normal
-
Normal
7
p.Thr293Thr (hetero)
+
+
-
Normal
+
-
-
Normal
6
5
p.Pro291Pro (hetero), p.Thr293Thr (hetero)
Moderate
+
-
-
Normal
Mild
-
-
-
Normal
EP
11
AC C
6
32
p.Pro291Pro (hetero),
TE D
5
Congenital Profound
M AN U
4
9
60
7
Severe
+
-
-
Normal
32
8
Moderate
+
-
+
Normal
7
6
Mild
-
-
-
Normal
35
Unknown
Severe
+
-
-
Normal
9
Congenital Severe
-
-
-
Normal
p.Pro291Pro
7
ACCEPTED MANUSCRIPT
(hetero), p.Thr293Thr (hetero)
10
34
Profound
+
-
+
Congenital Severe
-
-
-
45
10
Moderate
+
+
-
15
11
Moderate
+
-
-
14
10
Mild
-
-
40
12
Moderate
18
6
Moderate
Normal
RI PT
2
Normal
Normal
Normal
SC
9
60
-
M AN U
8
Normal
+
+
+
Normal
+
-
-
Normal
None of the 29 hearing impaired individuals exhibited any pathogenic variants in KCNQ4 gene,
TE D
including most commonp.Trp276Ser or any other disease-causing amino-acid variant. Only two synonymous amino acid changes, first one Proline>Proline at amino acid position 291 and
EP
another Threonine > Threonine at the position 293 were identified in the study (Table 2). It was observed that these two synonymous changes transmitted together in patients and control group
AC C
as they are situated in same linkage disequilibrium in south Asian population according to 1000 genome project. The minor allele frequency ofp.Pro291Pro and p.Thr293Thr among case (10.34%) and control (7.5%) individuals was almost same which indicates that these two synonymous changes are not the causal factor for ADNSHL in our population.
8
ACCEPTED MANUSCRIPT
Table 2: Nucleotide variants of KCNQ4 gene in patients and controls. Domain
Category
Reported/ novel
Numbers found in patients (n-29)
Numbers found in controls (n-40)
c.873G>A
Pro291Pro
P-loop region
Synonymous variant
rs:12117176
6
6
c.879G>A
Thr293Thr
P-loop region
Synonymous variant
rs:12143503
6
6
Allele frequency in patients(%)
Allele frequency in control (%)
RI PT
Amino acid change
p-value
10.34%
7.5%
0.78
10.34%
7.5%
0.78
M AN U
SC
Nucleotide Change
4. Discussion
In the present study, we have conducted a comprehensive genetic screening of KCNQ4 in10 Bengali families with ADNSHL to establish the mutation spectrum. The patients belong to mixed ethnic backgrounds, Indo-European and Austro-Asiatic [12]. It is noteworthy that this is
TE D
the first study of screening KCNQ4 gene among ADNSHL patients in Indian population. The majority of the families in our study (7/10) had post lingual progressive hearing loss. Moreover, audiometric evaluation of patients revealed that degree of hearing loss in most of the patients
EP
was moderate (55%). Absence of any inner ear malformation, indicates that hearing loss was not caused by alteration in inner ear structure. From genetic analysis of the KCNQ4 gene, two
AC C
synonymous changes in the p loop region of K+ channel p.Pro291Pro and p.Thr293Thr in the same linkage group were identified. These two variants were also found in control individuals with almost similar frequency. The finding of the current study indicates that KCNQ4 may not play a significant role in ADNSHL in our study. In a previous study Coucke et al., also failed to identify any KCNQ4mutation among large member of patients from an Indonesian family [4]. Contrary to our 9
ACCEPTED MANUSCRIPT
findings, KCNQ4 mutation is one of the common cause of ADNSHL among Dutch [4, 13, 14], American [15-17], Japanese [3, 18] and Chinese [19]. Thus far, 16 missense mutations and 4 deletions in KCNQ4 have been reported. Majority of these are private mutations found in a
RI PT
particular ADNSHL family in particular countries [19]. A missense mutation p.Gly285Ser in KCNQ4, was the first identified mutation associated with dominant deafness in a small French family [8]. Interestingly, Coucke et al., identified another mutation, p.Gly285Cys at the same
SC
amino acid position in an American family [4]. In contrast to recessive deafness, where recurrent mutations are common - such as c.235delC, c.35delG, c.167delT in GJB2 [20, 21], p.His723Arg
