575062

research-article2015

AORXXX10.1177/0003489415575062Annals of Otology, Rhinology & LaryngologyMiyagawa et al

Article

Germinal Mosaicism in a Family With BO Syndrome

Annals of Otology, Rhinology & Laryngology 2015, Vol. 124(5S) 118­S–122S © The Author(s) 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003489415575062 aor.sagepub.com

Maiko Miyagawa, MD, PhD1,2, Shin-ya Nishio, PhD1,2, Mitsuru Hattori, PhD1, Yutaka Takumi, MD, PhD1,2, and Shin-ichi Usami, MD, PhD1,2

Abstract Objectives: To clarify the existence of germinal mosaicism, we performed a genetic analysis of 2 siblings identified with an EYA1 mutation associated with branchiooto (BO) syndrome but who were born from normal parents. Methods: Detailed data from the 2 affected siblings were collected for clinical diagnosis, with haplotype analysis also performed to prove germinal mosaicism. Results: The 2 sisters showed characteristic clinical features of BO syndrome (middle and inner ear anomalies, microtia, and auditory canal stenosis/atresia). Haplotype analysis confirmed the genetic relationship between the affected sisters and their parents. The younger sister with auditory canal atresia received a bone-anchored hearing aid (Baha), a transcutaneous bone conduction hearing device, resulting in a good hearing outcome. Conclusions: Based on the results of haplotype analysis, we proved that the BO syndrome in these cases was caused by germinal mosaicism of the EYA1 gene in either the mother or father. We also demonstrated that the bone-conduction hearing implant is a good option for BO patients with complex outer, middle, and inner ear anomalies. Keywords EYA1, bronchiootorenal syndrome, Baha, next generation sequencing, germinal mosaicism

Introduction More than 400 forms of syndromic hearing loss have been described, with branchiootorenal (BOR) syndrome and branchiooto (BO) syndrome being 2 of the commonly encountered forms of syndromic hearing loss. BOR syndrome is an autosomal dominant inherited disorder that affects about 1 in 40 000 children, including 2% of profoundly deaf children.1 Typically, BOR patients show major characteristic malformations of the outer, middle, and inner ear and the branchial fistulae or cysts as well as renal malformations. BO syndrome patients have the same symptoms as BOR syndrome without the associated renal disorder. Further minor anomalies, such as preauricular tags, lacrimal duct aplasia, short palate, retrognathia, and cleft palate, have also been described in many reports.2-4 The severity of hearing impairment ranges from mild to profound and may be conductive, sensorineural, or of mixed type.2,5 Intrafamilial variations have also been observed in the symptoms associated with BOR/BO syndrome.6 It is well known that EYA1 mutations are present in a majority of BOR/BO syndrome patients. To date, more than 160 pathogenic mutations in the EYA1 gene have been reported. A list of the EYA1 mutations is available on the Pendred/BOR home page.7 Most of the mutations differ

from one family to another, while some of them were found in sporadic cases. Here, we describe 2 siblings with BO syndrome caused by an EYA1 mutation who were born from unaffected parents, suggesting germinal mosaicism of the EYA1 gene.

Subjects and Methods The patients in this case were identified through our previous screening program based on next generation sequencing (NGS) technology.8 In brief, we attempted to identify genomic variations responsible for deafness by massive sequencing of the exons of 112 target candidate genes. The analysis of 216 randomly selected Japanese deafness patients (120 early-onset and 96 late-detected), who had already been evaluated for common genes/mutations by 1

Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto, Japan 2 Department of Hearing Implant Sciences, Shinshu University School of Medicine, Matsumoto, Japan Corresponding Author: Shin-ichi Usami, MD, PhD, Department of Otorhinolaryngology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. Email: [email protected]

119S

Miyagawa et al Invader assay,9 with 46 previously diagnosed, allowed us to efficiently identify the causative mutations and/or mutation candidates in 57 genes. Computer analysis to predict the effect of missense variants on protein function was performed with wANNOVAR10 using the following functional prediction programs: PhyloP,11 Sorting Intolerant from Tolerant (SIFT),12 Polymorphism Phenotyping,13 LRT,14 and MutationTaster.15 Candidate mutations were confirmed by Sanger sequencing, and the responsible mutations were identified by segregation analysis using samples from among the patients’ family members.

