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PEDOT-7062; No. of Pages 10 International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Review Article
EYA1-related disorders: Two clinical cases and a literature review Alessandro Castiglione a,*, Salvatore Melchionda b, Massimo Carella b, Patrizia Trevisi a, Roberto Bovo c, Renzo Manara d, Alessandro Martini e a
Department of Neurosciences, University of Padua, Via Giustiniani, 2, Padua, Italy Unit of Medical Genetics, IRCCS, ‘‘Casa Sollievo della Sofferenza’’ Hospital, 71013 San Giovanni Rotondo, Italy c Operative Unit of Otolaryngology and Otosurgery, University of Padua, Via Giustiniani, 2, Padua, Italy d Neuroradiologic Unit, Padua University, Via Giustiniani, 2, Padua, Italy e Department of Neurosciences, Operative Unit of Otolaryngology and Otosurgery, University of Padua, Via Giustiniani, 2, Padua, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2013 Received in revised form 27 March 2014 Accepted 29 March 2014 Available online xxx
Objectives: To delineate the diagnostic and rehabilitative aspects of syndromes that have overlapping features, we present the cases of two unrelated Caucasian males affected by hearing impairment, preauricular pits and cervical fistulae. Specific findings that are helpful in the diagnosis and management of EYA1-related disorders are highlighted. Methods: Genetic, otologic, imaging, eye and renal evaluations were conducted to achieve a detailed and comprehensive assessment, leading to the most accurate diagnosis and appropriate treatment. A literature review was also carried out. Results: Diagnostic criteria indicated that the two patients were affected by BOS1 (Branchio-Otic Syndrome 1). We also identified a novel sporadic missense mutation in the EYA1 gene: p.G533R (c.1597G > A, NM_000503.4), a highly conserved, heterozygotic amino acid substitution. In the other case, we identified the p.X593QextX6 (c.1777T > A, NM_000503.4) substitution, known to be responsible for BO/BOR phenotypes. Both substitutions lead to isoform 1 (EYA1B and EYA1C) which is composed of 592 amino acids. Clinical and in silico evidence suggests a pathogenic role for the new, never before described p.G533R mutation. Imaging evaluation revealed a complex pathology, characterized by external, inner and middle ear malformations, without renal anomalies. Conclusions: Our results demonstrate the importance of considering the imaging evaluation and the complete DNA sequencing of the EYA1 gene for the differential diagnosis of deafness and related branchio-oto-renal disorders. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Branchio-oto-renal Branchio-otic Oto-Facio-Cervical Branchio-oto-ureteral Hearing loss EYA1 mutations
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . Clinical and audiological data . 2.1. Genetic and molecular analysis 2.2. Imaging data . . . . . . . . . . . . . . . 2.3. In silico analysis . . . . . . . . . . . . 2.4. Literature review . . . . . . . . . . . 2.5. Results . . . . . . . . . . . . . . . . . . . . . . . . . Clinical and audiological data . 3.1. Genetic and molecular analysis 3.2.
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* Corresponding author at: Department of Neurosciences, Operative Unit of Otolaryngology and Otosurgery, 2, Giustiniani, Padua 35100, Italy. Tel.: +39 328 6181047; fax: +39 049 821 1994. E-mail addresses: [email protected], [email protected] (A. Castiglione), [email protected] (S. Melchionda), [email protected] (M. Carella), [email protected] (P. Trevisi), [email protected] (R. Bovo), [email protected] (R. Manara), [email protected] (A. Martini).
http://dx.doi.org/10.1016/j.ijporl.2014.03.032 0165-5876/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: A. Castiglione, et al., EYA1-related disorders: Two clinical cases and a literature review, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.032
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PEDOT-7062; No. of Pages 10 A. Castiglione et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx
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4. 5.
3.3. Imaging data . . . . . . . In silico analysis . . . . 3.4. Literature review . . . 3.5. Discussion and conclusions . Consent . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .
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shortest isoform and corresponds to the variant EYA1D. Isoform 2 is reported to be composed of 559 amino acids and is widely known as variant EYA1A. This classification is not clear or helpful in the definition and collection of EYA1 mutations. Additionally, many allelic variants or polymorphisms are consistently reported during clinical practice and have been supported by genetic investigations. To avoid misunderstandings, it would be helpful to directly refer to the amino acidic length of the different isoforms or variants. To date, more than 160 pathologic mutations in the EYA1 gene have been reported in the literature. The EYA1 protein may be required for the normal development of branchial arches, ears and kidneys. The expression pattern of the murine EYA1 ortholog, Eya1, suggests a role in the development of all components of the inner ear, from the emergence of the otic placode. The expression pattern in the developing kidney indicates a role for Eya1 in the metanephric cells surrounding the ‘just-divided’ ureteric branches [2,8]. The EYA1 gene product is a tyrosine phosphatase that specifically dephosphorylates Tyr-142 of histone H2AX (H2AXY142ph). Tyr-142 phosphorylation of histone H2AX plays a central role in DNA repair and acts as a mark that distinguishes between apoptotic and repair responses to genotoxic stress. Its function as a histone phosphatase likely explains its role in transcription regulation during organogenesis. EYA1 appears to coactivate SIX2, SIX4 and SIX5 [2]. The structure of eya proteins includes the eya homologous region (eyaHR), a conserved carboxyl-terminal region, and the eya variable region (eyaVR), a divergent transactivation domain rich in tripeptide PST (proline– serine–threonine) [13]. The vertebrate orthologs of so (sine oculis) are six genes (SIX1, SIX2, SIX3, SIX4, SIX5, SIX6) and their protein products bind to eya proteins, inducing the nuclear translocation of the complex [9]. From a clinical standpoint, hearing loss is the most common and constant feature of EYA1 mutation and is detected in more than 90% of the affected individuals (Tables 3–5). In Caucasian populations, common EYA1 mutations are p.R440Q, c.867 + 5G > A and p.R297X [1–3,5–7,9]. There is no distinct relationships observed between the nature of the mutations and
