- Email: [email protected]
Novel COCH mutation in a family with autosomal dominant late onset sensorineural hearing impairment and tinnitus
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY 3 4 ( 2 0 13 ) 23 0–2 3 5
Available online at www.sciencedirect.com
www.elsevier.com/locate/amjoto
Case reports
Novel COCH mutation in a family with autosomal dominant late onset sensorineural hearing impairment and tinnitus Emily Gallant, BA a , Lauren Francey, MS a , Heather Fetting, BS a , Maninder Kaur, MS a , Hakon Hakonarson, MD a, b, c , Dinah Clark, MS a , Marcella Devoto, PhD a, b, d , Ian D. Krantz, MD a, b,⁎ a
Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA The Perelman School of Medicine at The University of Pennsylvania, Philadelphia, PA, USA c The Center for Applied Genomics at the Children's Hospital of Philadelphia, Philadelphia, PA, USA d Department of Molecular Medicine, University of Rome La Sapienza, Rome, Italy b
A R T I C LE I N FO
AB S T R A C T
Article history:
This report describes a three generation family with late onset bilateral sensorineural
Received 23 October 2012
hearing impairment (BLSNHI) and tinnitus in which a novel mutation in the COCH gene was identified after a genome-wide linkage approach. The COCH gene is one of the few genes clinically examined when investigating the etiology of autosomal dominant late onset hearing impairment. Initially mutations in the COCH gene were only reported in exons 4 and 5, coding for the LCCL protein domain. More recently, additional mutations have been identified in exon 12, the only mutations identified outside of the LCCL domain. Currently clinical genetic testing for the COCH gene primarily focuses on identifying mutations in these three exons. In this study, we identify a novel mutation in the COCH gene in exon 11, which, like the exon 12 mutations, falls within the vWFA2 protein domain. This finding reinforces the need for clinical genetic screening of the COCH gene to be expanded beyond the current limited exon screening, as there is now more evidence to support that mutations in other areas of this gene are also causative of a similar form of late onset BLSNHI. © 2013 Elsevier Inc. All rights reserved.
1.
Introduction
The COCH gene, located on the long arm of chromosome 14 (14q12-q13), is one of the few genes causative of late onset bilateral sensorineural hearing impairment (BLSNHI) when mutated. Mutations in this gene were first reported in 1996 [1]. The protein product of this gene, cochlin, is expressed in the cochlea and the vestibular system of the inner ear. The COCH protein makes up 70% of inner ear proteins [2]. It assists in structural support, sound processing and maintenance of
balance. Due to the large contribution COCH makes to inner ear proteins, it follows that mutations in this protein were found to interfere with disulfide bonds and proper protein folding and results in general structural disturbances affecting critical cochlear functions that are essential for hearing [3,4]. Effects of mutations in the COCH gene have been found to cause non-syndromic autosomal dominant sensorineural deafness type 9 (DFNA9). Mutations were initially identified in exon 4 [1], resulting in a form of progressive BLSNHI caused by disruption of neural receptors in the inner ear and nerve
⁎ Corresponding author. 1007-C ARC Division of Human Genetics, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA. Tel.: + 1 215 590 2931; fax: +1 215 590 3850. E-mail address: [email protected] (I.D. Krantz). 0196-0709/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.amjoto.2012.11.002
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI N E AN D SUR G E RY 3 4 ( 2 0 13 ) 23 0–2 3 5
pathways to the auditory centers of the brain receiving sound information. Mutant cochlin proteins may also lead to abnormal acidophilic deposits in the inner ear that could contribute to DFNA9 BLSNHI by cutting off, or causing degeneration of, dendrite fibers [3]. Individuals with DFNA9 BLSNHI exhibit progressive late onset hearing impairment with onset usually prior to the fourth or fifth decade. Onset typically is in the high frequencies, progressing to include lower frequencies, and generally resulting in severe hearing impairment by the sixth decade of life. COCH mutations also frequently lead to vestibular dysfunction and balance problems. The COCH gene is routinely screened for mutations in patients with late onset hearing impairment. Clinical mutational analysis for this gene until recently only encompassed exons 4 and 5, both of which encode the Limulus factor C, Coch5b2, and Lg11 [LCCL] domain, since these were the only exons in which mutations had been found. The first mutation found outside of the LCCL domain was a c.1625 G > T transversion in exon 12 which is in the von Willebrand factor A2 (vWFA2) domain [5]. Recent clinical mutational analysis has extended to include this exon [6]. This protein domain is best known for its involvement in cell adhesion in extracellular matrix proteins, such as collagen [7]. The von Willebrand Factor is itself a protein secreted from endothelial cells that works as a coagulation protein in blood [8]. Cysteines flanking the vWF domain, A1 and A2, form a disulfide bond and mutations in the vWFA2 domain are likely to disrupt this bond, interfering with normal platelet binding activity [5]. Mutations in this domain of the COCH gene would disrupt interactions with collagens and other extracellular matrix proteins [9] and affect structural stability in the inner ear. This is in keeping with the effect of other COCH mutations, which also demonstrate various deleterious effects on inner ear integrity.
