Novel partial duplication of EYA1 causes branchiootic syndrome in a ...

International Journal of Audiology 2015; Early Online: 1–6

Original paper

Novel partial duplication of EYA1 causes branchiootic syndrome in a large Brazilian family

Int J Audiol Downloaded from informahealthcare.com by University of Toronto on 06/05/15 For personal use only.

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Vitor G.L. Dantas*, Erika L. Freitas*, Valter A. Della-Rosa , Karina Lezirovitz*, , Ana Maria S.M. de Moraes , $ ‡ Silvia B. Ramos , Jeanne Oiticica , Leandro U. Alves*, Peter L. Pearson*, Carla Rosenberg* & Regina C. Mingroni-Netto* *Department of Genetics and Evolutionary Biology, Biosciences Institute, University of São Paulo, Brazil, †Department of Biotechnology, Genetics and Cellular Biology, State University of Maringá, Brazil, ‡Otolaryngology Lab, LIM32, School of Medicine Clinics Hospital, University of São Paulo, Brazil, #Department of Medicine, State University of Maringá, Brazil, $University Center of Maringá, Paraná, Brasil

Abstract Objective: To identify novel genetic causes of syndromic hearing loss in Brazil. Design: To map a candidate chromosomal region through linkage studies in an extensive Brazilian family and identify novel pathogenic variants using sequencing and array-CGH. Study sample: Brazilian pedigree with individuals affected by BO syndrome characterized by deafness and malformations of outer, middle and inner ear, auricular and cervical fistulae, but no renal abnormalities. Results: Whole genome microarray-SNP scanning on samples of 11 affected individuals detected a multipoint Lod score of 2.6 in the EYA1 gene region (chromosome 8). Sequencing of EYA1 in affected patients did not reveal pathogenic mutations. However, oligonucleotide-array-CGH detected a duplication of 71.8Kb involving exons 4 to 10 of EYA1 (heterozygous state). Real-time-PCR confirmed the duplication in fourteen of fifteen affected individuals and absence in 13 unaffected individuals. The exception involved a consanguineous parentage and was assumed to involve a different genetic mechanism. Conclusions: Our findings implicate this EYA1 partial duplication segregating with BO phenotype in a Brazilian pedigree and is the first description of a large duplication leading to the BOR/BO syndrome.

Key Words: Syndromic deafness; ear malformation; array-CGH; genetic mapping; copy number variation (CNV) Branchiootorenal syndrome (BOR) is an autosomal dominantly inherited syndrome characterized by malformations of the outer, middle, and inner ear, branchial cysts and fistulae associated with sensorineural, conductive, or mixed hearing loss. Renal abnormalities of various types, including renal agenesis and polycystic kidneys are frequently present. In a related but milder phenotype, known as branchiootic syndrome (BO), many features of BOR syndrome are present, excluding renal abnormalities. Prevalence of BOR/BO syndrome is ∼1:40 000 children and it has been estimated to be present in 2% of children with profound hearing loss (Fraser et al, 1980). Penetrance is high and expressivity of the phenotype is variable both between different families and individuals from the same family (Konig et al, 1994; Stratakis et al, 1998). The most frequently occurring features of the BO/BOR syndrome are hearing loss (93% of affected individuals), preauricular pits (82%), branchial fistulae (49%), outer-ear malformations (36%), and external auditory canal stenosis (29%) (Chen et al, 1995). Further, genetic heterogeneity characterizes the BO/BOR syndrome; three different loci have already been associated with the BO phenotype and two to the BOR phenotype. The BOS1/BOR1 locus was first mapped to 8q12-22 by Haan et al (1989), and its location narrowed down to 8q13.3 by Ni et al (1994) and Wang et al (1994). Many different types of mutations have been described in EYA1, the gene most frequently