1. Introduction Branchio-oto-renal (BOR), Branchio-otic (BO), Branchio-otoureteral (BOU) and Oto-Facio-Cervical (OFC) syndromes are dominant disorders characterized by variable hearing impairment (HI) and branchial defects [1–3]. BOR and BOU syndromes include additional kidney and urinary tract anomalies. All of these syndromes are genetically heterogeneous and caused by mutations in the EYA1, SIX1, and SIX5 genes [3,4]. The involvement of different genes was conducted to define different syndrome subtypes, without wide clinical variations (Table 1). It was also reported that DFNA23, a locus for non-syndromic hearing impairment, is due to mutations in SIX1 [5]. Nevertheless, mutations in the same gene can lead to different syndromes even if it is unclear why some EYA1 mutations are associated with kidney abnormalities and others are not. It was also previously thought that EYA1 mutations may be responsible for BranchioOculo-Facial (BOF) syndrome (MIM#113620) because of the presence of overlapping features; it is now clear that BOF syndrome is due to mutations in the TFAP2A gene (Table. 1) [6]. Approximately 40 percent of people with BO, BOR, BOU or OFC syndromes have mutations in the EYA1 gene. The prevalence of EYA1-related disorders is approximately 2.5:100,000 newborns and require a multidisciplinary approach in diagnosis, management and treatment (Tables 2–4) [7]. The EYA1 (Eyes absent homolog 1) gene is the human homologue of the Drosophila Eya1 gene, which is essential for eye development in that species. In humans, it consists of 16 coding exons and has been localized to chromosome 8q13.3 [1–3]. To date, at least 4 different isoforms are known (EYA1A, EYA1B, EYA1C, EYA1D) and are composed of 559 amino acids (AA), 592 AA, 592 AA and 557 AA, respectively. However, the general acceptance of these isoforms remains lacking because of additional transcriptional variants and an inconsistent definition in specialized online databases. It should be noted that isoforms B and C have the same length; however, they could be considered different variants because of change in amino acidic composition. Some authors grouped and named variants B and C as isoform 1, which is composed of 592 AA. Isoform 3 is composed of only 557 AA, is the
Table 1 Syndromes with similar phenotype sand overlapping findings; mutations in the EYA1 gene are responsible for three subtypes of those syndromes: BOR1, BOS1 and OFCS. Although these syndromes are often considered to be three different entities, many authors suggest that they should be considered different expressions of the same syndrome. Genes: EYA1, Eyes Absent 1 (drosophila), homolog of; SIX1, SINE OCULIS HOMEOBOX 1 (drosophila), homolog of; SIX5, SINE OCULIS HOMEOBOX 5, drosophila, homolog of; TFAP2A, Transcription FACTOR AP2-Alpha. Syndromes: BOR, Branchio-oto-renal syndrome; BO, Branchio-otic syndrome; OFC, Oto-Facio-Cervical syndrome; BOF, Branchio-oculo-facial syndrome. Syndrome
Molecular genetics
Name
Gene
Locus
MIM number
Inheritance pattern
Branchial defects
Ear defects
Kidney defects
Eye defects
MIM number
EYA1 SIX5 SIX1
8q13.3 19q13.32 14q23.1
601653 600963 601205
Dominant Dominant Dominant
+ + +
+ + +
+ +
+
113650 610896
EYA1 ? SIX1 EYA1 TFAP2A
8q13.3 1q31 14q23.1 8q13.3 6p24.3
601653 – 601205 601653 107580
Dominant Dominant Dominant Dominant Dominant
+ + + + +
+ + + +
+
602588 120502 608389 166780 113620
BOR BOR 1 BOR 2 BOR 3 (?) BO BOS1 BOS2 BOS3 OFC BOF
Phenotype
Please cite this article in press as: A. Castiglione, et al., EYA1-related disorders: Two clinical cases and a literature review, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.032
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PEDOT-7062; No. of Pages 10 A. Castiglione et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx Table 2 Estimated prevalence of EYA1, SIX1 and SIX5 mutations. Prevalence of Branchio-oto-renal spectrum disorders 2–3% of profoundly deaf children
3
Table 4 Phenotypic features of BO/BOR syndrome (Chen et al., 1995). Phenotypic features of BOR syndrome
30–40% with EYA1 mutations 5% with SIX5 mutations 20%) - Hearing loss (93%) - Preauricular pits (82%) - Renal anomalies (67%) - Branchial fistulae (49%) - Pinnae deformities (36%) - External auditory canal stenosis (29%)
Minor features ( T 184C > T 319G > A 321delT 348delA 402C > A 418G > A 430C > T 450_451del 466C > T
T55M P62S G107S A107fs G117fs Y134X G140S Q144X G151fs Q156X
4 4 4 6 6 6 6 5 7 5
BOR (?) BOR BOR BOR BOR BOR BOR BOR BOR BOR
NM_000503 NM_172060 NM_172060 NM_000503 NM_000503 NM_000503 NM_000503.4 NM_172060 NM_000503
Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat. (2011) Krug et al., Hum. Mutat. (2011) Lee et al., Ann. Clin. Lab Sci. (2009) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Kim et al., Mol. Biol. Rep. (2014) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Wang et al., Laryngoscope (2012)
553C > T 586_596dup (+) 636_644delInsTG 592G > T 602C > G 616dupT 634C > T 638A > T
Q185X [S200IfsX12 + S213GfsX112] G198X S201X Y206LfsX50 Q212X Q213L P216L
7 7
BOR BOR
NM_000503 NM_172060
Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011)
8 8 7 8 8 7
BOR BO BOR BOR BOR BOR (?)
NM_000503 NM_000503 NM_172060 NM_000503 NM_000503 NM_172058
639G > C 639 + 1G > C 639 + 2delT 640-15G > A 670delC c.699 + 5G > A 722delC 768C > A 777dupA 783delA 845_852del 851C > G 863_866del 866delA 867 + 5G > A
Q213H Invariant ‘gt’ Invariant ‘gt’ New splice acceptor Q224SfsX109
8 IVS8 IVS8 IVS8 7 IVS10 7 9 9 8 10 10 10 10 IVS8
BOR (?) BOR (?) BOR (?) BOR BOR BOR/BO BOR BO BOR BOR BOR (?) BOR BOR BOR BO/BOR/OFC
NM_000503 NM_000503 NM_000503 NM_000503 NM_172060
867_867 + 14del 868-2A > G 875dupC 920delG 922C > T
R290EfsX43
8 IVS8 10 10 10
BOR BO BOR (?) BOR BOR/BO
NM_172060
Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Hoskins et al., Nephrol Dial. Transplant. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Song et al., PloS ONE (2013) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat. (2011); Stockley et al., Am. J. Med. Genet. A (2009) Krug et al., Hum. Mutat (2011) Kwon et al., Acta Otolaryngol. (2009) Orten et al., Hum. Mutat. (2008) Sanggaard et al., Eur. J. Hum. Genet. (2007) Orten et al., Hum. Mutat. (2008)
965A > G 967-1G > A 977T > A 989A > T 1029delC 1039G > T 1050 + 1G > T 1051-12T > G 1051-1G > C 1081C > T 1100 + 1G > C 1118_1119del 1138_1140 +1delinsCTTC 1140 + 1G > A 1156delC
E322G Invariant ‘ag’ I326 N E330V Y344fs E374X Invariant ‘gt’ New splice acceptor Invariant ‘ag’ R361X
10 IVS10 11 10 11 10 IVS11 IVS11 IVS11 12 IVS11 12 12, IVS12 IVS12 13
BOR/BO BOR BOR (?) BOR BOR (?) BOR BOR BO BO/BOR (?) BOR BOR BO BOR BOR/BO BO
T241KfsX92 Y256X E260fs L262CfsX71 S282fs S284X K288fs D289fs
D293X R307fsX365 R308X
H373fs Invariant ‘gt’ H386fs
NM_172060 NM_000503 NM_000503 NM_172060 NM_000503 NM_000503 NM_000503 NM_000503 NM_172060
NM_000503 NM_005982 NM_000503
NM_000503 NM_000503 NM_172060 NM_000503 NM_172060 NM_000503 NM_000503 NM_000503 NM_000503 NM_172060 NM_000503 NM_000503 NM_000503
Song et al., PloS ONE (2013) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Song et al., PloS ONE (2013) Orten et al., Hum. Mutat. (2008)
Please cite this article in press as: A. Castiglione, et al., EYA1-related disorders: Two clinical cases and a literature review, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.032
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Table 6 (Continued ) Mutation (c.)
AA change (p.)
Exon
Phenotype
Reference
1216_1219dup 1231_1232dupAT 1268delG 1360 + 4 > G 1372_1375dupTCCC 1377_1378 delinsAT 1141-1G > A 1405delG 1420-1421delCC 1425delA 1434dup 1442T > C 1475G > C
R407QfsX13 Y412SfsX24 G423fs G454fsX461 R459LfsX41 K460X E380fsX387 A469fs
12 12 14 15 13 15 13 15 14 14 14 14 15
BOR BOR BO BOR BOR BOR BOR BO BOR BOR BOR BO BOR (?)
