Branchio-Oto-Renal Syndrome (BOR) associated with focal glomerulosclerosis in a patient with a novel EYA1 splice site mutation
© Gigante et al.; licensee BioMed Central Ltd. 2013
Received: 3 December 2012
Accepted: 13 March 2013
Published: 18 March 2013
Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial, ear, and renal anomalies. The most common gene mutated in BOR patients is EYA1, the human homolog of the Drosophila eyes absent gene, while mutations in SIX1 gene, the human homolog of sine oculis, encoding a DNA binding protein interacting with EYA1, have been reported less frequently. Recently, mutations in another SIX family member, SIX5, have been described in BOR patients, however, this association has not been confirmed by other groups.
In this study, we have clinically and genetically characterized a proband that displayed hearing loss, pre-auricular pits, branchial fistulae, hypoplasia of the left kidney, bilateral mild hydronephrosis, progressive proteinuria and focal glomerulosclerosis. Mutational analysis of EYA1 gene revealed a novel splice site mutation, c.1475 + 1G > C, that affects EYA1 splicing and produces an aberrant mRNA transcript, lacking exon 15, which is predicted to encode a truncated protein of 456 aa.
This report provided the functional description of a novel EYA1 splice site mutation and described for the first time a case of BOR syndrome associated with the atypical renal finding of focal glomerulosclerosis, highlighting the importance of molecular testing and detailed clinical evaluation to provide accurate diagnosis and appropriate genetic counselling.
KeywordsBOR syndrome EYA1 Focal Glomerulosclerosis Mutational analysis RNA analysis
Branchio-oto-renal (BOR) syndrome (MIM 113650) is an autosomal dominant disorder characterized by branchial, ear, and renal anomalies. This syndrome occurs with a frequency of approximately 1:40,000 in the general population and it is found in about 2% of profoundly deaf children . The major clinical signs are hearing loss, branchial fistulae and pre-auricular pits, malformations of the external ear, auditory canal and mid or inner ear, and renal anomalies ranging from hypoplasia to bilateral renal agenesis . Other associated clinical manifestations, although less frequent, include facial and palate anomalies, lacrimal duct aplasia and cataracts. In the absence of renal anomalies, it is defined as Branchio-oto syndrome (BO, MIM 601653) .
The most common gene mutated in BOR patients is EYA1 (eyes absent homolog 1; MIM 601653) , the human homolog of the Drosophila eyes absent gene. Over 130 different EYA1 disease-causing mutations, resulting in either BOR or branchial-otic syndrome (BO), have been published [5–10]. The vertebrate EYA gene family comprises four transcriptional activators that ensure normal branchial arch and epibranchial placode formation and sensory neurogenesis, including hair cell and neuron formation in the inner ear [11, 12]. In the kidney, EYA1 is a patterning gene essential for early metanephric mesenchyme development .
Mutations in SIX1 gene (MIM 601295), the human homolog of sine oculis, encoding a DNA binding protein that interacts with EYA1, have also been associated with BOR syndrome although less frequently than EYA1 mutations [14, 15]. Recently, mutations in another SIX family member, SIX5 (MIM 610896), have been reported in patients with BOR syndrome . SIX5 homologous interacts with eya-1 in Caenorhabditis elegans. However, the association of SIX5 mutations with BOR syndrome has not been confirmed by other groups and the pathogenetic role of some SIX5 mutations was reconsider. BOR syndrome has high penetrance, but incomplete and variable expressivity. The genetic heterogeneity and the spectrum of phenotypes associated with different mutations make the diagnosis of BOR sometimes difficult. Thus, molecular analysis would be a valuable and useful tool for the confirmation of a clinical diagnosis. Here, we report the clinical and genetic diagnosis of an Italian patient with BOR syndrome associated with focal segmental glomerulosclerosis and a novel EYA1 splice site mutation.