associated with BOR/BO phenotypes (Abdelhak et al, 1997b). EYA1 mutations were also associated with other phenotypes, similar but not identical to BOR/BO, for instance, the otofaciocervical syndrome 1 (OFC1, OMIM#166780); BOS2 was mapped to chromosomal region 1q31 (Kumar et al, 1998), but, to date, no gene was found to be associated to the syndrome in BOS2 locus. BOS3 was mapped to the chromosomal region 14q23.1 (Ruf et al, 2003), where the SIX1 gene was located and associated with the syndrome (Ruf et al, 2004). BOR2 is located in the chromosomal region 19q13.32 (Hoskins et al, 2007) and the SIX5 gene was found to be mutated in cases of the syndrome (Hoskins et al, 2007). Here we report a partial duplication comprising exons 4 to 10 of the EYA1 gene, in heterozygous state, segregating with BO syndrome in an extensive Brazilian family.

Materials and Methods Patients A large pedigree including 19 affected individuals with hearing loss was ascertained in Paraná state, South-Eastern Brazil. This study was approved by the National Committee for Ethics in Science (CONEP).

Correspondence: Regina Célia Mingroni Netto Departamento de Genética e Biologia Evolutiva, Universidade de São Paulo, SP, Brazil.E-mail: [email protected] Received: 14 August 2014 Accepted: 12 March 2015 ISSN 1499-2027 print/ISSN 1708-8186 online © 2015 British Society of Audiology, International Society of Audiology, and Nordic Audiological Society DOI: 10.3109/14992027.2015.1030511

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V. G. L. Dantas et al.


Abbreviations ADD Autosomal dominant disorder ADM-2 Aberration detection method 2 Array-CGH Comparative genomic hybridization array BO Branchiootic syndrome BOR Branchiootorenal syndrome BOR1 Branchiootorenal syndrome 1 BOR2 Branchiootorenal syndrome 2 BOS1 Branchiootic syndrome 1 BOS2 Branchiootic syndrome 2 BOS3 Branchiootic syndrome 3 CONEP Comissão Nacional de Ética em Pesquisa CT Computer tomography CNV Copy number variation D Deletion DGV Database of genomic variants ED EYA1 domain EYA1 Eyes absent 1 Eya Eyes absent protein GAPDH Glyceraldehyde-3-phosphate dehydrogenase GJB2 Gap junction protein, beta 2 GJB6 Gap junction protein, beta 6 GRCh37 Genome reference consortium human genome build 37 hg19 Human genome version 19 HL Hearing loss HPRT Hypoxanthine guanine phosphoribosyltransferase 1 Kb Kilobase OFC1 Otofaciocervical syndrome 1 PCR Polymerase chain reaction PR Paraná qPCR Real time quantitative polymerase chain reaction SG SYBR Green Six SIX gene family Six Six gene family proteins SIX1 Sine oculis homeobox, drosophila, homolog of, 1 SIX2 Sine oculis homeobox, drosophila, homolog of, 2 SIX5 Sine oculis homeobox, drosophila, homolog of, 5 SNP Single nucleotide polymorphism

After obtaining informed consent, samples from 15 affected individuals (ten affected by sensorineural hearing loss and five affected by mixed hearing loss) and 13 unaffected individuals from the family were collected. The pedigree is presented in Figure 1. The following hearing measurements were performed: acoustic emission, including tympanometry and acoustic reflexes thresholds, pure tonal and vocal audiometry with conditioning methods according to patient age. Computed tomography (CT) of the temporal bone was performed in five individuals with mixed hearing loss. Blood and urine samples were collected for blood count and urine analysis. Urea and creatinine levels were determined in blood. Ultrasonography was used to evaluate the presence of renal malformations.

DNA extraction DNA was extracted from peripheral blood lymphocytes by commercial kits including ‘Easy-DNAt Kit (Version D) Genomic DNA Isolation’ from Invitrogen (Carlsbad, USA), a GFX Genomic Blood DNA Purification Kit (Amersham Biosciences, Buckinghamshire, UK) or using an equipment Autopure LS (Gentra Systems, Minneapolis, USA). All samples were analysed for concentration and quality using a Nanodrop 1000 spectophotometer.