NM_172060 NM_172060 NM_000503 NM_005982 NM_172060 NM_000503 NM_005982 NM_000503
15 IVS15 16 16 16 16 15 15 16 16 16 16 Intron 16 17 16 17 17 16 17 16 16 17 17 18 16 16
BOR BOR BOR BOR BO BO BOR BOR BO BOR (?) BOR (?) BOR BOR/BO BOR BO BO BO BOR BOR BO BOR BOR BOR BO BOR BO
NM_000503.4 NM_000503 NM_000503 NM_000503 NM_000503 NM_000503 NM_172060 NM_172060 NM_000503 NM_000503 NM_000503 NM_000503
1475 + 1G > C 1476-2A > G 1496delT 1510C > T 1534G > T 1538T > C 1542_1546delAAAAG 1554T > G 1570G > T 1579T > A 1580A > G 1591A > T 1598-2 A > C 1603_1607del 1607T > C 1644delA 1649T > A 1655dup 1657_1659del 1667-1668insT 1678T > C 1697dupA 1698 + 1G > T 1716G > A 1735delG 1773C > G
L476WfsX9 V479SfsX20 L481P R492P
Invariant ‘ag’ L499X Q504X V512F L513P R514SfsX83 Y518X E524X Y527 N Y527 C K531X E535fs M536 T V549fs V550E H552QfsX47 V553del X560V (+38AA) X560Q H567fs Invariant ‘gt’ W572X D579fs Y591X
showed hearing impairment and renal diseases. Audiological tests revealed a bilateral low-frequency conductive hearing impairment. The young patient presented mild external ear deformities with otoscopically normal tympanic membranes and bilateral normal type A tympanograms and did not show stapedial reflexes in either ears (Figs. 1 and 2). Upon subsequent evaluation we found no progressive worsening in the pure tone threshold, and the boy did not require audiological or speech rehabilitation: during speech tests, he correctly repeated 20/20 disyllabic words in an open set. The unilateral cervical fistula was surgically removed due to recurrent infections. The results from routine blood and renal function tests were within the normal range. No anomalies were detected during the eye examination. Unexpectedly, the young boy showed only a mild bilateral conductive hearing impairment, which only required monitoring. CASE 2: This case is a thirty-year-old male suffering from mild congenital conductive HL in the low frequency range (125 Hz– 1000 Hz). No pre-, peri- or postnatal risk factors for hearing loss were identified. No other family member showed hearing impairment or renal diseases. Audiological tests revealed a bilateral low-frequency conductive hearing impairment, with mild progression over the last five years. The patient presented mild external ear deformities, particularly on the left ear; otoscopically normal tympanic membranes; and bilateral type As tympanograms without stapedial reflexes. The results from routine blood and renal function tests were within the normal range. No anomalies were detected during the eye examination, even if the patient expressed concerns with eye lacrimal ducts. In this case, the cervical fistula was also surgically
NM_172060 NM_172060 NM_172060 NM_000503
NM_000503 NM_172060 NM_000503 NM_000503 NM_172060 NM_000503 NM_172060 NM_172060 NM_000503 NM_000503 NM_000503 NM_005982
Krug et al., Hum. Mutat (2011) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Sanggaard et al., Eur. J. Hum. Genet. (2007) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Sanggaard et al., Eur. J. Hum. Genet. (2007) Orten et al., Hum. Mutat. (2008) Nardi et al., Clin. Nephrol. (2011) Krug et al., Hum. Mutat (2011) Krug et al., Hum. Mutat (2011) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Gigante et al. BMC Nephrol.(2013) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Song et al., 2013 PloS ONE (2013) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Matsunaga et al., Acta Otolaryngol. (2007) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Wang et al., Laryngoscope (2012) Sanggaard et al., Eur. J. Hum. Genet. (2007)
removed during childhood because of recurrent infections. For the same reason, the patient had undergone surgical exportation of preauricular fistula in the right ear (Fig. 3). 3.2. Genetic and molecular analysis CASE 1: Genetic analysis of the first patient revealed a novel sporadic missense mutation in exon 14; the proband’s genotype is heterozygous for the c.1597G > A (NM_000503.4) mutation in the EYA1 gene, corresponding to the substitution G533R of the 592 amino acid isoform. The G533R substitution, previously undescribed, is highly evident of being pathogenic and responsible for signs and symptoms of BO1/BOR1 syndromes. The genotypes of both parents were normal and they showed normal hearing without BO/BOR features. The genotype of the sister (a prenatal diagnosis) was normal. The family tree is shown in Fig. 1. CASE 2: Genetic analysis of the second patient revealed a mutation in exon 16: the proband’s genotype is heterozygous for the c.1777T > A mutation in the EYA1 gene, causing the p.X593QextX6 substitution in the isoform 1 (the 592 AA length isoform). The c.1777T > A mutation has never been described, even though analogous protein substitutions have previously been reported and are known to be related to pathological phenotypes. Furthermore, other genetic variants involving the carboxylterminal tract of EYA1 have been reported with strong evidence to be responsible for the signs and symptoms of BO1/BOR1 syndromes. Other family members showed normal hearing without BO/BOR features (Fig. 3).
Please cite this article in press as: A. Castiglione, et al., EYA1-related disorders: Two clinical cases and a literature review, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.032
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Fig. 1. The first patient showed preauricular pits ((A) black arrow), mild facial asymmetries, external ear malformations, and cup-shaped pinnae with low set ears. (B) Family tree and EYA1 genotypes (in brackets); (C) audiological assessment of the affected proband: tonal audiometry (on the left) shows a mild conductive hearing impairment in both ears, resembling conditions of a fixed hypomobile ossicular chain. Micro – otoscopy: normal in both ears; Rinne test in both ears: Bone Conduction (BC) > Air Conduction (AC); Weber: without lateralization; tympanogram: type A in both ears; stapedial reflexes: absent; tonal/speech audiometry: no dissociation. PTA Bone Conduction (BC) is normal in both ears. Left Air Conduction PTA: 32.5 dBHL; right Air Conduction PTA: 30 dBHL; despite the neuroradiological findings, the patient did not show sensorineural deafness. Legend: O = right air conduction; X = left air conduction;] = right bone conduction; [ = left bone conduction; Hz = Hertz; dBSPL = deciBel Sound Pressure Level; dBHL = deciBel Hearing Level; SRT = speech recognition threshold. (D) EYA1 protein: ClustalW2 multiple amino acid sequence alignment of 13 orthologs (Xenopus laevis, Xenopus Silurana tropicalis, Mus musculus, Sarcophilus harrisii, Pongo belii, Otolemur garnettii, Pan aniscus, Rattus norvegicus, Callithrix jacchus, Strongylocentrotus purpuratus, Macaca mulatta, Danio rerio, Anolis carolinensis); the Glycine (G) at position 533, corresponding to positions 500, 533, 533 and 498 of the human isoforms (EYA1A, EYA1B, EYA1C and EYA1D, respectively), is a highly conserved amino acid in a highly conserved region. The substitution score for Glycine ! Arginine substitution (G533R) is 0.13 (http:// biochem218.stanford.edu/Projects%202001/Yu.pdf).