M AN U
in SLC26A4 [22], and p.Pro204Leu in CDH23 [23]; these are rare for dominant deafness gene. So far, p.Trp276Ser is the missense mutation of KCNQ4 channel that has been detected in multiple ADNSHL families from different racial origin (Belgiam, Dutch, Japanese). This observation suggests that Trp276 is a mutational hot spot [24].The Trp276 is situated in the pore
TE D
region of KCNQ4 channel and play a crucial role in K+ channel function, presumably holding the pore open at a correct diameter. Therefore mutation at this position alters channel function and leads to hearing loss [25].In addition to p.Trp276Ser, other mutations in p loop region p.Leu274His [13,
EP
14], p.Leu281Ser [15], p.Gly296Ser [26] and p.Trp.275Arg [19] were found to contribute in ADNSHL in different geographic locations around the world. Mutations in KCNQ4 were also
AC C
found in the positions other than the P-loop region in ADNSHL patients. The mutation p.Phe182Leu in third transmembrane domain was reported in a Chinese family [27] while three different missense mutations p.Trp241stop, p.Glu260Lys and p.Asp262Val were found by Hildebrand et al., at fifth transmembrane domain in American family [16]. Coucke et al., also reported p.Gly321Ser mutation in sixth transmembrane domain in a Dutch family [4]. In a recent study with 287 probands from independent Japanese ADNSHL families, Naito et al., found four missense mutations (p.Val230Glu, p.Pro291Ser, p.Pro291Leu and p.Arg291Leu), and concluded 10
ACCEPTED MANUSCRIPT
that KCNQ4 is a common cause of ADNSHL with a frequency of 6.62% in Japanese population [3]. Besides common missense mutations, one recurrent deletion - c.211delC was observed in 13 unrelated Japanese families. The frequency of c.211delC among Japanese was 4.53% and it
RI PT
accounted for 68.4% of all KCNQ4 mutations and was predicted to exert pathogenic effects on hearing physiology [3].
SC
KCNQ4gene has been reported as the major contributor to ADNSHL among many populations in the world including East Asian - Japanese and Chinese population. Although, present study
M AN U
conducted in South Asia, did not identify any pathogenic variants in KCNQ4. Population studies clearly displayed, Japanese can be classified under two main ethnic groups: the Hondo and the Ryukyu [28] and Chinese belong to Han Chinese [29].While, the individuals included in the present study, belong to mixed ethnic background - Indo European and Austro Asiatic [12]. These observations highlight, that distinct ethnicity variation altered KCNQ4 mutations spectrum
TE D
in non-syndromic hearing loss.
It is generally noted that in India, information which is important for proper diagnosis of
EP
autosomal dominant non-syndromic deaf individuals like - detailed health data, pedigree, family history; are often not available to the physicians. Therefore, identification of genes associated
AC C
with hearing loss, and identification of families with autosomal dominant deafness is difficult. The development of proper diagnostic services and improvement in collecting clinical data and pedigree for genetic deafness would prevent the frustration of incomplete etiological diagnosis among dominant deaf individuals in India.
11
ACCEPTED MANUSCRIPT
5. Conclusion In summary, our finding shows that the mutation in KCNQ4 is likely to be rare (<9%) in our studied families from West Bengal. Possibly mutations in other known/unknown gene may be
genes associated with ADNSHL in patients from West Bengal.
SC
Conflict of interest There is no conflict of interest.
M AN U
Acknowledgement
RI PT
responsible for the phenotype. Therefore, further study is required to screen full spectrum of
The authors would like to thank the participants and their families who gave their consent and collaborated in this study. Funding
TE D
This research was supported by grant from the Department of Biotechnology, Govt. of West Bengal, India [Sanction No-106-BT (Estt.)/RD-20/11].
