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Mar 4, 2014 - Abstract The EYA1 gene is known as the causative gene of. BOR (Branchio-oto-renal) syndrome which is a genetic disorder associated with ...
Mol Biol Rep (2014) 41:4321–4327 DOI 10.1007/s11033-014-3303-6

Identification of a novel nonsynonymous mutation of EYA1 disrupting splice site in a Korean patient with BOR syndrome Hui Ram Kim • Mee Hyun Song • Min-A Kim • Ye-Ri Kim • Kyu-Yup Lee • Jong Kyung Sonn • Jaetae Lee • Jae Young Choi • Un-Kyung Kim

Received: 26 July 2013 / Accepted: 14 February 2014 / Published online: 4 March 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The EYA1 gene is known as the causative gene of BOR (Branchio-oto-renal) syndrome which is a genetic disorder associated with branchial cleft cysts of fistulae, hearing loss, ear malformation, and renal anomalies. Although approximately 40 % of patients with BOR syndrome have mutations in the EYA1 gene and over 130 disease-causing mutations in EYA1 have been reported in various populations, only a few mutations have been reported in Korean families. In this study, genetic analysis of the EYA1 gene was performed in a Korean patient diagnosed with BOR syndrome and his parents. A de novo novel missense mutation, c.418G[A, located at the end of exon 6, changed glycine to serine at amino acid position 140

Hui Ram Kim and Mee Hyun Song contributed equally to this work. H. R. Kim  M.-A. Kim  Y.-R. Kim  J. K. Sonn  U.-K. Kim (&) Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Korea e-mail: [email protected] M. H. Song Department of Otorhinolaryngology, Kwandong University College of Medicine, Myongji Hospital, Goyang, South Korea K.-Y. Lee Department of Otorhinolaryngology-Head and Neck Surgery, College of Medicine, Kyungpook National University, Daegu, South Korea J. Lee Department of Nuclear Medicine, Kyungpook National University School of Medicine, Daegu, South Korea J. Y. Choi (&) Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, South Korea e-mail: [email protected]

(p.G140S) and was suspected to affect normal splicing. Our in vitro splicing assay demonstrated that this mutation causes exon 6 skipping leading to frameshift and truncation of the protein to result in the loss of eyaHR. To the best of our knowledge, this is the first report revealing that a missense mutation in the exon disturbs normal splicing as a result of a substitution of the last nucleotide of an exon in EYA1. Keywords BOR syndrome  EYA1  Missense mutation  Splicing  Korean

Introduction Branchio-oto-renal (BOR) syndrome (OMIM 113650) is a hereditary disorder showing autosomal dominant inheritance pattern that is associated with branchial cleft cysts or fistulae, hearing loss, ear malformation, and renal anomalies [1]. The incidence of BOR syndrome is estimated to be approximately 1 case per 40,000, accounting for 2 % of children with profound hearing loss [2]. Hearing loss is the most common clinical feature of BOR syndrome reported in more than 90 % of the patients, followed by preauricular pits or tags (82 %), renal anomalies (67 %), branchial fistulae (49 %), pinnae deformity (36 %), and external auditory canal stenosis (29 %) [3]. Since BOR syndrome has high but incomplete penetrance with variable expressivity and the clinical spectrum involves a broad range of phenotypes commonly overlapping with features of other syndromes, genetic analysis is an important part of the diagnosis in these patients [4–8]. The causative gene of BOR syndrome was identified by positional cloning in the early 1990s [6]. The EYA1 gene, the human homologue of the Drosophila eye absent (eya) gene, consists of 16 coding exons spanning 156 kb on

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chromosome 8q13.3, and has three isoforms and four transcripts resulting from alternative splicing [9–12]. This gene belongs to the EYA family characterized by a highly conserved, 271 amino acid, C-terminal domain called eyaHR, also known as the Eya domain(ED), which is encoded by exons 9–16 of the gene [12, 13]. Mutations in the EYA1 gene have been identified in approximately 40 % of patients with BOR syndrome [4]. Until now, over 130 disease-causing mutations in EYA1 have been reported including missense, nonsense, frameshift, aberrant splicing, small duplication, deletion and rearrangements in various populations [4, 12, 14–17]. More than 80 % of EYA1 mutations have been reported in Caucasians, while only a few mutations have been reported in the East Asian population, including Korea [18–22]. In this study, we report a Korean patient showing typical features of BOR syndrome who was found to carry a de novo novel missense mutation at the 30 end of exon 6 of EYA1 gene that affected normal splicing resulting in a truncated protein.