3.3. Imaging data Interestingly, the two unrelated probands showed similar neuroradiological findings (Figs. 1–3). In particular, CT scanning
revealed various middle and inner ear anomalies: (1) foreshortened dysplastic incus and hypoplastic long process; (2) dilated/patulous Eustachian tube; (3) hypoplastic/dysplastic cochlea with enlarged cochlear aqueducts; (4) hypoplastic/
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Fig. 2. Case 1. Temporal bone CT scan; (A) foreshortened dysplastic incus and hypoplastic long process (white arrows); (B) dilated/patulous Eustachian tube (white asterisk); (C) hypoplastic/dysplastic cochleas (white arrows) with enlarged cochlear aqueducts (white asterisks) and (D) hypoplastic/dysplastic semi-circular canals (white arrow), dilated/bulbous internal auditory canals (white asterisks), facial nerve deviated to the medial side of the cochlea (black arrow) and lateralized in the intratympanic portion (not visible in this slice); left large mastoid emissary vein (black asterisk). (A–D): axial slices.
dysplastic semi-circular canals; (5) dilated/bulbous internal auditory canals; (6) facial nerve deviated to the medial side of the cochlea and lateralized in the intratympanic portion; and (7) large left mastoid emissary vein (Tables 3–5). Sonography revealed normal kidneys and urinary tracts. Both parents underwent renal and urinary tract ultrasound, and the results were interpreted as normal. It is important to note that in our experience, the involvement of the semi-circular canals and ossicular chain is quite constant. 3.4. In silico analysis The evolutionary conservation of EYA1 residues among thirteen EYA1 orthologs was investigated at ClustalW2. The EYA1 gene is conserved in the frog, mouse, Tasmanian devil, orangutan, chimpanzee, monkey, rat, purple urchin, macaque, zebrafish and lizard. This gene encodes a member of the eyes absent (EYA) family of proteins. The bioinformatic analysis conducted assigned a 99% probability that the amino acid substitution G533R is pathogenic. The Glycine to Arginine substitution, corresponding to positions
500, 533, 533 and 498 of the human isoforms (EYA1A, EYA1B, EYA1C and EYA1D), respectively, is a significant change indicated by a substitution score of 0.13. The results are compatible with a disease-causing mutation (Fig. 1) with a dominant inheritance pattern. The C-terminal region of the protein is also highly conserved and amino acidic substitution of this region may be considered as a pathogenic variation. To the best of our knowledge, the p.X593QextX6 substitution in exon 16 of isoform 1 (592 AA) is responsible for signs and symptoms of a dominant inheritance pattern (Fig. 3): similar mutations involving the terminal peptidic sequence were associated with BO/BOR phenotypes. 3.5. Literature review A total of 323 articles were including in our analysis, 30 of which best matched our inclusion criteria. Novel mutations that we found in the literature (not yet reported at http://www.healthcare.uiowa.edu/labs/pendredandbor/, last update December 1st, 2013) are shown in Table 6 [1,13–22].
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Fig. 3. Case 2. Left (A) and right (B) ear of the second patient. Note that external ear malformations are not directly related with middle and inner malformation entity and are also not directly predictable for hearing impairment. (C) Coronal CT scan showing ossicular chain defects; (D) axial CT scan of the left ear showing bulbous inner ear canal and hypoplastic semi-circular canals; (E) family tree of the proband and (F) tonal and speech audiometry.
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4. Discussion and conclusions In the present study, we present two cases of EYA1-related branchiotorenal spectrum disorders. The first case, a young boy affected by mild bilateral conductive hearing loss (not due to otitis), preauricular pits and mild deformities of the external auditory canal, presented conditions that may be sufficient to require additional genetic and imaging investigations to achieve an accurate diagnosis. Using PCR and sequencing, we identified one novel sporadic mutation in the EYA1 gene: c.1597G > A, causing the G533R substitution in the isoform 1 (592 aa). This mutation appears to display a dominant inheritance pattern. Clinical findings and in silico analysis supported the pathogenic role of the new variant.
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In the second clinical case, we found the c. 1777T > A mutation, causing the p.X593QextX6 substitution, a C-terminal variant which is known to be involved in branchio-otorenal spectrum disorders. Therefore, mutations in the EYA1 gene are responsible for similar disorders with variable phenotypes: BOR syndrome (subtypes 1), BO syndrome (subtype 1) and OFC syndrome. These conditions can be referred to as EYA1-related disorders and should be included in branchiotorenal spectrum disorders. The present results and previously reported mutations in the EYA1 gene demonstrate a great variety, including nonsense mutations, deletions and insertions, resulting in frameshifts, aberrant splicing and deletions. It is thought that these mutation types have unpredictable phenotypes (Table 6). Moreover, available data about genotype-phenotype
Fig. 4. EYA1-related disorders: diagnostic flow chart.
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correlations in clinical studies are limited, especially those concerning the most common symptom: hearing loss. It has been reported that hypoacusis may be either conductive (30% of cases), sensorineural (20% of cases) or mixed (50% of cases), ranging from mild to profound in degree. Imaging evaluations should include renal function tests, kidney/urinary tract ultrasound and voiding cystourethrogram (with contrast) to exclude vesicoureteral reflux. Generally, this exam is not routinely performed due to the X-ray exposure and contrast use, particularly in young patients. In such cases, as well in case of contraindication to the contrast use, additional urine/serum analyses may be useful. The following conditions must be investigated: (1) renal agenesis, hypoplasia or dysplasia; (2) uretero-pelvic junction (UPJ) obstruction; (3) calyceal cyst/diverticulum; and (4) calyectasis, pelviectasis, hydronephrosis and vesicoureteral reflux. Even if the renal involvement is not always present (13–67% of affected), when a renal dysfunction of any degree is documented, it must be monitored and eventually treated. This is because it could be responsible for severe renal failure, leading to renal transplantation. A temporal bone CT scan without contrast is mandatory to study the middle and internal ear structures. A temporal bone MRI (without contrast) may be necessary to complete the imaging assessment. Interestingly, the entity of external ear malformations does not relate to inner and middle ear malformations, and the entity of hearing loss is not directly proportional to the neuroradiological findings. In summary, an effective clinical management effort should consider: (1) surgery to treat branchial defects (cleft cysts, cleft palate, fistulae or sinuses) and, when possible, hearing loss (by canaloplasty, ossiculoplasty or cochlear implantation); (2) an appropriate auditory habilitation/rehabilitation; (3) medical and surgical treatment for renal abnormalities; (4) neuroimaging; (5) genetic investigation; (6) monitoring (semiannual/annual examinations especially to assess stability of hearing loss and renal function); (7) screening of relatives for hearing loss and renal involvement; and, finally, (8) nephrotoxic and ototoxic agents should be avoided. Regardless of the potential necessity of further studies to establish prognostic conditions or genotype/phenotype correlations, the syndrome subtypes are generally associated with different clinical features; an accurate differential diagnosis may contribute to defining the evolution or progression of symptoms. Furthermore, it is important to note that in our cases, the neuroimaging pictures were not directly proportional to the audiological pictures. Similar conditions are expected to have severe/profound hearing impairment. In conclusion, these results add to the spectrum of EYA1-related disorders and indicate that the BO/BOR/BOU/OFC/BOF phenotypes are an indication for additional molecular studies. Nevertheless, a comprehensive multidisciplinary approach is mandatory to obtain an accurate differential diagnosis (Fig. 4). 5. Consent Written informed consent was obtained from the patients and other family members according to the current national rules and laws for the publication of these cases and any accompanying images. Competing interests The authors declare that they have no competing interests.