Genetic mapping Genome scanning was performed in samples from 10 affected and one unaffected individual using an array from Affymetrix containing ∼50 000 SNPs (GeneChip® Human Mapping K Array Xba 240), as described by the manufacturer. The arrays were analysed on a Genechip 3000 Scanner and data by the GeneChip Command Console Software (Affymetrix, Santa Clara, USA). The Merlin linkage program (Abecassis et al, 2002) was used to calculate multiple point Lod scores for each autosome. The population disease allele frequency was set at an arbitrary, but likely value of 0.0001; similarly the disease penetrance value was set at 90%; population SNP frequencies were calculated using the various SNP genotypes observed within the family.

EYA1 sequencing PCR amplification of all coding exons was performed and primer sequences were the same as described by Abdelhak et al, 1997(b).

Figure 1. Pedigree showing individuals with EYA1 partial duplication. Individuals marked with (-) were tested for the duplication. The individuals marked with (*) carried the duplication. Affected individual IV:11 did not carry the duplication but had consanguinous parents.

Dantas – EYA1 partial duplication causes BO Syndrome

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(Microsoft Corp, Redmond, USA), which assumes that the calibrator DNA has two copies of the control genes (Livak et al, 2001). The EYA1 qPCR pair primer sequences are available on request.


Results and Discussion

Figure 2. Multipoint Lod score values for chromosome 8. Sequencing reactions for all exons were performed with the kit BigDye Terminator v3.1 (Applied Biosystems, Foster City, USA) and analysed on an Applied Biosystems 3730 DNA analyser.

Array-CGH Array-CGH was performed in DNA samples from three patients (IV-3, IV-18, and index case, V-6) using an 180K oligo-platform from Agilent Technologies, as described by the manufacturer. Data were processed with Feature Extraction software and subsequently analysed with the Genomic Workbench software (Agilent Technologies, Santa Clara, USA). Gains and losses of genomic sequences were determined using the aberration detection statistical algorithm ADM-2, with a sensitivity threshold of 6.7. For each sample, we used two reversed labeled hybridizations; alterations not detected in both dye-swap experiments were disregarded. The detected copy number variation (CNVs) was compared to data from oligoarray studies documented in the Database of Genomic Variants (DGV; http://projects.tcag.ca/variation/).

Real-time quantitative PCR (qPCR) Confirmation of the duplication and its segregation in the pedigree were performed by real-time quantitative qPCR using primers pairs to amplify exons 4, 5, and 11 of the EYA1 gene and using the SYBR Green system (Applied Science, Indianapolis, USA) on a 7500 Fast Real-Time PCR System apparatus (Applied Biosystems, Foster City, USA). As controls and for copy number calibration, we used DNA samples obtained from healthy donors and the qPCR values for the GAPDH and HPRT genes for normalization. All samples were run in triplicate, and the data analysed using the comparative DDCt cycle threshold method (Applied Biosystems) running on Microsoft Excel