Haplotype Analysis The haplotype pattern within the 2Mbp region surrounding position NM_000503: c.634C>T (p. R212X) was analyzed using a set of 48 single nucleotide polymorphisms (SNPs) (10 sites upstream and 38 sites downstream) for both of the patients and their parents. Haplotype analysis was performed by the direct sequencing method, and the haplotypes were determined using all of the family data together with the reference haplotype data from the HapMap Japan data set.

A

SNS5265

SNS5266

I 2

1

c.[=];[=]

c.[=];[=]

4203

4202

II 1

2

c.[634C>T];[=]

c.[634C>T];[=]

B GTTCA C AGCAG

GTTCA Y AGCAG

WT

c.634C>T

C

Frequency (Hz)

-20

125

250

500

Frequency (Hz)

1,000 2,000 4,000 8,000

-20

0

0

10

10

Hearing threshold (dB)

The 2 sisters identified with the same mutation were found to have the characteristic clinical features of BO syndrome as described in the following (Figure 1).

20 30 40 50 60 70 80

40 50 60 70 80 90

100

110

110

I-1

Case 1: Patient ID 4203

120

I-2

Frequency (Hz) 125

250

500

Frequency (Hz)

1,000 2,000 4,000 8,000

-20

Hearing threshold (dB)

-10

125

250

500

1,000 2,000 4,000 8,000

-10

0

0

10

10

20 30 40 50 60 70 80

20 30 40 50 60 70 80

90

90

100

100

110

110

120

1,000 2,000 4,000 8,000

30

90

-20

500

20

100 120

The elder sister (43 years old) with a c.[634C>T];[=] (p.[R212X];[=]) mutation showed bilateral conductive hearing loss with malformation of the auricle, external auditory canal stenosis, preauricular pits, branchial fistulae, and retrognathia. She showed otoscopically small tympanic membranes, normal type A tympanograms, and a loss of stapedial reflexes. Temporal bone computed tomography (CT) scanning revealed cochlear hypoplasia, hypoplasia of the semicircular canals, a severe middle ear anomaly, and a dilated eustachain tube (Figure 2). Her hearing impairment was congenital and progressive, and she began wearing hearing aids in early childhood. The results of renal screening by echography and renal function were normal.

250

-10

Hearing threshold (dB)

Results

Hearing threshold (dB)

-10

125

II-1

120

II-2

Figure 1.  (A) The patients (4203, 4202) showed heterozygous EYA1 mutations, c.[ 634C>T];[=] (p.[R212X];[=]). The father also developed age-related hearing loss with a different type of audiogram. (B) The results of Sanger sequencing. (C) Audiograms of the family.

Case 2: Patient ID 4202 The younger sister (41 years old) was identified with the same mutation, c.[634C>T];[=] (p.[R212X];[=]). She showed external auditory canal atresia, microtia, preauricular pits, branchial fistulae, retrognathia, and mixed severe

hearing loss. She began using a bone-conductive hearing aid in childhood and later underwent auricular reconstruction otoplasty for microtia. Her temporal bone CT scanning revealed various ear anomalies, hypoplasia of the cochlea

120S

Annals of Otology, Rhinology & Laryngology 124(5S)

A 4203

B 4202

Figure 2.  Temporal bone computed tomography (CT) scan: (A) the elder sister (4203) and (B) the younger sister (4202). Malformation of the ossicles, a dilated eustachain tube, and hypoplasia of the cochlea can be observed.

and semicircular canals, a middle ear anomaly, and a dilated eustachain tube (Figure 2). The results of renal screening by echography and renal function were normal. She underwent bone-anchored hearing aid (Baha) implantation, which is a transcutaneous bone-conduction hearing device, at 34 years of age (details of the surgery were reported in Takumi et al16). Her hearing threshold with Baha was improved. Her ability to understand speech was improved using the Baha device, and she was satisfied with the aesthetic outcome. Haplotype Analysis.  The haplotype pattern within the 1Mbp region surrounding the position of the most frequent mutation, c.634C>T (p.R212X), was characterized using a set of 48 SNPs (10 sites upstream and 38 sites downstream). The

haplotype analysis indicated the parents were the biological parents. Although the 2 sisters showed identical haplotypes, both parents showed a wild-type allele with c.634C homozygous (Figure 3).