Materials and methods Subjects A 4-year-old boy who was clinically diagnosed with BOR syndrome according to the criteria proposed by Chang et al. [4] was analyzed in this study (Table 1). None of the family members were affected by any of the common features of BOR syndrome. After obtaining written informed consent from the proband and his parents, mutational analysis for the EYA1 gene was performed in these subjects by direct DNA sequencing. This study was approved by the Institutional Review Board of the Yonsei University College of Medicine. Gene screening Genomic DNA was isolated from peripheral blood of the proband and his parents using a Flexigene DNA kit Table 1 Diagnostic criteria for BOR syndrome Major criteria

Minor criteria

Branchial anomalies

External ear anomalies

Deafness

Middle ear anomalies

Preauricular pits

Inner ear anomalies

Renal anomalies

Preauricular tags Other: facial asymmetry, palate abnormalities

For diagnosis of BOR syndrome, one must have at least three major criteria; two major criteria and at least two minor criteria; or one major criteria and an affected first-degree relative meeting criteria for BOR. [4]

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following the manufacturer’s instructions (Qiagen, Hilden, Germany). To identify any mutations of the EYA1 gene causing BOR syndrome in the proband, all 18 exons including the 30 and 50 un-translated regions (UTR) regions and exon/intron flanking regions were amplified by polymerase chain reaction (PCR) with specific primers which were used in our previous report [22] designed using Primer 3 software (http://frodo.wi.mit.edu/) and H taq polymerase (Solgent, Daejeon, South Korea). The amplified products were purified by using Shrimp alkaline phosphatase and exonuclease I (USB, Cleveland, USA). Cycle sequencing was performed with Bigdye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) on 3130xl Genetic Analyzer (Applied Biosystems, Foster City, USA). The sequence data were analyzed using Sequencing analysis v5.2 (Applied Biosystems, Foster City, USA) and Chromas Pro v1.5 software (Technelysium Pty Ltd, Tewantin, Australia) compared to the reference sequence (GenBank Accession Nos. NG_011735.1, NM_000503.4, NP_000494.2). The prediction programs, Polyphen 2 (http://genetics.bwh.harvard.edu/pph2), mutationtaster (http://www.mutationtaster.org/), and SIFT (http://sift.jcvi.org/) were used to predict the pathogenic effect of the mutation.

Splicing assay To evaluate the splicing pattern, a pSPL3 vector was constructed so that exon 6 and its 300 bp 30 - and 50 -intronic flanking regions with either normal or mutant (c.418G[A) allele of the EYA1 gene were inserted between exons A and B. For the in vitro splicing assay, HeLa cells were transfected with the hybrid minigene using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) cultured in complete DMEM (10 % FBS, 1 % penicillin/streptomycin) at 37 °C, in 5 % CO2 concentration. Then, the transfected cells were harvested 24 h after transfection. Total RNA was isolated from the transfected cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. One microgram of total RNA was subjected to synthesize complimentary DNA (cDNA) by a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA). The cDNA was used as a template and PCR was performed with pSPL3 specific primers: SD6, 50 -TCTGAGTCACCTGGACAACC-30 and SA2, 50 -ATCTCAGTGGTATTTGTGAGC-30 . The amplified products were separated by electrophoresis on a 1.5 % agarose gel to identify the size of products. Sequence of products was also confirmed by direct sequencing using Bigdye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) on the 3130xl Genetic Analyzer (Applied Biosystems, Foster City, USA).