Web resources The URLs for data presented herein are as follows: Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm. ih.gov/Omim/, http://www.healthcare.uiowa.edu/labs/pendredandbor/, http://www.genecards.org/ and http://www.uniprot.org/. References [1] D.J. Orten, S.M. Fischer, J.L. Sorensen, U. Radhakrishna, C.W. Cremers, H.A. Marres, et al., Branchio-oto-renal syndrome (BOR): novel mutations in the EYA1 gene, and a review of the mutational genetics of BOR, Hum. Mutat. 29 (4) (2008) 537–544. [2] S. Abdelhak, V. Kalatzis, R. Heilig, S. Compain, D. Samson, C. Vincent, et al., A human homologue of the Drosophila eyes absent gene underlies branchiooto-renal (BOR) syndrome and identifies a novel gene family, Nat. Genet. 15 (2) (1997) 157–164. [3] M.A. Mosrati, B. Hammami, I.B. Rebeh, L. Ayadi, L. Dhouib, K. Ben Mahfoudh, et al., A novel dominant mutation in SIX1, affecting a highly conserved residue, result in only auditory defects in humans, Eur. J. Med. Genet. 54 (5) (2011) e484–e488. [4] S.H. Wang, C.C. Wu, Y.C. Lu, Y.H. Lin, Y.N. Su, W.L. Hwu, et al., Mutation screening of the EYA1, SIX1, and SIX5 genes in an East Asian cohort with branchio-oto-renal syndrome, Laryngoscope 122 (5) (2012) 1130–1136. [5] A.A. Salam, F.M. Hafner, T.E. Linder, T. Spillmann, A.A. Schinzel, S.M. Leal, A novel locus (DFNA23) for prelingual autosomal dominant nonsyndromic hearing loss maps to 14q21-q22 in a Swiss German kindred, Am. J. Hum. Genet. 66 (6) (2000) 1984–1988. [6] T. Trummer, D. Muller, A. Schulze, W. Vogel, W. Just, Branchio-oculo-facial syndrome and branchio-otic/branchio-oto-renal syndromes are distinct entities, J. Med. Genet. 39 (1) (2002) 71–73. [7] A. Castiglione, M. Busi, A. Martini, Syndromic hearing loss: an update, Hearing, Balance Commun. 11 (3) (2013) 146–159. [8] B.E. Hoskins, C.H. Cramer 2nd, V. Tasic, E.O. Kehinde, S. Ashraf, R. Bogdanovic, et al., Missense mutations in EYA1 and TCF2 are a rare cause of urinary tract malformations, Nephrol. Dial. Transplant.: Off. Publ. Eur. Dial. Transplant Assoc. – Eur. Ren. Assoc. 23 (2) (2008) 777–779. [9] H. Ohto, S. Kamada, K. Tago, S.I. Tominaga, H. Ozaki, S. Sato, et al., Cooperation of six and eya in activation of their target genes through nuclear translocation of Eya, Mol. Cell. Biol. 19 (10) (1999) 6815–6824. [10] E.H. Chang, M. Menezes, N.C. Meyer, R.A. Cucci, V.S. Vervoort, C.E. Schwartz, et al., Branchio-oto-renal syndrome: the mutation spectrum in EYA1 and its phenotypic consequences, Hum. Mutat. 23 (6) (2004) 582–589. [11] S. Ceruti, C. Stinckens, C.W. Cremers, J.W. Casselman, Temporal bone anomalies in the branchio-oto-renal syndrome: detailed computed tomographic and magnetic resonance imaging findings, Otol. Neurotol. 23 (2) (2002) 200–207. [12] E. Senel, B.N. Gulleroglu, S. Senel, Additional temporal bone findings on computed tomography imaging in branchio-oto-renal syndrome, Laryngoscope 119 (4) (2009) 832. [13] P. Krug, V. Moriniere, S. Marlin, V. Koubi, H.D. Gabriel, E. Colin, et al., Mutation screening of the EYA1, SIX1, and SIX5 genes in a large cohort of patients harboring branchio-oto-renal syndrome calls into question the pathogenic role of SIX5 mutations, Hum. Mutat. 32 (2) (2011) 183–190. [14] J.D. Lee, S.C. Kim, Y.W. Koh, H.J. Lee, S.Y. Choi, U.K. Kim, A novel frameshift mutation in the EYA1 gene in a Korean family with branchio-oto-renal syndrome, Ann. Clin. Lab. Sci. 39 (3) (2009) 303–306. [15] T.L. Stockley, R. Mendoza-Londono, E.J. Propst, S. Sodhi, L. Dupuis, B.C. Papsin, A recurrent EYA1 mutation causing alternative RNA splicing in branchio-oto-renal syndrome: implications for molecular diagnostics and disease mechanism, Am. J. Med. Genet. Part A 149 (3) (2009) 322–327. [16] E. Nardi, A. Palermo, P. Cusimano, G. Mule, G. Cerasola, Young woman with branchio-oto-renal syndrome and a novel mutation in the EYA-1 gene, Clin. Nephrol. 76 (4) (2011) 330–333. [17] M.J. Kwon, S.H. Boo, H.J. Kim, Y.S. Cho, W.H. Chung, S.H. Hong, A novel splice site mutation in the EYA1 gene in a Korean family with branchio-oto (BO) syndrome, Acta Otolaryngol. 129 (6) (2009) 688–693. [18] T. Matsunaga, M. Okada, S. Usami, T. Okuyama, Phenotypic consequences in a Japanese family having branchio-oto-renal syndrome with a novel frameshift mutation in the gene EYA1, Acta Otolaryngol. 127 (1) (2007) 98–104. [19] M. Gigante, M. d’Altilia, E. Montemurno, S. Diella, F. Bruno, G.S. Netti, et al., Branchio-Oto-Renal Syndrome (BOR) associated with focal glomerulosclerosis in a patient with a novel EYA1 splice site mutation, BMC Nephrol. 14 (2013) 60. [20] K.M. Sanggaard, N.D. Rendtorff, K.W. Kjaer, H. Eiberg, T. Johnsen, S. Gimsing, et al., Branchio-oto-renal 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.: EJHG 15 (11) (2007) 1121–1131. [21] M.H. Song, T.J. Kwon, H.R. Kim, J.H. Jeon, J.I. Baek, W.S. Lee, et al., Mutational analysis of EYA1, SIX1 and SIX5 genes and strategies for management of hearing loss in patients with BOR/BO syndrome, PloS ONE 8 (6) (2013) e67236. [22] H.R. Kim, M.H. Song, M.A. Kim, Y.R. Kim, K.Y. Lee, J.K. Sonn, et al., Identification of a novel nonsynonymous mutation of EYA1 disrupting splice site in a Korean patient with BOR syndrome, Mol. Biol. Rep. (2014).
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International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Review Article
EYA1-related disorders: Two clinical cases and a literature review Alessandro Castiglione a,*, Salvatore Melchionda b, Massimo Carella b, Patrizia Trevisi a, Roberto Bovo c, Renzo Manara d, Alessandro Martini e a
Department of Neurosciences, University of Padua, Via Giustiniani, 2, Padua, Italy Unit of Medical Genetics, IRCCS, ‘‘Casa Sollievo della Sofferenza’’ Hospital, 71013 San Giovanni Rotondo, Italy c Operative Unit of Otolaryngology and Otosurgery, University of Padua, Via Giustiniani, 2, Padua, Italy d Neuroradiologic Unit, Padua University, Via Giustiniani, 2, Padua, Italy e Department of Neurosciences, Operative Unit of Otolaryngology and Otosurgery, University of Padua, Via Giustiniani, 2, Padua, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2013 Received in revised form 27 March 2014 Accepted 29 March 2014 Available online xxx
Objectives: To delineate the diagnostic and rehabilitative aspects of syndromes that have overlapping features, we present the cases of two unrelated Caucasian males affected by hearing impairment, preauricular pits and cervical fistulae. Specific findings that are helpful in the diagnosis and management of EYA1-related disorders are highlighted. Methods: Genetic, otologic, imaging, eye and renal evaluations were conducted to achieve a detailed and comprehensive assessment, leading to the most accurate diagnosis and appropriate treatment. A literature review was also carried out. Results: Diagnostic criteria indicated that the two patients were affected by BOS1 (Branchio-Otic Syndrome 1). We also identified a novel sporadic missense mutation in the EYA1 gene: p.G533R (c.1597G > A, NM_000503.4), a highly conserved, heterozygotic amino acid substitution. In the other case, we identified the p.X593QextX6 (c.1777T > A, NM_000503.4) substitution, known to be responsible for BO/BOR phenotypes. Both substitutions lead to isoform 1 (EYA1B and EYA1C) which is composed of 592 amino acids. Clinical and in silico evidence suggests a pathogenic role for the new, never before described p.G533R mutation. Imaging evaluation revealed a complex pathology, characterized by external, inner and middle ear malformations, without renal anomalies. Conclusions: Our results demonstrate the importance of considering the imaging evaluation and the complete DNA sequencing of the EYA1 gene for the differential diagnosis of deafness and related branchio-oto-renal disorders. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Branchio-oto-renal Branchio-otic Oto-Facio-Cervical Branchio-oto-ureteral Hearing loss EYA1 mutations
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Introduction . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . Clinical and audiological data . 2.1. Genetic and molecular analysis 2.2. Imaging data . . . . . . . . . . . . . . . 2.3. In silico analysis . . . . . . . . . . . . 2.4. Literature review . . . . . . . . . . . 2.5. Results . . . . . . . . . . . . . . . . . . . . . . . . . Clinical and audiological data . 3.1. Genetic and molecular analysis 3.2.