Discussion Our previous exome sequencing of selected genes using NGS technology allowed us to detect an EYA1 mutation, c.634C>T (p.R212X), in 2 sisters with BOR/BO syndrome.8 This mutation has already been reported to cause BOR syndrome in the American population.4 In a previous report, c.634C>T (p.R212X) was shown to cause a familial case of renal disorder. Therefore, this mutation is a possible mutational hot spot.

121S

Miyagawa et al

SNS5265

rs16936880 rs6998291 rs10504477 rs13259607 rs12679081 rs268579 rs406703 rs1477943 rs1481804 rs7830811 mutation rs6982031 rs11990714 rs6472569 rs1900079 rs6472575 rs13264845 rs900126 rs920966 rs1424874 rs10110535 rs7016612 rs13279612 rs13272889 rs3779757 rs7831158 rs1481847 rs6472653 rs9298192 rs7827617 rs2701454 rs4738209 rs6991655

SNS5266

C T T C A T C G A G C T C A G T G G G G T A C T A G A G C G C A A

C T C C A T C G A G C T C A G T G G G G T G C T C G G T C A T C A

rs16936880 rs6998291 rs10504477 rs13259607 rs12679081 rs268579 rs406703 rs1477943 rs1481804 rs7830811 mutation rs6982031 rs11990714 rs6472569 rs1900079 rs6472575 rs13264845 rs900126 rs920966 rs1424874 rs10110535 rs7016612 rs13279612 rs13272889 rs3779757 rs7831158 rs1481847 rs6472653 rs9298192 rs7827617 rs2701454 rs4738209 rs6991655

C C C C A C T G A G C T C A G C G G A A T G C C C G G T C A C A G

T T C T A C T G A G C T C C G T G G G G C G A T C G G T C A C A A

4203

rs16936880 rs6998291 rs10504477 rs13259607 rs12679081 rs268579 rs406703 rs1477943 rs1481804 rs7830811 mutation rs6982031 rs11990714 rs6472569 rs1900079 rs6472575 rs13264845 rs900126 rs920966 rs1424874 rs10110535 rs7016612 rs13279612 rs13272889 rs3779757 rs7831158 rs1481847 rs6472653 rs9298192 rs7827617 rs2701454 rs4738209 rs6991655

4202

C T T C A T C G A G T/C T C A G T G G G G T A C T A G A G C G C A A

T T C T A C T G A G C/T T C C G T G G G G C G A T C G G T C A C A A

rs16936880 rs6998291 rs10504477 rs13259607 rs12679081 rs268579 rs406703 rs1477943 rs1481804 rs7830811 mutation rs6982031 rs11990714 rs6472569 rs1900079 rs6472575 rs13264845 rs900126 rs920966 rs1424874 rs10110535 rs7016612 rs13279612 rs13272889 rs3779757 rs7831158 rs1481847 rs6472653 rs9298192 rs7827617 rs2701454 rs4738209 rs6991655

C T T C A T C G A G T/C T C A G T G G G G T A C T A G A G C G C A A

T T C T A C T G A G C/T T C C G T G G G G C G A T C G G T C A C A A

We diagnosed the patients with BOR/BO syndrome based on their symptoms (hearing loss, preauricular pits, branchial fistulae, pinnae deformities, and external auditory canal stenosis/atresia), which fulfilled the major criteria associated with the BOR syndrome phenotype.2 The severity of the phenotype is not correlated with the genotype of EYA1 mutation.6 Our study identified clear variations in symptoms between the sisters. In fact, as the older sister (ID 4203) exhibited external auditory canal stenosis, she could use in-the-ear aids. Conversely, the younger sister (ID 4202) showed external auditory canal atresia and microtia, making meatoplasty difficult. When she was 34 years old, she underwent Baha implantation, with good hearing outcomes and a better aesthetic outcome than that observed for classical born-conductive hearing aids. Although the 2 sisters presented with BO syndrome resulting from EYA1 mutations, their parents had no symptoms. As their pedigree indicated recessive inheritance, it is unlikely that typical autosomal dominant inherited EYA1 mutations are involved. However, NGS is helpful in excluding other possible causative genes. As a result of NGS, we detected heterozygous mutations in the EYA1 gene in the 2 sisters. In contrast, no mutations were detected in their parents. One possible explanation is germinal mosaicism, and we were able to confirm the presence of the germinal mosaicism phenomenon through haplotype analysis of the family members. However, in this study, it was impossible to prove that the germinal mosaicism was either maternal or paternal as the haplotypes of the 2 sisters were identical. Germinal mosaicism in non-twin sibs whose parents are unaffected is thought to be rare.17 We believe that this is the first report of germinal mosaicism of a gene associated with BOR/BO syndrome. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a Health and Labour Sciences Research Grant for Research on Rare and Intractable Diseases (H24Nanchito(Nan)-Ippan-032) and Comprehensive Research on Disability Health and Welfare (H25-Kanakaku-Ippan-002) from the Ministry of Health, Labour and Welfare, Japan (S.U.) and by a Grant-in-Aid for Scientific Research (A) (22249057) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (S.U.).