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The patient had hearing loss on both sides and exhibited bilateral branchial cleft fistulae and preauricular pits, satisfying three of four major criteria proposed by Chang et al. [4] for the clinical diagnosis of BOR syndrome (Fig. 1). On pure tone audiometry performed at 4 years of age, the hearing threshold calculated by averaging the thresholds at 0.5, 1, 2, and 4 kHz was 40 dB HL on the right side and 86 dB HL on the left side (Fig. 1). The bone conduction thresholds were within the normal range on both sides. Since the patient could not fully understand the masking process during audiometry testing, mixed hearing loss may have been present on the left side unlike the conductive hearing loss on the right. Among the minor criteria, cup ear deformity was seen bilaterally and inner ear malformations were identified on the temporal bone computed tomography (Fig. 1). Cochlear hypoplasia with defective modiolus,

facial nerve deviation, and slightly dilated vestibule were demonstrated while no definite middle ear pathology could be found despite the conductive hearing loss (Fig. 1). Renal sonography revealed no abnormal pathology. Analysis of the nucleotide sequences of all EYA1 exons revealed a novel heterozygous missense mutation (c.418G[A based on the reference mRNA sequence, NM_000503.4; p.G140S) in the proband. The amino acid at protein position 140 changed from glycine to serine by c.418G[A in exon 6. This mutation occurred de novo since neither of the parents carried the same mutation (Fig. 2). The mutation, c.418G[A, was also not detected in 95 unrelated healthy Korean individuals with normal hearing. The pathogenicity of this nonsynonymous amino acid variant (p.G140S) was predicted using three prediction programs, Polyphen2, Mutationtaster, and SIFT. The score means that the mutation would have a deleterious effect on the phenotype if it is closer to 1 for Polyphen2 or 0 for

Fig. 1 Clinical manifestations of a 4-year-old boy diagnosed with BOR syndrome. a Pure tone audiometry revealed conductive hearing loss on both sides with hearing thresholds of 40 dB HL on the right and 86 dB HL on the left. O right air conduction thresholds, X left air conduction thresholds, \right bone conduction thresholds, unmasked, [ left bone conduction thresholds, unmasked. b Preauricular pit (black arrow) and cup ear deformity were identified on both ears

(only left ear shown). c Temporal bone computed tomography of the left ear revealed cochlear hypoplasia (thick black arrow) and defective modiolus (thin white arrow). d The vestibule was slightly dilated (thin black arrow) and the internal auditory canal was enlarged (white asterisk), while the ossicles (white arrowhead) were relatively intact. e The labyrinthine segment of the facial canal (thick white arrow) was widened and deviated

Results and discussion

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Fig. 2 Identification of a novel missense mutation, c.418G[A. a Pedigree of a 4-year-old patient diagnosed with BOR syndrome consisted of two generations. Squares and circles represent males and females, respectively. Filled symbols represent affected individuals

and the arrow marks the proband of the family. b Nucleotide sequence of exon 6 in EYA1 demonstrates the mutation c.418G[A in the proband (II-2) which was not found in the parents (I-2 and I-3). The arrow indicates the location of the nucleotide substitution

Fig. 3 In vitro splicing assay of EYA1 mutation, c.418G[A. a Amplified products of the hybrid minigene transcripts separated on 1.5 % agarose gel demonstrated different sizes for the wild type (409 bp) and the mutant type (263 bp). The different splicing processes are shown in schematic graphics for the wild type and

the mutant type. b Partial DNA sequences of the hybrid minigene transcripts demonstrated skipping of exon 6 for the mutant type. WT wild type, MT mutant type, RTN reverse transcription negative control, NCT negative control

SIFT. This mutation was predicted to be probably damaging with a Polyphen2 score of 0.987. Also, Mutationtaster predicted the mutation to be disease causing with

amino acid sequence change, protein features affected, and splice site change. However, the SIFT score was 0.39, meaning that the mutation would be tolerated. Overall, two