Patient and clinical data
Analysis of EYA1gene
This study have been performed in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of University Hospital in Foggia. Informed consent for genetic studies was obtained from proband and all family members. Genomic DNA was purified from peripheral blood samples of proband and all available family members (two siblings and father), using standard procedures. Mutational analysis of EYA1 gene (NM_000503.4; GeneID: 2138) was performed by polymerase chain reaction (PCR) and bidirectional sequencing of the coding exons and intron/exon flanking regions. EYA1 flanking intronic primers were designed using primer3 program (http://primer3.wi.mit.edu/). PCR products were sequenced using the Big Dye Terminator v3.1 cycle sequencing kit on 3130 Genetic Analyzer (Life Technologies, Ltd). EYA1 mutation was named according to Human Genome Variation Society recommendations (http://www.hgvs.org/mutnomen). The potential effect of splice site mutation on mRNA splicing was analyzed using Splice Site Prediction server (http://www.fruitfly.org/seq_tools/splice.html). A skin biopsy was performed in order to get fibroblasts for RNA analysis. Total RNA was extracted from normal and proband’s cultured skin fibroblasts by Qiagen’s RNA Mini Kit (Qiagen) and cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Ltd). RT-PCR and sequencing analysis were performed by specific primer pairs (F: 5′-CCGCTACAGACGGGTAAAAG-3′; R: 5′-CCCATACAGCAGGACTTTCG-3′) surrounding the region of exon 15.
BOR syndrome was first described in 1975 by Melnick et al. and it is characterized by hearing loss, renal anomalies and branchial cysts or fistulae . Renal abnormalities are present in 66% of affected individuals, and about 6% of them progress to renal failure. Major renal alterations include monolateral renal agenesis, monolateral or bilateral hypodisplasia and hydronephosis caused by ureteropelvic obstruction or vesicoureteral reflux .
We report here a case of BOR syndrome presenting typically with bilateral conductive hearing loss, associated with a complex dysplasia of external, middle and inner ear; pre-auricular pits; branchial fistulae; hypoplasia of the left kidney; bilateral mild hydronephrosis and progressive proteinuria associated with atypical renal histological pattern compatible with focal glomerulosclerosis (Figure 1). However, in the absence of overt nephrotic syndrome, we can not exclude a form of secondary focal glomerulosclerosis, due to obstructive uropathy related to the presence of left renal hypoplasia. Mutational analysis of EYA1 gene revealed a novel EYA1 splice site mutation, c.1475 + 1G > C, in the donor site of exon 15. RNA analysis on skin biopsy sample showed that this mutation affects EYA1 splicing, producing an aberrant mRNA transcript, lacking exon 15, that is predicted to encode an EYA1 truncated protein of 456 aa respect to the wild type protein of 592 aa (Figure 2). The transcriptional effects of EYA1 mutations are often unknown due to the difficulty of obtaining appropriate samples for RNA analysis. To date, EYA1 transcript analysis have been reported only for five other patients with BOR syndrome, two of which presented unstable EYA1 transcripts  and three aberrant transcripts [15, 18]. Clinical features of the latter three patients included renal hypoplasia, similar to our patient with the c.1475 + 1G > C mutation, however focal glomerulosclerosis associated with IgM and C3 positive immunofluorescence was never been reported.
EYA1 is a dual-function transcription factor, with an amino terminal transcriptional co-activator region that interacts with SIX1 and DACH, and a highly conserved 271 aa carboxy terminal Eya Domain (ED) that dephosphorylates SIX1–DACH complexes to switch from repression . In vivo analysis showed that ED mutations impaired the catalytic activity (i.e., dephosphorylation) of the EYA1 protein, suggesting that the loss of phosphatase activity may contribute to impaired EYA1 activity and in turn to BOR phenotype .
The c.1475 + 1G > C mutation produces an aberrant mRNA transcript that predicts a truncated version of EYA1 protein containing only 135 aa of the conserved carboxyterminal Eya domain. The predicted protein would retain the ability to interact with SIX1–DACH, but would be unable to dephosphorylate such complexes to allow gene activation . In agreement with other reports [8, 18], our findings confirm that some mutant EYA1 proteins, lacking or with a disrupted phosphatase domain, might have a dominant-negative gain-of-function activity, suggesting another possible model for the pathogenesis of BOR syndrome, in addition to the previously reported haploinsufficiency model. BOR syndrome is often misdiagnosed or not diagnosed in the presence of mild clinical symptoms . The hearing of BOR children with malformations of the inner ear can be exacerbated by minor environment and playground injuries (i.e., head trauma), as well as nutrition and nephrotoxic drugs might contribute to kidney failure in patients with kidney malformations. Our case is an example of a late diagnosis (27 years) of BOR syndrome, characterized by bilateral conductive hearing loss, bilateral mild hydronephrosis and progressive proteinuria associated with focal glomerulosclerosis. An accurate medical history associated with clinical, instrumental and genetic analyses lead us to diagnose BOR syndrome, even though a more timely clinical and molecular diagnosis would allow to implement nutritional and lifestyle strategies that would have prevented the most severe effects of BOR syndrome. This case suggests the opportunity of timely researching mutations of EYA1 gene in patient affected by deafness associated with urinary anomalies and/or branchial cysts or fistulae.
Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
MG: PhD, Post-graduate school in Medical Genetics, permanent position as Biologist, University of Foggia; Md’A and FB: post-graduate school in Nephrology, University of Foggia; EM: Biologist, University of Bari; SD: laboratory technician, University of Foggia; BI, GSN and GS: MD, Nephrology, University of Foggia; ER: Associate Professor of Clinical Pathology, University of Foggia; GG: Associate Professor of Nephrology, University of Foggia; LG: Full Professor of Nephrology, University of Bari.
Polymerase chain reaction
We thank patient and his family members for consent to publish the data. This work was supported in part by grant: “Premio di ricerca Gianluca Montel, 2011/2012” from University of Foggia, awarded to Maddalena Gigante.
- Fraser FC, Sproule JR, Halal F: Frequency of the branchio-oto-renal (BOR) syndrome in children with profound hearing loss. Am J Med Genet. 1980, 7: 341-349. 10.1002/ajmg.1320070316.View ArticlePubMedGoogle Scholar
- Chen A, Francis M, Ni L, Cremers CW, Kimberling WJ, Sato Y, Phelps PD, Bellman SC, Wagner MJ, Pembrey M, Smith RJH: Phenotypic manifestations of branchio - oto - renal syndrome. Am J Med Genet. 1995, 58: 365-370. 10.1002/ajmg.1320580413.View ArticlePubMedGoogle Scholar
- Vincent C, Kalatzis V, Abdelhak S, Chaib H, Compain S, Helias J, Vaneecloo FM, Petit C: BOR and BO syndromes are allelic defects of EYA1. Eur J Hum Genet. 1997, 5: 242-246.PubMedGoogle Scholar
- Abdelhak S, Kalatzis V, Heilig R, Compain S, Samson D, Vincent C, Weil D, Cruaud C, Sahly I, Leibovici M, Bitner-Glindzicz M, Francis M, Lacombe D, Vigneron J, Charachon R, Boven K, Bedbeder P, Van RN, Weissenbach J, Petit C: A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. Nat Genet. 1997, 15: 157-164. 10.1038/ng0297-157.View ArticlePubMedGoogle Scholar
- Kumar S, Deffenbacher K, Cremers CW, Van Camp G, Kimberling WJ: Branchio-oto-renal syndrome: identification of novel mutations, molecular characterization, mutation distribution, and prospects for genetic testing. Genet Test. 1997–1998, 1: 243-251. 10.1089/gte.1997.1.243.View ArticleGoogle Scholar
- Abdelhak S, Kalatzis V, Heilig R, Compain S, Samson D, Vincent C, Levi-Acobas F, Cruaud C, Le MM, Mathieu M, Konig R, Vigneron J, Weissenbach J, Petit C, Weil D: Clustering of mutations responsible for branchio-oto-renal (BOR) syndrome in the eyes absent homologous region (eyaHR) of EYA1. Hum Mol Genet. 1997, 6: 2247-2255. 10.1093/hmg/6.13.2247.View ArticlePubMedGoogle Scholar
- Chang EH, Menezes M, Meyer NC, Cucci RA, Vervoort VS, Schwartz CE, Smith RJ: Branchio-oto-renal syndrome: the mutation spectrum in EYA1 and its phenotypic consequences. Hum Mutat. 2004, 23: 582-589. 10.1002/humu.20048.View ArticlePubMedGoogle Scholar
- Sanggaard KM, Rendtorff ND, Kjaer KW, Eiberg H, Johnsen T, Gimsing S, Dyrmose J, Nielsen KO, Lage K, Tranebjaerg L: Branchio-otorenal syndrome: detection of EYA1 and SIX1 mutations in five out of six Danish families by combining linkage, MLPA and sequencing analyses. Eur J Hum Genet. 2007, 15: 1121-1131. 10.1038/sj.ejhg.5201900.View ArticlePubMedGoogle Scholar