Given the locus heterogeneity related to BO/BOR phenotypes, genomic scanning was performed with samples from 10 affected and one unaffected individual (proband, III:9, III:11, III:13, IV:1, IV:3, IV:5, IV:6, IV:16, IV:18 and V:1). After Lod score calculations using Merlin, a peak Lod score of 2.6 was obtained for chromosomal region 8q13.3 (Figure 2) which was the only location exhibiting positive Lod scores. Since this location is compatible with the EYA1 gene, sequencing of all exons was performed, but no putative pathogenic variants were detected. DNA samples from three affected individuals (proband, IV:3, IV:18, and V-6) were analysed by array-CGH. A duplication at 8q13.3 (chr8:72,174,526-72,246,351; GRCh37/hg19) of approximately 72 Kb, encompassing exons 4 to 10 of the 18 exons of the EYA1 gene was detected, in heterozygous state. No other gene was included in the duplicated segment. The duplication detected by array-CGH in the proband, V-6, is shown in Figure 3. The presence of the partial EYA1 duplication was confirmed by quantitative real-time PCR in samples from the same three individuals analysed by array-CGH. Subsequently, this feature was investigated in samples from another 12 affected individuals (III:1, III:3, III:9, III:11, III:13, IV:5, IV:6, IV:11 , IV:15, IV:16, IV-19, and V:11). All results were compatible with the partial duplication being present in all but one affected, individual (IV-11), whose parents were consanguineous and not affected themselves by hearing loss. Further, Individual IV-11 is only affected by hearing loss and does not present other BOS syndrome features. We hypothesize that his hearing loss is due to an autosomal recessive mechanism independent of the EYA1 partial duplication. Screening of other frequent deafness mutations, such as GJB2 sequencing and deletions of GJB6 D13S1830 and D13S1854 were performed in the patient without deletion, but no other mutations were found. The partial duplication was absent in all 13 unaffected individuals from the family studied. Accordingly, real-time quantitative PCR analysis confirmed that the duplication segregated only with the disease phenotype with the exception for one individual, IV-11 (Figure 4). Branchiootic syndrome 1 (BOS1) is a congenital disease characterized by several ear and branchial anomalies due to mutations in EYA1, located on chromosome band 8q13.3 (Abdelhak et al, 1997a; Vincent et al, 1997). EYA1 protein is required for regulating genes that modulate cell proliferation. In mice, EYA1 is expressed in the otic placode, located in the developing membranous inner ear and surrounding mesenchyme otic capsule (Abdelhak et al, 1997a), suggesting a central role in the development of many, if not all,

Figure 3. Array-CGH profile of the 8q13.3 region (red bar in the ideogram) showing a 71.8 Kb duplication encompassing exons 4–10 of the EYA1 gene in the proband.


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Figure 4. Quantitative real time PCR of exons 4 and exon 11 showing duplication in affected individuals and the absence of duplication in individual IV-11. Control + is a control sample, known to present the duplication in a previous experiment. inner-ear components. Disruptions in EYA1 were also reported to cause BOR1 syndrome. The phenotype is distinguished from BOS by the absence of renal abnormalities. It is estimated that about 40% of people with BOR, BO, or OFC syndromes have mutations in EYA1 with more than 160 different mutations reported. These were mostly single nucleotide substitutions, small inversions or deletions, and some splicing mutations (Abdelhak et al, 1997b; Castiglioni et al, 2014; Gigante et al, 2013; Shimasaki et al, 2004; Spruijt et al, 2006; Stockley et al, 2009 and Wang et al, 2012) Abdelhak et al (1997a) studied 21 unrelated probands affected by BOR1 syndrome using Southern Blotting. In one sporadic proband, they found a deletion of about 7 Kb comprising exons 11 to 15 of the EYA1 gene. In a further study, Abdelhak et al (1997b) analysed all EYA1 exons in 20 probands affected by BOR1 syndrome using Southern Blotting. They found two deletions and one Alu insertion. One of the deletions, found in two related affected individuals, was about 5.6 Kb and comprised the region spanning exon 9 and intron IX; the second deletion was about 20–37 Kb size and extending between introns X to exon 16. An Alu insertion was found in two related individuals in exon 10. Accordingly, both deletions and insertions are a recognized mechanism of mutation leading to the BOS1/ BOR1 syndromes, but duplications similar to the one described by us have not been reported previously. Bilateral hearing loss, sensorineural or mixed, in all frequencies was present in all the individuals carrying the duplication, although