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Fig. 4 Schematic overwiew of the human EYA1 gene and the position of the mutation, c.418G[A. The EYA1 gene located at chromosome 8q13.3 has 18 exons including the EYA1 homologous region spanning from exon 11 to 18. The dotted and double lines

indicate EYA1 respectively. The located at the end the EYA1 gene. E

homologous region and EYA1 coding region, arrowhead designates the mutation, c.418G[A, of exon 6. The square boxes refer to each exon of exon

of three programs predicted this variation to have a deleterious effect. We performed RNA splicing assay in vitro to verify the pathogenic effect of c.418G[A, because a mutation located at the last base of an exon had a potential to change the splicing site [23]. The wild type amplicon showed normal size (409 bp) that included exon 6 between exons A and B. However, the mutant type demonstrated a smaller sized amplicon (263 bp) that included only exons A and B without exon 6 (Fig. 3a). The wild type and mutant products were confirmed by directed sequencing (Fig. 3b). These results demonstrate that the mutation (c.418G[A) destroyed the splicing donor site in vitro resulting in production of an abnormal transcript (Fig. 4). A single nucleotide change at the end of an exon has been shown to induce aberrant splicing in previous reports, such that changes at the last nucleotide of exon 11 in the TAT gene and exon 8 in the ATM gene were found to result in skipping of exons 11 and 8, respectively [23, 24]. This case corresponds to the above mentioned studies and is the first report to demonstrate that a missense mutation occurring at the end of an exon disrupted splicing in the EYA1 gene. The mutation c.418G[A induced aberrant splicing leading to skipping of exon 6, which caused a frameshift (p.N91KfsX10) resulting in a truncated protein. This frameshift mutation was predicted to produce a mutant EYA1 protein that is 99 amino acids shorter than the normal EYA1 protein (592 amino acids), lacking eyaHR. Because EYA1 protein does not contain a DNA binding domain, its eyaHR interacts with SIX1 so that EYA1 can function as a co-activator in the regulation of the downstream genes [25–27]. The SIX1 gene, the human homolog of sine oculis, which is co-expressed with the EYA1 gene during early development, has also been identified as a causative gene of BOR syndrome [15, 28–30]. In mammals, EYA1 plays an important role as a transcription factor that functions in the development of many organ systems and tissues such as inner ear and kidney [31].

Accordingly, EYA1 protein without a functioning eyaHR cannot form EYA1-SIX1-DNA complexes, resulting in disruption of downstream gene regulation and failure of normal development of many organ systems and tissues. Over 130 different mutations of EYA1 associated BOR syndrome have been reported worldwide to date (http:// www.hgmd.cf.ac.uk/, last updated January, 2013). In the East Asian population, a total of 16 EYA1 mutations have been reported in Japanese BOR/BO families, while only nine EYA1 mutations have been associated with BOR/BO syndrome in the Korean population [7, 18–22, 32–40]. The EYA1 mutations identified in Korean BOR/BO families include four splice-site, two frameshift, one missense, and one nonsense mutations, as well as one large deletion including the whole EYA1 gene (Table 2). All of these mutations were found scattered throughout the entire EYA1 gene. In the Korean population, splice-site mutations were the most common type of EYA1 mutations found in 50 % of BOR/BO families, which is much higher than 17 %

Table 2 Mutation of EYA1 in Korean patients with BOR syndrome No.

Nucleotide change

Amino acid change

Type

Reference

1

c.1474_1475insC

p.R492PfsX40

Frameshift

[21]

2

c.430C[T

p.G144X

Nonsense

[18]

3

c.868-2A[G



Splicing

[20]

4

c.321delT

p.A107fs

Frameshift

[19]

5

c.965A[G

p.E332G

Missense

[22]

6

c.699?5G[A



Splicing

[22]

7

c.1140?1G[A



Splicing

[22]

8

c.1598-2A[C



Splicing

[22]

9





Large deletion including EYA1

[22]

10

c.418G[A

p.G140S

Missense/ Splicing

This study

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reported in previous studies (http://www.hgmd.cf.ac.uk/, last updated January, 2013). In summary, a novel missense mutation (c.418G[A) that disrupts normal splicing was identified in a Korean patient with BOR syndrome. Furthermore, our study demonstrated that this mutation caused BOR syndrome by inducing a truncated protein lacking eyaHR. To the best of our knowledge, this is the first report revealing that a missense mutation could disturb normal splicing as a result of a substitution of the last nucleotide of an exon in EYA1. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A1015761 for M.H.Song, and 2011-0028066 for U. K. Kim and J. Y. Choi) the Korea Health Technology R&D Project, Ministry of Health &Welfare, Republic of Korea (A111345).

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