Materials and methods
2.1.
Research subjects and controls
All affected individuals from the studied family (Fig. 1) and controls were enrolled in the study under a protocol of informed consent approved by the Institutional Review Board at The Children's Hospital of Philadelphia. Blood
2
1
4*
3*
2
1*
3*
2
1
5*
4
IV
V
2.
2
1
II
III
To date nine mutations have been identified in the COCH gene, all localized to within exons 4, 5, and 12 [1,5,6,10–14]. In this study, we report a 3 generation extended family in which a novel mutation was identified in the COCH gene in exon 11, in the vWFA2 protein domain, the same domain also encoded by exon 12. The mutation is a heterozygous 18 base pair deletion, NM_001135058:c.1196_1213del, resulting in an inframe deletion of 6 amino acids, p.Ile399_Ala404del. This novel mutation has not previously been implicated in DFNA9 related BLSNHI and is not currently screened for by most clinically available tests for COCH mutations. Additionally, all previously reported mutations in the COCH gene have been point mutations, while this mutation is an in frame deletion of 6 amino acids. Consistent with other mutations in the COCH gene, the hearing impairment present in the family reported here is late onset, first occurring in the third decade, and progressive, initially involving the higher frequencies and progressing to severe encompassing all frequencies over the next several decades. Tinnitus is also a common finding in affected family members. They had no noted balance problems or other common medical problems; all other mutations in this gene have led to some sort of vestibular impairment, making this family an exception.
1
I
2
3
4
5
6
3
5*
7
6
7
231
8
8*
6*
9*
10*
7*
11* 12*
9 10 11 12 13 14 15 16 17
13
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18
Fig. 1 – Family pedigree. Black squares/circles indicate affected individuals, all of whom presented with the mutation when sequenced. One circle with slashes represents a family member with hearing impairment believed to not be caused by the same mutation. She did not show the mutation when sequenced. * indicates samples available for linkage studies and mutational analysis. Affected status of generation V is unknown due to all members of this generation being below the age of 20 years (COCH-related hearing loss usually does not manifest below this age), and therefore none were used in the linkage analysis.
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samples were obtained from a total of 17 members of an extended family with hereditary hearing impairment. The DNA was extracted and DNA samples were prepared using the Puregene genomic DNA isolation kit (Qiagen, cat. 158467). Three affected subjects, known to have hearing impairment, are all from one nuclear family (III 6, IV 10, IV 12). One member of the extended family, subject (III 4), had an atypical form of hearing impairment from the rest of the family. Four individuals with available samples are below the expected age of onset of hearing impairment as seen in the affected family members. Samples were also available from nine individuals from the extended family who are above the age of expected onset, but do not exhibit symptoms. Additionally, 49 ethnically matched unrelated controls were sequenced.
2.2.
Medical history
Medical histories and examinations were initially taken at a consultation of the three adults with hearing impairment: the proband (IV 10), his father (III 6), and his sister (IV 12). These histories included details on the progression of their hearing impairment, as well as any other significant medical concerns that may or may not have been related to their hearing impairment, and results of brain imaging. An extended family history was obtained and subsequently additional family members were evaluated and samples collected (Fig. 1).
2.3.
Preliminary mutation analysis
The proband underwent clinical mutational analysis of a hearing impairment panel that included the GJB2, GJB6, and SLC26A4 genes, the mtA1555G point mutations, and COCH gene screening (inclusive of exons 4 and 5). No mutations were identified.
2.4.
Linkage analysis
All samples were analyzed using HumanHap610K SNP genotyping array (Illumina, San Diego CA) at the Center for Applied Genomics (CAG) at The Children's Hospital of Philadelphia, using the manufacturer's and CAG's recommended quality control protocols as previously described [15]. Linkage analysis was performed on the SNP array data as follows: 1) PLINK software package [16,17] was used to remove Mendelian errors and SNPs in high linkage disequilibrium. 2) Merlin [18] was used to try to identify loci of significant linkage. LOD scores were determined, using model-free (non-parametric) as well as model-based (parametric) linkage analysis. Model-based analysis assumed full penetrance under an autosomal dominant mode of inheritance. Individuals who were younger than the minimum age of onset of the hearing impairment were not included in the linkage analysis. Regions with negative LOD scores were eliminated.