EP
References
[1]http://hereditaryhearingloss.org
AC C
[2] N. Hilgert, R.J. Smith, G. Van Camp, Forty-six genes causing nonsyndromichearing impairment: which ones should be analyzed in DNA diagnostics?,MutatRes. 681 (2009) 189– 196. [3] T .Naito, S.Y. Nishio, Y. Iwasa, T. Yano, K. Kumakawa,S. Abe et al., Comprehensive genetic screening of KCNQ4 in a large autosomal dominant nonsyndromic hearing loss cohort: genotype-phenotype correlations and a founder mutation, PLoS One. 8 (2013) e63231. [4] P.J. Coucke, P. Van Hauwe, P.M. Kelley, H. Kunst, I. Schatteman, D. Van Velzen et al., Mutations in the KCNQ4 gene are responsible for autosomal dominant deafness in four DFNA2 families, Hum Mol Genet. 8(1999) 1321–1328. 12
ACCEPTED MANUSCRIPT
[5] H.S. Wang, Z. Pan, W. Shi, B.S.Brown, R.S.Wymore, I.S. Cohen, J.E. Dixon and D.McKinnon,KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the Mchannel, Science 282(1998) 1890–1893.
RI PT
[6] B.C.Schroeder, C.Kubisch, V. Stein and T.J. Jentsch, Moderate loss of function of cyclicAMP-modulated KCNQ2/KCNQ3 K+ channels causes epilepsy, Nature 396(1998) 687–690. [7]W.P. Yang, P.C. Levesque, W.A. Little, M.L.Conder, P.Ramakrishnan, M.G.Neubauer and M.A. Blanar, Functional expression of two KvLQT1-related potassium channels responsible for inherited idiopathic epilepsy, J. Biol. Chem. 273(1998) 19419–19423.
M AN U
SC
[8]C. Kubisch, B.C. Schroeder, T. Friedrich, B. Lutjohann, A. El-Amraoui, et al., KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness, Cell 96 (1999) 437–446. [9] L.M. Dominguez, K.M. Dodson, Geneticsof hearing loss: focus on DFNA2, The Application of Clinical Genetics 5(2012)97–104.
TE D
[10] A.Pavithra , J.Chandru, J.M.Jeffrey, N.P.Karthikeyen, C.R.Srisailapathy, Rare compound heterozygosity involving dominant and recessive mutations of GJB2 gene in an assortative mating hearing impaired Indian family, Eur Arch Otorhinolaryngol.(2016) 274(1)119-125. [11]A.Pavithra, M.Selvakumari, V.Nityaa, N.Sharanya, R.Ramakrishnan, M.Narasimhan, C.R.Sr isailapathy, Autosomal dominant hearing loss resulting from p.R75Q mutation in the GJB2 gene: nonsyndromic presentation in a South Indian family, Ann Hum Genet. 79(1) (2015) 76-82.
AC C
EP
[12] A.Basu , N.Mukherjee, S.Roy, S.Sengupta, S.Banerjee, M.Chakraborty, B.Dey, M.Roy, B.Roy, N.P.Bhattacharyya, S.Roychoudhury, P.P.Majumder, Ethnic India: a genomic view, with special reference to peopling and structure, Genome Res. 13(10) (2003) 2277-2290. [13] P. Van Hauwe, P. J. Coucke, R. J. Ensink, P. Huygen, C.W.Cremers, G. V. Camp, Mutations in the KCNQ4 K+ channel gene, responsible for autosomal dominant hearing loss, cluster in the channel pore region, Am. J. Med. Genet. 93 (2000) 184-187. [14] A.M. de Heer , M. Schraders, J. Oostrik, L. Hoefsloot, P.L. Huygen, C.W. Cremers, Audioprofile-directed successful mutation analysis in a DFNA2/KCNQ4 (p.Leu274His) family,AnnOtolrhinolaryngol. 120(4)(2011)243-248.