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* Corresponding author at: Department of Neurosciences, Operative Unit of Otolaryngology and Otosurgery, 2, Giustiniani, Padua 35100, Italy. Tel.: +39 328 6181047; fax: +39 049 821 1994. E-mail addresses: [email protected], [email protected] (A. Castiglione), [email protected] (S. Melchionda), [email protected] (M. Carella), [email protected] (P. Trevisi), [email protected] (R. Bovo), [email protected] (R. Manara), [email protected] (A. Martini).
http://dx.doi.org/10.1016/j.ijporl.2014.03.032 0165-5876/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: A. Castiglione, et al., EYA1-related disorders: Two clinical cases and a literature review, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.032
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3.3. Imaging data . . . . . . . In silico analysis . . . . 3.4. Literature review . . . 3.5. Discussion and conclusions . Consent . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .
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000 000 000 000 000 000
shortest isoform and corresponds to the variant EYA1D. Isoform 2 is reported to be composed of 559 amino acids and is widely known as variant EYA1A. This classification is not clear or helpful in the definition and collection of EYA1 mutations. Additionally, many allelic variants or polymorphisms are consistently reported during clinical practice and have been supported by genetic investigations. To avoid misunderstandings, it would be helpful to directly refer to the amino acidic length of the different isoforms or variants. To date, more than 160 pathologic mutations in the EYA1 gene have been reported in the literature. The EYA1 protein may be required for the normal development of branchial arches, ears and kidneys. The expression pattern of the murine EYA1 ortholog, Eya1, suggests a role in the development of all components of the inner ear, from the emergence of the otic placode. The expression pattern in the developing kidney indicates a role for Eya1 in the metanephric cells surrounding the ‘just-divided’ ureteric branches [2,8]. The EYA1 gene product is a tyrosine phosphatase that specifically dephosphorylates Tyr-142 of histone H2AX (H2AXY142ph). Tyr-142 phosphorylation of histone H2AX plays a central role in DNA repair and acts as a mark that distinguishes between apoptotic and repair responses to genotoxic stress. Its function as a histone phosphatase likely explains its role in transcription regulation during organogenesis. EYA1 appears to coactivate SIX2, SIX4 and SIX5 [2]. The structure of eya proteins includes the eya homologous region (eyaHR), a conserved carboxyl-terminal region, and the eya variable region (eyaVR), a divergent transactivation domain rich in tripeptide PST (proline– serine–threonine) [13]. The vertebrate orthologs of so (sine oculis) are six genes (SIX1, SIX2, SIX3, SIX4, SIX5, SIX6) and their protein products bind to eya proteins, inducing the nuclear translocation of the complex [9]. From a clinical standpoint, hearing loss is the most common and constant feature of EYA1 mutation and is detected in more than 90% of the affected individuals (Tables 3–5). In Caucasian populations, common EYA1 mutations are p.R440Q, c.867 + 5G > A and p.R297X [1–3,5–7,9]. There is no distinct relationships observed between the nature of the mutations and
1. Introduction Branchio-oto-renal (BOR), Branchio-otic (BO), Branchio-otoureteral (BOU) and Oto-Facio-Cervical (OFC) syndromes are dominant disorders characterized by variable hearing impairment (HI) and branchial defects [1–3]. BOR and BOU syndromes include additional kidney and urinary tract anomalies. All of these syndromes are genetically heterogeneous and caused by mutations in the EYA1, SIX1, and SIX5 genes [3,4]. The involvement of different genes was conducted to define different syndrome subtypes, without wide clinical variations (Table 1). It was also reported that DFNA23, a locus for non-syndromic hearing impairment, is due to mutations in SIX1 [5]. Nevertheless, mutations in the same gene can lead to different syndromes even if it is unclear why some EYA1 mutations are associated with kidney abnormalities and others are not. It was also previously thought that EYA1 mutations may be responsible for BranchioOculo-Facial (BOF) syndrome (MIM#113620) because of the presence of overlapping features; it is now clear that BOF syndrome is due to mutations in the TFAP2A gene (Table. 1) [6]. Approximately 40 percent of people with BO, BOR, BOU or OFC syndromes have mutations in the EYA1 gene. The prevalence of EYA1-related disorders is approximately 2.5:100,000 newborns and require a multidisciplinary approach in diagnosis, management and treatment (Tables 2–4) [7]. The EYA1 (Eyes absent homolog 1) gene is the human homologue of the Drosophila Eya1 gene, which is essential for eye development in that species. In humans, it consists of 16 coding exons and has been localized to chromosome 8q13.3 [1–3]. To date, at least 4 different isoforms are known (EYA1A, EYA1B, EYA1C, EYA1D) and are composed of 559 amino acids (AA), 592 AA, 592 AA and 557 AA, respectively. However, the general acceptance of these isoforms remains lacking because of additional transcriptional variants and an inconsistent definition in specialized online databases. It should be noted that isoforms B and C have the same length; however, they could be considered different variants because of change in amino acidic composition. Some authors grouped and named variants B and C as isoform 1, which is composed of 592 AA. Isoform 3 is composed of only 557 AA, is the
Table 1 Syndromes with similar phenotype sand overlapping findings; mutations in the EYA1 gene are responsible for three subtypes of those syndromes: BOR1, BOS1 and OFCS. Although these syndromes are often considered to be three different entities, many authors suggest that they should be considered different expressions of the same syndrome. Genes: EYA1, Eyes Absent 1 (drosophila), homolog of; SIX1, SINE OCULIS HOMEOBOX 1 (drosophila), homolog of; SIX5, SINE OCULIS HOMEOBOX 5, drosophila, homolog of; TFAP2A, Transcription FACTOR AP2-Alpha. Syndromes: BOR, Branchio-oto-renal syndrome; BO, Branchio-otic syndrome; OFC, Oto-Facio-Cervical syndrome; BOF, Branchio-oculo-facial syndrome. Syndrome
Molecular genetics
Name
Gene
Locus
MIM number
Inheritance pattern
Branchial defects
Ear defects
Kidney defects
Eye defects
MIM number
EYA1 SIX5 SIX1
8q13.3 19q13.32 14q23.1
601653 600963 601205
Dominant Dominant Dominant
+ + +
+ + +
+ +
+
113650 610896
EYA1 ? SIX1 EYA1 TFAP2A
8q13.3 1q31 14q23.1 8q13.3 6p24.3
601653 – 601205 601653 107580
Dominant Dominant Dominant Dominant Dominant
+ + + + +
+ + + +
+
602588 120502 608389 166780 113620
BOR BOR 1 BOR 2 BOR 3 (?) BO BOS1 BOS2 BOS3 OFC BOF
Phenotype
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PEDOT-7062; No. of Pages 10 A. Castiglione et al. / International Journal of Pediatric Otorhinolaryngology xxx (2014) xxx–xxx Table 2 Estimated prevalence of EYA1, SIX1 and SIX5 mutations. Prevalence of Branchio-oto-renal spectrum disorders 2–3% of profoundly deaf children
3
Table 4 Phenotypic features of BO/BOR syndrome (Chen et al., 1995). Phenotypic features of BOR syndrome
30–40% with EYA1 mutations 5% with SIX5 mutations 20%) - Hearing loss (93%) - Preauricular pits (82%) - Renal anomalies (67%) - Branchial fistulae (49%) - Pinnae deformities (36%) - External auditory canal stenosis (29%)
Minor features ( T 184C > T 319G > A 321delT 348delA 402C > A 418G > A 430C > T 450_451del 466C > T
T55M P62S G107S A107fs G117fs Y134X G140S Q144X G151fs Q156X
4 4 4 6 6 6 6 5 7 5
BOR (?) BOR BOR BOR BOR BOR BOR BOR BOR BOR
NM_000503 NM_172060 NM_172060 NM_000503 NM_000503 NM_000503 NM_000503.4 NM_172060 NM_000503
Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat. (2011) Krug et al., Hum. Mutat. (2011) Lee et al., Ann. Clin. Lab Sci. (2009) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Kim et al., Mol. Biol. Rep. (2014) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Wang et al., Laryngoscope (2012)
553C > T 586_596dup (+) 636_644delInsTG 592G > T 602C > G 616dupT 634C > T 638A > T
Q185X [S200IfsX12 + S213GfsX112] G198X S201X Y206LfsX50 Q212X Q213L P216L
7 7
BOR BOR
NM_000503 NM_172060
Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011)
8 8 7 8 8 7
BOR BO BOR BOR BOR BOR (?)