2.5.
Polymerase chain reaction (PCR) primers were designed and all 12 exons were screened using PCR. DNA of affected individuals was screened initially. Once a mutation had been identified unaffected family members and family members with unknown clinical status (e.g. those too young to manifest hearing impairment) were screened for the mutation. The sequencing data were analyzed using Sequencher software (Sequencher 4.9, Gene Codes Corporation, Ann Arbor, MI). PCR was performed on a cDNA sample synthesized from RNA from the proband, using Invitrogen's SuperScriptTM First-Strand Synthesis System (Grand Island, NY), to further characterize the findings. DNA samples of 49 additional ethnically matched control samples were sequenced for the exon of interest.
COCH gene mutational analysis
Mutation analysis of the COCH gene was performed for all exons and flanking intron/exon boundaries as determined by the genomic reference sequence (NM_001135058) obtained from the UCSC Genome Browser (http://genome.ucsc.edu).
3.
Results
3.1.
High frequency, progressive hearing loss
The proband (IV 10), examined at age 38, had a normal audiogram at age 21. He developed a moderate high frequency hearing impairment in his mid to late 20s that has progressed to involve the lower frequencies as well more recently. He reported having experienced bilateral tinnitus since his early thirties. He had a brain and temporal bone MRI that showed no abnormalities. The proband's father (III 6) first noticed hearing impairment in his 20s. This progressed to being mild sloping to severe bilaterally. He has not experienced tinnitus. A brain and temporal bone MRI was within normal limits. The proband's sister (IV 12), age 31 when seen, also has bilateral sensorineural hearing impairment that was first diagnosed in her 20s, is more severe in the higher frequencies, and is progressive. Her hearing loss was not as severe as other family members, likely due to her younger age. She does not require hearing aids. She, too, experiences tinnitus, which is more pronounced in her right ear. There is no history of vertigo or vestibular dysfunction. The proband's grandfather (II 2) and great grandfather (I 1) were also suspected of having had the same pattern of hearing impairment.
3.2.
Preliminary mutation analysis
Clinical screening results for mutations in the GJB2, GJB6, and SLC26A4 genes, the A1555G mitochondrial point mutation, and exons 4 and 5 of the COCH gene were all negative.
3.3. Linkage analysis and possible linkage to the COCH locus Linkage analysis was limited due to the small size of the core family and unknown affection status of some of the younger individuals (due to the late-onset of the clinical phenotype). The highest potential LOD score was estimated to equal 0.609 using non-parametric analysis, and 1.622 using parametric analysis under an autosomal dominant model with full penetrance including all affected as well as unaffected individuals with ages greater than 20, slightly below the
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI N E AN D SUR G E RY 3 4 ( 2 0 13 ) 23 0–2 3 5
Fig. 2 – Chromatograms of identified c.1196_1213del mutation (A) in the COCH gene and normal control samples (B). Arrow indicates start of deletion.
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youngest observed age of onset (Fig. 1). Obtained LOD scores were all less than or equal to 1.33 when looking at the entire family. Eliminating all negative LOD scores allowed us to narrow down the amount of possible regions to 8.2% of the total genome. By correlating loci associated with autosomal dominant forms of nonsyndromic hearing impairment to the positive LOD score regions, only two loci remained. One encompassed the COL11A2 gene, on chromosome 5 and the other encompassed the COCH gene, on chromosome 14. Both had LOD scores of 1.33 when looking at the whole family (with parametric analysis), and an LOD score of 0.301 when looking at affected individuals (with nonparametric analysis). Given our initial suspicion that the etiology of the hearing impairment in this family was likely due to mutations in the COCH gene (but with available clinical testing for exons 4 and 5 of this gene being normal) complete sequencing of the COCH gene was undertaken.
3.4.
Identification of a novel mutation
A novel heterozygous c.1196_1213del mutation in exon 11 of the COCH gene was identified. This 18 base pair deletion results in the in-frame deletion of 6 amino acids from the protein. All affected individuals on whom samples were available (indicated by an asterisk in Fig. 1) were positive for
this mutation. All unaffected individuals in the pedigree on whom we had samples to test (including the proband's aunt with hearing loss presumed to be of a different etiology, and the proband's son, whose status remained unknown as he was younger than the age of onset of hearing impairment), did not carry this mutation (Fig. 2). Confirmation of the presence of the mutation was undertaken with RT-PCR screening on a cDNA sample from the proband, successfully identifying the expressed 18 base pair deletion in a heterozygous state. 49 ethnically matched control samples were sequenced for exon 11 and none carried the identified mutation.