the severity of hearing loss was variable. The age of onset of hearing loss varied from birth to 33 years. An audiogram from one individual with mixed hearing loss is presented in Figure 5. A summary of clinical findings in ten affected individuals is shown in Table 1. In addition to hearing abnormalities, the proband of our family also presented cleft palate. Computer tomography was performed in five individuals presenting mixed hearing loss and malformations of outer, middle and inner ear, auricular and cervical fistulae were detected. The results are summarized in Table 2. A critical comparison of clinical findings of our family with other reports of structural abnormalities in the EYA1 is limited, since large duplications in EYA1 gene have never previously been described. However, an overall comparison with the clinical findings in the cases investigated by Abdelhak et al, (1997b), exhibiting large deletions or small duplication plus an Alu insertion revealed no remarkable features distinguishing our patients from those previously described. The protein product of EYA1 acts as a protein tyrosine phosphatase and transcriptional co-activator. The N-terminal domain is required for the co-activator function (Xu et al, 1997) and the C-terminal domain for the tyrosine phosphatase function (Li et al, 2003), which is highly conserved (Buller et al, 2001). The conserved Eya domain (ED), in the C terminal portion of the protein, is required for interaction with proteins of the Six family (Chen et al, 1997; Fan et al, 2000; Landgraf et al, 2010; Tootle et al, 2003). It is known that mutations in the SIX1 and SIX5, members of this gene family, also cause BOR syndrome; mutations in SIX2 are related to renal hypoplasia (Hoskins et al, 2007; Ruf et al, 2004; Weber et al, 2008). Missense mutations S454P, L472R and L550P in EYA1 gene lead to enhanced proteasomal degradation, probably because they disrupt the interaction with SIX proteins, which protect the Eya protein from degradation (Musharraf et al, 2014). Accordingly, haploinsufficiency seems to be the most likely explanation for BOR-related phenotypes in cases with missense mutations, and large deletions leading to similar disease phenotypes. The exon 4-10 duplication detected by us was not reported in DGV, among normal individuals, and its segregation confirms that we identified a novel molecular mechanism leading to BOS1 syndrome. The duplication is about 72 Kb and comprises EYA1 exons 4 to 10 and there are no other genes involved in the duplication. In fact, the first amino acids of the ED domain are coded by exon 10. The linkage studies indicate that the most probable location of the duplication overlaps the wild type gene chromosomal position. Two simple presentations could be imagined for the duplication: one in tandem, with exon 10 being followed by an extra copy of exons 4 to 10, or in an inverted position with exon 10 being followed by an extra copy of exons 10 to 4. In either situation, no complete ED

Figure 5. Pure-tone audiogram showing thresholds of one individual affected by mixed hearing loss. ( ) represents bone conduction and (´; •) air conduction.

Dantas – EYA1 partial duplication causes BO Syndrome

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Table 1. Summary of clinical findings in 10 affected individuals (NI: not investigated). * are individuals with mixed hearing loss.


Frequent findings in BOR Deafness Branchial cysts Auricular dysplasia Renal dysplasia External auditory channel stenosis Lacrimal duct stenosis or aplasia Branchial fistulae Preauricular pits Internal ear dysplasia Middle ear dysplasia Occasional findings Anomalous facial nerve Preauricular appendix Euthyroid goiter Pancreatic cysts Congenital cholesteatoma Narrow andlong face Cleft palate Gustatory lacrimation Congenital hip dislocation Malocclusion of teeth Microdontia of permanent teeth Intestinal malrotation Narrow palate Facial paralysis Benign intracranial tumor Bifid uvula

III:1*

III:11*

III:13

IV:3

IV:5*

IV:15

IV:16

IV:18*

IV:19

Proband*

⫹ ⫹ ⫹ NI ⫺

⫹ ⫺ ⫹ NI ⫺

⫹ ⫺ ⫺ ⫺ ⫺

⫹ ⫹ ⫹ NI ⫺

⫹ ⫹ ⫹ ⫺ ⫺

⫹ ⫹ ⫺ ⫺ ⫺

⫹ ⫺ ⫹ NI ⫺

⫹ ⫺ ⫹ ⫺ ⫺

⫹ ⫺ ⫺ ⫺ ⫺

⫹ ⫺ ⫹ ⫺ ⫺

⫺

⫺

⫺

⫺

⫺

⫺

⫺

⫺

⫺

⫺

⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ NI NI ⫹ ⫺ ⫺ ⫺ ⫹ Dental prosthesis ⫺ ⫺ ⫺ NI NI

⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ NI NI ⫹ ⫺ ⫺ ⫺ ⫹ Superior prosthesis ⫺ ⫺ ⫺ NI ⫺

⫺ ⫹ NI NI ⫺ ⫺ ⫺ ⫺ NI NI ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ NI ⫺

⫺ ⫹ NI NI ⫺ ⫺ ⫺ ⫺ NI NI ⫹ ⫺ ⫺ ⫺ NI NI ⫺ NI ⫺ NI NI

⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ NI NI ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ NI NI

⫹ ⫹ NI NI ⫺ ⫺ ⫺ ⫺ NI NI ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ NI ⫺

⫹ ⫹ NI NI ⫺ ⫺ ⫺ ⫺ NI NI ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ NI ⫺

⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ NI NI ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ NI ⫺

⫺ ⫹ NI NI ⫺ ⫺ ⫹ ⫺ NI NI ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ NI ⫺

⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ NI NI ⫹ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ NI NI

domain would be translated. The tandem presentation would result in a frameshift, because the first codon of exon 4 starts with the last base of exon 3 (exon 4 is 1-1) and exon 10 ends with a complete codon (exon 10 is 0-0). In the second, the reading frame would be interrupted by an inverted segment. Either situation would result in an abnormal interaction with Six proteins and EYA1 degradation as the most likely outcome; either interpretation predicts EYA1 haploinsufficiency as the cause of the disease phenotype. Unfortunately, it was not possible to investigate the structure of the abnormal transcript in affected patients, since the EYA1 transcript from individuals cannot be detected in blood, lymphoblastoid cell lines or cells obtained from buccal mucosa (data not shown).

Conclusion In summary, this is the first description of a partial duplication in the EYA1 gene, which segregates with the BO phenotype in a large Brazilian family.

Acknowledgements We thank Maria Teresa Balester de Mello Auricchio (Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Brazil) for technical assistance. We thank Maria Cristina C. Braga Scorzafave (from Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de Maringá), for helping with collection of genetic data and samples. We also thank Dr. Jeferson Cedaro de Mendonça for his analysis of CT scanning and Dr. Sergio Seiji Yamada for interpretation of tests of renal function, both from the Department of Medicine, Universidade Estadual de Maringá. We thank FAPESP (process 2003/04780-9, 2009/00898-1 and 2009/05620-1), CEPID-FAPESP Human Genome and Stem Cell Research Center (HGRC- FAPESP/CEPID process 98/14254-2, Coordinator: Mayana Zatz) and CAPES for financial support. This study was presented as an abstract at the following scientific meeting: Mingroni-Netto, Regina Célia, Dantas VGL, Freitas EL, Moraes AMM, Braga MCC, Ramos SB, Rosenberg C, Della-Rosa VA. Duplication of EYA1 causes branchiootic syndrome

Table 2. Summary of computer tomography (CT) scanning of temporal bones in five affected individuals with mixed hearing loss (NI: not investigated). III:1

III:11

IV:5

IV:18

Proband

Cochlea dysplasia Bilateral volume reduction Bilateral volume reduction Bilateral volume reduction Dysplasia to the right ⫺ Vestibular dysplasia To the right To the left To the right ⫺ ⫺ Semicircular channel dysplasia To the right To the right Bilateral To the right Bilateral Auditory channel dysplasia NI NI ⫺ ⫹ ⫺ Enlarged vestibular aqueducts To the left To the left To the right ⫺ ⫺

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in a Brazilian family. American Society of Human Genetics 2013 Annual Meeting, 2013. p.2938F–2938F. Declaration of interest: The authors declare no conflicts of interest.


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