NM_000503 NM_000503 NM_172060 NM_000503 NM_000503 NM_172058
639G > C 639 + 1G > C 639 + 2delT 640-15G > A 670delC c.699 + 5G > A 722delC 768C > A 777dupA 783delA 845_852del 851C > G 863_866del 866delA 867 + 5G > A
Q213H Invariant ‘gt’ Invariant ‘gt’ New splice acceptor Q224SfsX109
8 IVS8 IVS8 IVS8 7 IVS10 7 9 9 8 10 10 10 10 IVS8
BOR (?) BOR (?) BOR (?) BOR BOR BOR/BO BOR BO BOR BOR BOR (?) BOR BOR BOR BO/BOR/OFC
NM_000503 NM_000503 NM_000503 NM_000503 NM_172060
867_867 + 14del 868-2A > G 875dupC 920delG 922C > T
R290EfsX43
8 IVS8 10 10 10
BOR BO BOR (?) BOR BOR/BO
NM_172060
Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Hoskins et al., Nephrol Dial. Transplant. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Song et al., PloS ONE (2013) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat. (2011); Stockley et al., Am. J. Med. Genet. A (2009) Krug et al., Hum. Mutat (2011) Kwon et al., Acta Otolaryngol. (2009) Orten et al., Hum. Mutat. (2008) Sanggaard et al., Eur. J. Hum. Genet. (2007) Orten et al., Hum. Mutat. (2008)
965A > G 967-1G > A 977T > A 989A > T 1029delC 1039G > T 1050 + 1G > T 1051-12T > G 1051-1G > C 1081C > T 1100 + 1G > C 1118_1119del 1138_1140 +1delinsCTTC 1140 + 1G > A 1156delC
E322G Invariant ‘ag’ I326 N E330V Y344fs E374X Invariant ‘gt’ New splice acceptor Invariant ‘ag’ R361X
10 IVS10 11 10 11 10 IVS11 IVS11 IVS11 12 IVS11 12 12, IVS12 IVS12 13
BOR/BO BOR BOR (?) BOR BOR (?) BOR BOR BO BO/BOR (?) BOR BOR BO BOR BOR/BO BO
T241KfsX92 Y256X E260fs L262CfsX71 S282fs S284X K288fs D289fs
D293X R307fsX365 R308X
H373fs Invariant ‘gt’ H386fs
NM_172060 NM_000503 NM_000503 NM_172060 NM_000503 NM_000503 NM_000503 NM_000503 NM_172060
NM_000503 NM_005982 NM_000503
NM_000503 NM_000503 NM_172060 NM_000503 NM_172060 NM_000503 NM_000503 NM_000503 NM_000503 NM_172060 NM_000503 NM_000503 NM_000503
Song et al., PloS ONE (2013) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Song et al., PloS ONE (2013) Orten et al., Hum. Mutat. (2008)
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Table 6 (Continued ) Mutation (c.)
AA change (p.)
Exon
Phenotype
Reference
1216_1219dup 1231_1232dupAT 1268delG 1360 + 4 > G 1372_1375dupTCCC 1377_1378 delinsAT 1141-1G > A 1405delG 1420-1421delCC 1425delA 1434dup 1442T > C 1475G > C
R407QfsX13 Y412SfsX24 G423fs G454fsX461 R459LfsX41 K460X E380fsX387 A469fs
12 12 14 15 13 15 13 15 14 14 14 14 15
BOR BOR BO BOR BOR BOR BOR BO BOR BOR BOR BO BOR (?)