References Figure 3.  The results of haplotype analysis are shown. Maternal haplotypes are indicated in pink and paternal haplotypes in blue. The haplotypes of the 2 sisters are a complete match.

1. Fraser FC, Sproule JR, Halal F. Frequency of the Branchiooto-renal (BOR) syndrome in children with profound hearing loss. Am J Med Genet. 1980;7:341-349.

122S 2. Chang EH, Menezes M, Meyer NC, et al. 2004. Branchiooto-renal syndrome: the mutation spectrum in EYA1 and its phenotypic con- sequences. Hum Mutat. 2004;6:582-589. 3. Kochhar A, Fischer SM, Kimberling WJ, Smith RJ. Branchio-oto-renal syndrome. Am J Med Genet A. 2007;15:143A(14):1671-1678. 4. Brophy PD, Alasti F, Darbro BW, et al. Genome-wide copy number variation analysis of a Branchio-oto-renal syndrome cohort identifies a recombination hotspot and implicates new candidate genes. Hum Genet. 2013;132(12):1339-1350. 5. Fitch N, Sorolovitz H. Severe renal dysgenesis produced by a dominant gene. Am J Dis Child. 1976;130:1356-1357. 6. Orten DJ, Fischer SM, Sorensen JL, et al. Branchio-otorenal syndrome (BOR): novel mutations in the EYA1 gene, and a review of the mutational genetics of BOR. Hum Mutat. 2008;29(4):537-544. 7. Pendred/BOR Homepage. http://www.healthcare.uiowa.edu/ labs/pendredandbor/. Accessed December 12, 2014. 8. Miyagawa M, Naito T, Nishio SY, Kamatani N, Usami S. Targeted exon sequencing successfully discovers rare causative genes and clarifies the molecular epidemiology of Japanese deafness patients. PLoS One. 2013;8:e71381. 9. Usami S, Nishio SY, Nagano M, Abe S, Yamaguchi T. Deafness Gene Study Consortium: simultaneous screening of multiple mutations by invader assay improves molecular

Annals of Otology, Rhinology & Laryngology 124(5S) diagnosis of hereditary hearing loss: a multicenter study. PLoS One. 2012;7:e31276. 10. wANNOVAR homepage. http://wannovar.usc.edu. Accessed December 12, 2014. 11. Pollard KS, Hubisz MJ, Rosenbloom KR, et al. Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res. 2010;20(1):110-121. 12. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009;4(7):1073-1081. 13. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7(4):248-249. 14. Chun S, Fay JC. Identification of deleterious mutations within three human genomes. Genome Res 2009;19(9):1553-1561. 15. Schwarz JM, Rodelsperger C, Schuelke M, et al. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods. 2010;7(8):575-576. 16. Takumi Y, Suzuki N, Moteki H, Kobayashi K, Usami S. PreBaha operation three dimensional computed tomography with markers for determining optimal implant site. Laryngoscope. 2008;118(10):1824-1826. 17. Sun Y, Xia W, Xing X, et al. Germinal mosaicism of GATA3 in a family with HDR syndrome. Am J Med Genet A. 2009;149A(4):776-778.

Copyright of Annals of Otology, Rhinology & Laryngology is the property of Sage Publications Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.

Germinal mosaicism in a family with BO syndrome.

To clarify the existence of germinal mosaicism, we performed a genetic analysis of 2 siblings identified with an EYA1 mutation associated with branchi...
389KB Sizes 5 Downloads 12 Views