4.
Discussion
In this study, we identified a heterozygous 18 base pair deletion on exon 11 of the COCH gene in a large multigenerational family segregating late onset progressive bilateral sensorineural hearing impairment and tinnitus. The mutation was seen to segregate appropriately in this family. This novel mutation results in an in-frame deletion of 6 amino acids from the COCH protein (Fig. 3.). This c.1196_1213del mutation lies in the vWFA2 domain of the COCH protein. The only other mutation identified in this domain, C254F, is a missense mutation that replaces one of
A
B
Previously found mutations:
AA Scale
Exon 12
Exon 4 Exon 5
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300
350
400
450
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Exons Cysteines
C
Protein Domains
LCCL domain vWFA1 domain
vWFA2 domain
D Fig. 3 – Diagrammatic representation of the localization of the c.1196_1213del mutation in the COCH gene. (A) Idiogram of chromosome 14. (B) Alignment of exons and introns of the COCH gene. (C) Alignment of relevant protein domains in COCH. (D) Amino acid sequence of exon 11, showing the affected amino acids; deleted amino acids denoted in red, altered amino acids crossed out.
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the critical cysteines of the domain with a tyrosine. It was hypothesized that in addition to affecting the function of the COCH protein by inhibiting disulfide bond formation, this mutation likely disrupts protein–protein interactions [6]. The mutation identified in this report is the first deletion mutation found in this gene, however, as it is an in-frame deletion, presumably resulting in a protein product, the mechanism by which it might be affecting its normal function and the integrity of the inner ear is likely similar to that of the missense mutation. The significance of the localization of this mutation to the vWFA2 domain as opposed to the more commonly reported mutations involving the LCCL domain is unclear. All previously reported mutations in the COCH gene have caused late onset hearing impairment with onset beginning anywhere from the late teens to slightly above 40 years of age. The earliest reported onset was at age 17, in an individual with a mutation in exon 12, in the vWFA2 domain [5]. While this study shows individuals with age of onset later than 17 years, the subjects with mutations in the vWFA2 domain overall seem to have earlier onset, in their 20s, than that generally reported for mutations in the LCCL domain. Additionally, while there is some evidence of vestibular impairment in each of the previously noted mutations in exons in the LCCL domain and not in the case of the vWFA2 domain mutations, the data are too limited to extrapolate clear genotype– phenotype correlations at this time. The identification of a novel mutation in exon 11 of the COCH gene that was missed on clinical testing suggests that additional mutations in this gene may be present in families and individuals with autosomal dominant late onset hearing impairment and that testing for this gene should be expanded to include the entire gene to allow for improved diagnostics and counseling.
Acknowledgments We are grateful for the support of the family described in this report as well as Institutional Development Funds from the Children's Hospital of Philadelphia (IDK).
[2]
[3] [4] [5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15] [16] [17]
REFERENCES [18] [1] Manolis EN, Yandavi N, Nadol JB, et al. A gene for nonsyndromic autosomal dominant progressive postlingual