- Orten DJ, Fischer SM, Sorensen JL, Radhakrishna U, Cremers CW, Marres HA, Van Camp G, Welch KO, Smith RJ, Kimberling WJ: Branchio-Oto-Renal Syndrome (BOR): novel mutations in the EYA1 gene, and a review of the mutational genetics of BOR. Hum Mutat. 2008, 29: 537-544. 10.1002/humu.20691.View ArticlePubMedGoogle Scholar
- Krug P, Morinière V, Marlin S, Koubi V, Gabriel HD, Colin E, Bonneau D, Salomon R, Antignac C, Heidet L: Mutation screening of the EYA1, SIX1, and SIX5 genes in a large cohort of patients harbouring branchio-oto-renal syndrome calls into question the pathogenic role of SIX5 mutations. Hum Mutat. 2011, 32: 183-190. 10.1002/humu.21402.View ArticlePubMedGoogle Scholar
- Zou D, Silvius D, Rodrigo-Blomqvist S, Enerbäck S, Xu PX: Eya1 regulates the growth of otic epithelium and interacts with Pax2 during the development of all sensory areas in the inner ear. Dev Biol. 2006, 298: 430-441. 10.1016/j.ydbio.2006.06.049.View ArticlePubMedGoogle Scholar
- Brugmann SA, Moody SA: Induction and specification of the vertebrate ectodermal placodes: precursors of the cranial sensory organs. Biol Cell. 2005, 97: 303-319. 10.1042/BC20040515.View ArticlePubMedGoogle Scholar
- Dressler GR: The cellular basis of kidney development. Annu Rev Cell Dev Biol. 2006, 22: 509-529. 10.1146/annurev.cellbio.22.010305.104340.View ArticlePubMedGoogle Scholar
- Li X, Oghi KA, Zhang J, Krones A, Bush KT, Glass CK, Nigam SK, Aggarwal AK, Maas R, Rose DW, Rosenfeld MG: Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature. 2003, 426: 247-254. 10.1038/nature02083.View ArticlePubMedGoogle Scholar
- Okada M, Fujimaru R, Morimoto N, Satomura K, Kaku Y, Tsuzuki K, Nozu K, Okuyama T, Iijima K: EYA1 and SIX1 gene mutations in Japanese patients with branchio-oto-renal (BOR) syndrome and related conditions. Pediatr Nephrol. 2006, 21: 475-481. 10.1007/s00467-006-0041-6.View ArticlePubMedGoogle Scholar
- Hoskins BE, Cramer CH, Silvius D, Zou D, Raymond RM, Orten DJ, Kimberling WJ, Smith RJ, Weil D, Petit C, Otto EA, Xu PX, Hildebrandt F: Transcription factor SIX5 is mutated in patients with branchio-oto-renal syndrome. Am J Hum Genet. 2007, 80: 800-804. 10.1086/513322.View ArticlePubMedPubMed CentralGoogle Scholar
- Melnick M, Bixler D, Silk K, Nance WE: Autosomal dominant branchio-oto-renal dysplasia. Birth defects. Birth Defects Orig Artic Ser. 1975, 11: 121-128.PubMedGoogle Scholar
- Stockley TL, Mendoza-Londono R, Propst EJ, Sodhi S, Dupuis L, Papsin BC: A recurrent EYA1 mutation causing alternative RNA splicing in branchio-oto-renal syndrome: implications for molecular diagnostics and disease mechanism. Am J Med Genet A. 2009, 149A: 322-327. 10.1002/ajmg.a.32679.View ArticlePubMedGoogle Scholar
- Mutsuddi M, Chaffee B, Cassidy J, Silver SJ, Tootle TL, Rebay I: Using Drosophila to decipher how mutations associated with human branchio-oto-renal syndrome and optical defects compromise the protein tyrosine phosphatase and transcriptional functions of eyes absent. Genetics. 2005, 170: 687-695. 10.1534/genetics.104.039156.View ArticlePubMedPubMed CentralGoogle Scholar
- Bellini C, Piaggio G, Massocco D, Perfumo F, Bertini , Gusmano R, Serra G: Branchio-oto-renal syndrome: a report on nine family groups. Am J Kidney Dis. 2001, 37: 505-509. 10.1053/ajkd.2001.22074.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/60/prepub
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