13
ACCEPTED MANUSCRIPT
[15] Z.Talebizadeh , P.M.Kelley, J.W.Askew, K.W.Beisel, S.D.Smith, Novel mutation in the KCNQ4 gene in a large kindred with dominant progressive hearing loss, Hum Mutat. 14(6)(1999) 493-501.
RI PT
[16]M.S.Hildebrand, D.Tack, S.J.McMordie, A.DeLuca, I.A.Hur, et al., Audioprofile-directed screening identifies novel mutations in KCNQ4causing hearing loss at the DFNA2 locus, Genet Med. 10(2008) 797–804.
SC
[17] J.Arnett, S.B.Emery, T.B.Kim, A.K.Boerst,K.Lee, et al., Autosomal dominant progressive sensorineural hearing loss due to a novel mutation in the KCNQ4 gene, Arch Otol Head Neck Surg. 137(2011) 54–59.
M AN U
[18] J. Akita, S. Abe, H. Shinkawa, W.J. Kimberling, S. Usami, Clinical and genetic features of nonsyndromic autosomal dominant sensorineural hearing loss: KCNQ4 is a gene responsible in Japanese, J Hum Genet. 46(2001) 355–361. [19] H. Wang, Y. Zhao, Y. Yi, Y. Gao, Q. Liu, D. Wang, et al., Targeted highthroughput sequencing identifies pathogenic mutations in KCNQ4 in two large Chinesefamilies with autosomal dominant hearing loss.PLoS One (2014) 9(8) e103133.
TE D
[20]L.VanLaer, P.Coucke, R.F.Mueller, G. Caethoven, K.Flothmann, et al., Acommon founder for the 35delG GJB2 gene mutation in connexin 26 hearing impairment, J Med Genet. 38(2001)515–518.
EP
[21] D. Yan, H.J. Park, X.M. Ouyang, A. Pandya, K. Doi, et al., Evidence of a founder effect for the 235delC mutation of GJB2 (connexin 26) in east Asians, Hum Genet. 114(2003) 44–50.
AC C
[22] H.J. Park, S. Shaukat, X.Z. Liu, S.H. Hahn, S. Naz, et al., Origins and frequencies of SLC26A4 (PDS) mutations in east and south Asians: global implications for the epidemiology of deafness, J Med Genet. 40(2003) 242–248. [23] M. Miyagawa, S.Y. Nishio, S. Usami, Prevalence and clinical features of hearing losspatients with CDH23 mutation: a large cohort study, Plos one (2012) 7(8) e40366. [24] G. Van Camp, P.J. Coucke, J. Akita, E. Fransen, S. Abe, et al., A mutational hot spot in the KCNQ4 gene responsible for autosomal dominant hearing impairment, Hum Mutat. 20(2002)15–19.
14
ACCEPTED MANUSCRIPT
[25] D.A. Doyle, J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, et al., The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity, Science 280 (1998) 69– 77.
RI PT
[26] A. Mencia, D. Gonzalez-Nieto, S. Modamio-Hoybjor, A. Etxeberria, G. Aranguez, et al., A novel KCNQ4 pore-region mutation (p.G296S) causes deafness by impairing cell-surface channel expression, Hum Genet. 123 (2008) 41–53.
SC
[27] C.C. Su, J.J. Yang, J.C. Shieh, M.C. Su, S.Y.Li, Identification of novel mutations in the KCNQ4 gene of patients with nonsyndromic deafness from Taiwan, AudiolNeurotol. 12(2007)20
M AN U
[28]Y.YamaguchiKabata, K.Nakazono, A.Takahashi, S.Saito, N.Hosono, M.Kubo, Y.Nakamura,N. Kamatani, Japanese population structure, based on SNP genotypes from 7003 individuals compared to other ethnic groups: effects on population-based association studies,Am J Hum Genet.83(4)(2008)44556.
AC C
EP
TE D
[29] J.Chen, H.Zheng,J.X. Bei, L.Sun, W.H.Jia,T. Li , F.Zhang, M.Seielstad, Y.X.Zeng, X.Zhang, J. Liu , Genetic structure of the Han Chinese population revealed by genome-wide SNP variation, Am J Hum Genet.85(6)(2009)775-85.
15