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sensorineural hearing loss maps to chromosome 14q12-13. Hum Mol Genet 1996;5:1047–50. Ikenzono T, Omori A, Ichinose S, et al. Identification of the protein product of the Coch gene (hereditary deafness gene) as the major component of bovine inner ear protein. Biochim Biophys Acta 2001;1535:258–65. Kent WJ, Sugnet CW, Furey TS, et al. The human genome browser at UCSC. Genome Res 2002;Vol. 2011. Yao J, Py BF, Zhu H, et al. Role of protein misfolding in DFNA9 hearing loss. J Biol Chem 2010;285:14909–19. Street VA, Kallman JC, Robertson NG, et al. A novel DFNA9 mutation in the vWFA2 domain of COCH alters a conserved cysteine residue and intrachain disulfide bond formation resulting in progressive hearing loss and site-specific vestibular and central oculomotor dysfunction. Am J Med Genet 2005;139A:86–95. Yuan HJ, Han DY, Sun Q, et al. Novel mutations in the vWFA2 domain of COCH in two Chinese DFNA9 families. Clin Genet 2008;73:391–4. Whittaker CA, Hynes RO. Distribution and evolution of von Willebrand/integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere. MBoC 2002;13: 3369–87. Flaumenhaft R. vWF feels the force. Hemotologist 2009;6:11. Khetarpal U. DFNA9 is a progressive audiovestibular dysfunction with a microfibrillar deposit in the inner ear. Laryngoscope 1000: 110: 1379–1384. de Kok YJM, Bom SH, Brunt TM, et al. A pro51-to-ser mutation in the COCH gene is associated with late onset autosomal dominant progressive sensorineural hearing loss with vestibular defects. Hum Mol Genet 1999;8:261–366. Grabski R, Szul T, Sasaki T, et al. Mutation in COCH that result in non-syndromic autosomal dominant deafness (DFNA9) affect matrix deposit of cochlin. Hum Genet 2003;113:406–16. Hildebrand MS, Tack D, DeLuca A, et al. Mutation in the COCH gene is associated with superior semicircular canal dehiscence. Am J Med Genet 2009;149A:280–5. Robertson NG, Lu L, Heller S, et al. Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction. Nat Genet 1998;20: 299–303. Usami S, Takahasi K, Yuge I, et al. Mutations in the COCH gene are a frequent cause of autosomal dominant progressive cochleo-vestibular dysfunction, but not of Meniere's disease. EJHG 2003;11:744–8. Steemers FJ, Gunderson KL. Whole genome genotyping technologies on the BeadArray platform. Biotechnol J 2007;2:9. Purcell S. PLINK v1.07. http://pngu.mgh.harvard.edu/purcell/ plink/. Purcell S, Nealt B, Todd-Brown K, et al. PLINK: a toolset for whole-genome association and population-based linkage analysis. Am J Hum Genet 2007:81. Abecasis GR, Cherny SS, Cookson WO, et al. Merlin—rapid analysis of dense genetic maps using sparse gene flow grees. Nat Genet 2002;30:97–101.
Available online at www.sciencedirect.com
www.elsevier.com/locate/amjoto
Case reports
Novel COCH mutation in a family with autosomal dominant late onset sensorineural hearing impairment and tinnitus Emily Gallant, BA a , Lauren Francey, MS a , Heather Fetting, BS a , Maninder Kaur, MS a , Hakon Hakonarson, MD a, b, c , Dinah Clark, MS a , Marcella Devoto, PhD a, b, d , Ian D. Krantz, MD a, b,⁎ a
Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA The Perelman School of Medicine at The University of Pennsylvania, Philadelphia, PA, USA c The Center for Applied Genomics at the Children's Hospital of Philadelphia, Philadelphia, PA, USA d Department of Molecular Medicine, University of Rome La Sapienza, Rome, Italy b
A R T I C LE I N FO
AB S T R A C T
Article history:
This report describes a three generation family with late onset bilateral sensorineural
Received 23 October 2012
hearing impairment (BLSNHI) and tinnitus in which a novel mutation in the COCH gene was identified after a genome-wide linkage approach. The COCH gene is one of the few genes clinically examined when investigating the etiology of autosomal dominant late onset hearing impairment. Initially mutations in the COCH gene were only reported in exons 4 and 5, coding for the LCCL protein domain. More recently, additional mutations have been identified in exon 12, the only mutations identified outside of the LCCL domain. Currently clinical genetic testing for the COCH gene primarily focuses on identifying mutations in these three exons. In this study, we identify a novel mutation in the COCH gene in exon 11, which, like the exon 12 mutations, falls within the vWFA2 protein domain. This finding reinforces the need for clinical genetic screening of the COCH gene to be expanded beyond the current limited exon screening, as there is now more evidence to support that mutations in other areas of this gene are also causative of a similar form of late onset BLSNHI. © 2013 Elsevier Inc. All rights reserved.
1.
Introduction
The COCH gene, located on the long arm of chromosome 14 (14q12-q13), is one of the few genes causative of late onset bilateral sensorineural hearing impairment (BLSNHI) when mutated. Mutations in this gene were first reported in 1996 [1]. The protein product of this gene, cochlin, is expressed in the cochlea and the vestibular system of the inner ear. The COCH protein makes up 70% of inner ear proteins [2]. It assists in structural support, sound processing and maintenance of
balance. Due to the large contribution COCH makes to inner ear proteins, it follows that mutations in this protein were found to interfere with disulfide bonds and proper protein folding and results in general structural disturbances affecting critical cochlear functions that are essential for hearing [3,4]. Effects of mutations in the COCH gene have been found to cause non-syndromic autosomal dominant sensorineural deafness type 9 (DFNA9). Mutations were initially identified in exon 4 [1], resulting in a form of progressive BLSNHI caused by disruption of neural receptors in the inner ear and nerve
⁎ Corresponding author. 1007-C ARC Division of Human Genetics, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA. Tel.: + 1 215 590 2931; fax: +1 215 590 3850. E-mail address: [email protected] (I.D. Krantz). 0196-0709/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.amjoto.2012.11.002
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI N E AN D SUR G E RY 3 4 ( 2 0 13 ) 23 0–2 3 5
pathways to the auditory centers of the brain receiving sound information. Mutant cochlin proteins may also lead to abnormal acidophilic deposits in the inner ear that could contribute to DFNA9 BLSNHI by cutting off, or causing degeneration of, dendrite fibers [3]. Individuals with DFNA9 BLSNHI exhibit progressive late onset hearing impairment with onset usually prior to the fourth or fifth decade. Onset typically is in the high frequencies, progressing to include lower frequencies, and generally resulting in severe hearing impairment by the sixth decade of life. COCH mutations also frequently lead to vestibular dysfunction and balance problems. The COCH gene is routinely screened for mutations in patients with late onset hearing impairment. Clinical mutational analysis for this gene until recently only encompassed exons 4 and 5, both of which encode the Limulus factor C, Coch5b2, and Lg11 [LCCL] domain, since these were the only exons in which mutations had been found. The first mutation found outside of the LCCL domain was a c.1625 G > T transversion in exon 12 which is in the von Willebrand factor A2 (vWFA2) domain [5]. Recent clinical mutational analysis has extended to include this exon [6]. This protein domain is best known for its involvement in cell adhesion in extracellular matrix proteins, such as collagen [7]. The von Willebrand Factor is itself a protein secreted from endothelial cells that works as a coagulation protein in blood [8]. Cysteines flanking the vWF domain, A1 and A2, form a disulfide bond and mutations in the vWFA2 domain are likely to disrupt this bond, interfering with normal platelet binding activity [5]. Mutations in this domain of the COCH gene would disrupt interactions with collagens and other extracellular matrix proteins [9] and affect structural stability in the inner ear. This is in keeping with the effect of other COCH mutations, which also demonstrate various deleterious effects on inner ear integrity.
Materials and methods
2.1.
Research subjects and controls
All affected individuals from the studied family (Fig. 1) and controls were enrolled in the study under a protocol of informed consent approved by the Institutional Review Board at The Children's Hospital of Philadelphia. Blood
2
1
4*
3*
2
1*
3*
2
1
5*
4
IV
V
2.
2
1
II
III
To date nine mutations have been identified in the COCH gene, all localized to within exons 4, 5, and 12 [1,5,6,10–14]. In this study, we report a 3 generation extended family in which a novel mutation was identified in the COCH gene in exon 11, in the vWFA2 protein domain, the same domain also encoded by exon 12. The mutation is a heterozygous 18 base pair deletion, NM_001135058:c.1196_1213del, resulting in an inframe deletion of 6 amino acids, p.Ile399_Ala404del. This novel mutation has not previously been implicated in DFNA9 related BLSNHI and is not currently screened for by most clinically available tests for COCH mutations. Additionally, all previously reported mutations in the COCH gene have been point mutations, while this mutation is an in frame deletion of 6 amino acids. Consistent with other mutations in the COCH gene, the hearing impairment present in the family reported here is late onset, first occurring in the third decade, and progressive, initially involving the higher frequencies and progressing to severe encompassing all frequencies over the next several decades. Tinnitus is also a common finding in affected family members. They had no noted balance problems or other common medical problems; all other mutations in this gene have led to some sort of vestibular impairment, making this family an exception.
1
I
2
3
4
5
6
3
5*
7
6
7
231
8
8*
6*
9*
10*
7*
11* 12*
9 10 11 12 13 14 15 16 17
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15
18
Fig. 1 – Family pedigree. Black squares/circles indicate affected individuals, all of whom presented with the mutation when sequenced. One circle with slashes represents a family member with hearing impairment believed to not be caused by the same mutation. She did not show the mutation when sequenced. * indicates samples available for linkage studies and mutational analysis. Affected status of generation V is unknown due to all members of this generation being below the age of 20 years (COCH-related hearing loss usually does not manifest below this age), and therefore none were used in the linkage analysis.
232
AM ER IC AN JOUR NA L OF OTOLARY NG OLOG Y –H EA D A N D N E CK ME D I CI NE AN D SUR G E RY 3 4 ( 2 0 13 ) 23 0–2 3 5
samples were obtained from a total of 17 members of an extended family with hereditary hearing impairment. The DNA was extracted and DNA samples were prepared using the Puregene genomic DNA isolation kit (Qiagen, cat. 158467). Three affected subjects, known to have hearing impairment, are all from one nuclear family (III 6, IV 10, IV 12). One member of the extended family, subject (III 4), had an atypical form of hearing impairment from the rest of the family. Four individuals with available samples are below the expected age of onset of hearing impairment as seen in the affected family members. Samples were also available from nine individuals from the extended family who are above the age of expected onset, but do not exhibit symptoms. Additionally, 49 ethnically matched unrelated controls were sequenced.
2.2.
Medical history
Medical histories and examinations were initially taken at a consultation of the three adults with hearing impairment: the proband (IV 10), his father (III 6), and his sister (IV 12). These histories included details on the progression of their hearing impairment, as well as any other significant medical concerns that may or may not have been related to their hearing impairment, and results of brain imaging. An extended family history was obtained and subsequently additional family members were evaluated and samples collected (Fig. 1).
2.3.
Preliminary mutation analysis
The proband underwent clinical mutational analysis of a hearing impairment panel that included the GJB2, GJB6, and SLC26A4 genes, the mtA1555G point mutations, and COCH gene screening (inclusive of exons 4 and 5). No mutations were identified.
2.4.
Linkage analysis
All samples were analyzed using HumanHap610K SNP genotyping array (Illumina, San Diego CA) at the Center for Applied Genomics (CAG) at The Children's Hospital of Philadelphia, using the manufacturer's and CAG's recommended quality control protocols as previously described [15]. Linkage analysis was performed on the SNP array data as follows: 1) PLINK software package [16,17] was used to remove Mendelian errors and SNPs in high linkage disequilibrium. 2) Merlin [18] was used to try to identify loci of significant linkage. LOD scores were determined, using model-free (non-parametric) as well as model-based (parametric) linkage analysis. Model-based analysis assumed full penetrance under an autosomal dominant mode of inheritance. Individuals who were younger than the minimum age of onset of the hearing impairment were not included in the linkage analysis. Regions with negative LOD scores were eliminated.
2.5.
Polymerase chain reaction (PCR) primers were designed and all 12 exons were screened using PCR. DNA of affected individuals was screened initially. Once a mutation had been identified unaffected family members and family members with unknown clinical status (e.g. those too young to manifest hearing impairment) were screened for the mutation. The sequencing data were analyzed using Sequencher software (Sequencher 4.9, Gene Codes Corporation, Ann Arbor, MI). PCR was performed on a cDNA sample synthesized from RNA from the proband, using Invitrogen's SuperScriptTM First-Strand Synthesis System (Grand Island, NY), to further characterize the findings. DNA samples of 49 additional ethnically matched control samples were sequenced for the exon of interest.
COCH gene mutational analysis
Mutation analysis of the COCH gene was performed for all exons and flanking intron/exon boundaries as determined by the genomic reference sequence (NM_001135058) obtained from the UCSC Genome Browser (http://genome.ucsc.edu).
3.
Results
3.1.
High frequency, progressive hearing loss
The proband (IV 10), examined at age 38, had a normal audiogram at age 21. He developed a moderate high frequency hearing impairment in his mid to late 20s that has progressed to involve the lower frequencies as well more recently. He reported having experienced bilateral tinnitus since his early thirties. He had a brain and temporal bone MRI that showed no abnormalities. The proband's father (III 6) first noticed hearing impairment in his 20s. This progressed to being mild sloping to severe bilaterally. He has not experienced tinnitus. A brain and temporal bone MRI was within normal limits. The proband's sister (IV 12), age 31 when seen, also has bilateral sensorineural hearing impairment that was first diagnosed in her 20s, is more severe in the higher frequencies, and is progressive. Her hearing loss was not as severe as other family members, likely due to her younger age. She does not require hearing aids. She, too, experiences tinnitus, which is more pronounced in her right ear. There is no history of vertigo or vestibular dysfunction. The proband's grandfather (II 2) and great grandfather (I 1) were also suspected of having had the same pattern of hearing impairment.
3.2.
Preliminary mutation analysis
Clinical screening results for mutations in the GJB2, GJB6, and SLC26A4 genes, the A1555G mitochondrial point mutation, and exons 4 and 5 of the COCH gene were all negative.
3.3. Linkage analysis and possible linkage to the COCH locus Linkage analysis was limited due to the small size of the core family and unknown affection status of some of the younger individuals (due to the late-onset of the clinical phenotype). The highest potential LOD score was estimated to equal 0.609 using non-parametric analysis, and 1.622 using parametric analysis under an autosomal dominant model with full penetrance including all affected as well as unaffected individuals with ages greater than 20, slightly below the
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Fig. 2 – Chromatograms of identified c.1196_1213del mutation (A) in the COCH gene and normal control samples (B). Arrow indicates start of deletion.
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youngest observed age of onset (Fig. 1). Obtained LOD scores were all less than or equal to 1.33 when looking at the entire family. Eliminating all negative LOD scores allowed us to narrow down the amount of possible regions to 8.2% of the total genome. By correlating loci associated with autosomal dominant forms of nonsyndromic hearing impairment to the positive LOD score regions, only two loci remained. One encompassed the COL11A2 gene, on chromosome 5 and the other encompassed the COCH gene, on chromosome 14. Both had LOD scores of 1.33 when looking at the whole family (with parametric analysis), and an LOD score of 0.301 when looking at affected individuals (with nonparametric analysis). Given our initial suspicion that the etiology of the hearing impairment in this family was likely due to mutations in the COCH gene (but with available clinical testing for exons 4 and 5 of this gene being normal) complete sequencing of the COCH gene was undertaken.
3.4.
Identification of a novel mutation
A novel heterozygous c.1196_1213del mutation in exon 11 of the COCH gene was identified. This 18 base pair deletion results in the in-frame deletion of 6 amino acids from the protein. All affected individuals on whom samples were available (indicated by an asterisk in Fig. 1) were positive for
this mutation. All unaffected individuals in the pedigree on whom we had samples to test (including the proband's aunt with hearing loss presumed to be of a different etiology, and the proband's son, whose status remained unknown as he was younger than the age of onset of hearing impairment), did not carry this mutation (Fig. 2). Confirmation of the presence of the mutation was undertaken with RT-PCR screening on a cDNA sample from the proband, successfully identifying the expressed 18 base pair deletion in a heterozygous state. 49 ethnically matched control samples were sequenced for exon 11 and none carried the identified mutation.
4.
Discussion
In this study, we identified a heterozygous 18 base pair deletion on exon 11 of the COCH gene in a large multigenerational family segregating late onset progressive bilateral sensorineural hearing impairment and tinnitus. The mutation was seen to segregate appropriately in this family. This novel mutation results in an in-frame deletion of 6 amino acids from the COCH protein (Fig. 3.). This c.1196_1213del mutation lies in the vWFA2 domain of the COCH protein. The only other mutation identified in this domain, C254F, is a missense mutation that replaces one of
A
B
Previously found mutations:
AA Scale
Exon 12
Exon 4 Exon 5
1
50
100
150
200
250
300
350
400
450
500
550
Exons Cysteines
C
Protein Domains
LCCL domain vWFA1 domain
vWFA2 domain
D Fig. 3 – Diagrammatic representation of the localization of the c.1196_1213del mutation in the COCH gene. (A) Idiogram of chromosome 14. (B) Alignment of exons and introns of the COCH gene. (C) Alignment of relevant protein domains in COCH. (D) Amino acid sequence of exon 11, showing the affected amino acids; deleted amino acids denoted in red, altered amino acids crossed out.
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the critical cysteines of the domain with a tyrosine. It was hypothesized that in addition to affecting the function of the COCH protein by inhibiting disulfide bond formation, this mutation likely disrupts protein–protein interactions [6]. The mutation identified in this report is the first deletion mutation found in this gene, however, as it is an in-frame deletion, presumably resulting in a protein product, the mechanism by which it might be affecting its normal function and the integrity of the inner ear is likely similar to that of the missense mutation. The significance of the localization of this mutation to the vWFA2 domain as opposed to the more commonly reported mutations involving the LCCL domain is unclear. All previously reported mutations in the COCH gene have caused late onset hearing impairment with onset beginning anywhere from the late teens to slightly above 40 years of age. The earliest reported onset was at age 17, in an individual with a mutation in exon 12, in the vWFA2 domain [5]. While this study shows individuals with age of onset later than 17 years, the subjects with mutations in the vWFA2 domain overall seem to have earlier onset, in their 20s, than that generally reported for mutations in the LCCL domain. Additionally, while there is some evidence of vestibular impairment in each of the previously noted mutations in exons in the LCCL domain and not in the case of the vWFA2 domain mutations, the data are too limited to extrapolate clear genotype– phenotype correlations at this time. The identification of a novel mutation in exon 11 of the COCH gene that was missed on clinical testing suggests that additional mutations in this gene may be present in families and individuals with autosomal dominant late onset hearing impairment and that testing for this gene should be expanded to include the entire gene to allow for improved diagnostics and counseling.
Acknowledgments We are grateful for the support of the family described in this report as well as Institutional Development Funds from the Children's Hospital of Philadelphia (IDK).
[2]
[3] [4] [5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15] [16] [17]
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