NM_172060 NM_172060 NM_000503 NM_005982 NM_172060 NM_000503 NM_005982 NM_000503
15 IVS15 16 16 16 16 15 15 16 16 16 16 Intron 16 17 16 17 17 16 17 16 16 17 17 18 16 16
BOR BOR BOR BOR BO BO BOR BOR BO BOR (?) BOR (?) BOR BOR/BO BOR BO BO BO BOR BOR BO BOR BOR BOR BO BOR BO
NM_000503.4 NM_000503 NM_000503 NM_000503 NM_000503 NM_000503 NM_172060 NM_172060 NM_000503 NM_000503 NM_000503 NM_000503
1475 + 1G > C 1476-2A > G 1496delT 1510C > T 1534G > T 1538T > C 1542_1546delAAAAG 1554T > G 1570G > T 1579T > A 1580A > G 1591A > T 1598-2 A > C 1603_1607del 1607T > C 1644delA 1649T > A 1655dup 1657_1659del 1667-1668insT 1678T > C 1697dupA 1698 + 1G > T 1716G > A 1735delG 1773C > G
L476WfsX9 V479SfsX20 L481P R492P
Invariant ‘ag’ L499X Q504X V512F L513P R514SfsX83 Y518X E524X Y527 N Y527 C K531X E535fs M536 T V549fs V550E H552QfsX47 V553del X560V (+38AA) X560Q H567fs Invariant ‘gt’ W572X D579fs Y591X
showed hearing impairment and renal diseases. Audiological tests revealed a bilateral low-frequency conductive hearing impairment. The young patient presented mild external ear deformities with otoscopically normal tympanic membranes and bilateral normal type A tympanograms and did not show stapedial reflexes in either ears (Figs. 1 and 2). Upon subsequent evaluation we found no progressive worsening in the pure tone threshold, and the boy did not require audiological or speech rehabilitation: during speech tests, he correctly repeated 20/20 disyllabic words in an open set. The unilateral cervical fistula was surgically removed due to recurrent infections. The results from routine blood and renal function tests were within the normal range. No anomalies were detected during the eye examination. Unexpectedly, the young boy showed only a mild bilateral conductive hearing impairment, which only required monitoring. CASE 2: This case is a thirty-year-old male suffering from mild congenital conductive HL in the low frequency range (125 Hz– 1000 Hz). No pre-, peri- or postnatal risk factors for hearing loss were identified. No other family member showed hearing impairment or renal diseases. Audiological tests revealed a bilateral low-frequency conductive hearing impairment, with mild progression over the last five years. The patient presented mild external ear deformities, particularly on the left ear; otoscopically normal tympanic membranes; and bilateral type As tympanograms without stapedial reflexes. The results from routine blood and renal function tests were within the normal range. No anomalies were detected during the eye examination, even if the patient expressed concerns with eye lacrimal ducts. In this case, the cervical fistula was also surgically
NM_172060 NM_172060 NM_172060 NM_000503
NM_000503 NM_172060 NM_000503 NM_000503 NM_172060 NM_000503 NM_172060 NM_172060 NM_000503 NM_000503 NM_000503 NM_005982
Krug et al., Hum. Mutat (2011) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Sanggaard et al., Eur. J. Hum. Genet. (2007) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Sanggaard et al., Eur. J. Hum. Genet. (2007) Orten et al., Hum. Mutat. (2008) Nardi et al., Clin. Nephrol. (2011) Krug et al., Hum. Mutat (2011) Krug et al., Hum. Mutat (2011) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Gigante et al. BMC Nephrol.(2013) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Song et al., 2013 PloS ONE (2013) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Matsunaga et al., Acta Otolaryngol. (2007) Krug et al., Hum. Mutat (2011) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Orten et al., Hum. Mutat. (2008) Wang et al., Laryngoscope (2012) Sanggaard et al., Eur. J. Hum. Genet. (2007)
removed during childhood because of recurrent infections. For the same reason, the patient had undergone surgical exportation of preauricular fistula in the right ear (Fig. 3). 3.2. Genetic and molecular analysis CASE 1: Genetic analysis of the first patient revealed a novel sporadic missense mutation in exon 14; the proband’s genotype is heterozygous for the c.1597G > A (NM_000503.4) mutation in the EYA1 gene, corresponding to the substitution G533R of the 592 amino acid isoform. The G533R substitution, previously undescribed, is highly evident of being pathogenic and responsible for signs and symptoms of BO1/BOR1 syndromes. The genotypes of both parents were normal and they showed normal hearing without BO/BOR features. The genotype of the sister (a prenatal diagnosis) was normal. The family tree is shown in Fig. 1. CASE 2: Genetic analysis of the second patient revealed a mutation in exon 16: the proband’s genotype is heterozygous for the c.1777T > A mutation in the EYA1 gene, causing the p.X593QextX6 substitution in the isoform 1 (the 592 AA length isoform). The c.1777T > A mutation has never been described, even though analogous protein substitutions have previously been reported and are known to be related to pathological phenotypes. Furthermore, other genetic variants involving the carboxylterminal tract of EYA1 have been reported with strong evidence to be responsible for the signs and symptoms of BO1/BOR1 syndromes. Other family members showed normal hearing without BO/BOR features (Fig. 3).
Please cite this article in press as: A. Castiglione, et al., EYA1-related disorders: Two clinical cases and a literature review, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.032
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Fig. 1. The first patient showed preauricular pits ((A) black arrow), mild facial asymmetries, external ear malformations, and cup-shaped pinnae with low set ears. (B) Family tree and EYA1 genotypes (in brackets); (C) audiological assessment of the affected proband: tonal audiometry (on the left) shows a mild conductive hearing impairment in both ears, resembling conditions of a fixed hypomobile ossicular chain. Micro – otoscopy: normal in both ears; Rinne test in both ears: Bone Conduction (BC) > Air Conduction (AC); Weber: without lateralization; tympanogram: type A in both ears; stapedial reflexes: absent; tonal/speech audiometry: no dissociation. PTA Bone Conduction (BC) is normal in both ears. Left Air Conduction PTA: 32.5 dBHL; right Air Conduction PTA: 30 dBHL; despite the neuroradiological findings, the patient did not show sensorineural deafness. Legend: O = right air conduction; X = left air conduction;] = right bone conduction; [ = left bone conduction; Hz = Hertz; dBSPL = deciBel Sound Pressure Level; dBHL = deciBel Hearing Level; SRT = speech recognition threshold. (D) EYA1 protein: ClustalW2 multiple amino acid sequence alignment of 13 orthologs (Xenopus laevis, Xenopus Silurana tropicalis, Mus musculus, Sarcophilus harrisii, Pongo belii, Otolemur garnettii, Pan aniscus, Rattus norvegicus, Callithrix jacchus, Strongylocentrotus purpuratus, Macaca mulatta, Danio rerio, Anolis carolinensis); the Glycine (G) at position 533, corresponding to positions 500, 533, 533 and 498 of the human isoforms (EYA1A, EYA1B, EYA1C and EYA1D, respectively), is a highly conserved amino acid in a highly conserved region. The substitution score for Glycine ! Arginine substitution (G533R) is 0.13 (http:// biochem218.stanford.edu/Projects%202001/Yu.pdf).
3.3. Imaging data Interestingly, the two unrelated probands showed similar neuroradiological findings (Figs. 1–3). In particular, CT scanning
revealed various middle and inner ear anomalies: (1) foreshortened dysplastic incus and hypoplastic long process; (2) dilated/patulous Eustachian tube; (3) hypoplastic/dysplastic cochlea with enlarged cochlear aqueducts; (4) hypoplastic/
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Fig. 2. Case 1. Temporal bone CT scan; (A) foreshortened dysplastic incus and hypoplastic long process (white arrows); (B) dilated/patulous Eustachian tube (white asterisk); (C) hypoplastic/dysplastic cochleas (white arrows) with enlarged cochlear aqueducts (white asterisks) and (D) hypoplastic/dysplastic semi-circular canals (white arrow), dilated/bulbous internal auditory canals (white asterisks), facial nerve deviated to the medial side of the cochlea (black arrow) and lateralized in the intratympanic portion (not visible in this slice); left large mastoid emissary vein (black asterisk). (A–D): axial slices.
dysplastic semi-circular canals; (5) dilated/bulbous internal auditory canals; (6) facial nerve deviated to the medial side of the cochlea and lateralized in the intratympanic portion; and (7) large left mastoid emissary vein (Tables 3–5). Sonography revealed normal kidneys and urinary tracts. Both parents underwent renal and urinary tract ultrasound, and the results were interpreted as normal. It is important to note that in our experience, the involvement of the semi-circular canals and ossicular chain is quite constant. 3.4. In silico analysis The evolutionary conservation of EYA1 residues among thirteen EYA1 orthologs was investigated at ClustalW2. The EYA1 gene is conserved in the frog, mouse, Tasmanian devil, orangutan, chimpanzee, monkey, rat, purple urchin, macaque, zebrafish and lizard. This gene encodes a member of the eyes absent (EYA) family of proteins. The bioinformatic analysis conducted assigned a 99% probability that the amino acid substitution G533R is pathogenic. The Glycine to Arginine substitution, corresponding to positions
500, 533, 533 and 498 of the human isoforms (EYA1A, EYA1B, EYA1C and EYA1D), respectively, is a significant change indicated by a substitution score of 0.13. The results are compatible with a disease-causing mutation (Fig. 1) with a dominant inheritance pattern. The C-terminal region of the protein is also highly conserved and amino acidic substitution of this region may be considered as a pathogenic variation. To the best of our knowledge, the p.X593QextX6 substitution in exon 16 of isoform 1 (592 AA) is responsible for signs and symptoms of a dominant inheritance pattern (Fig. 3): similar mutations involving the terminal peptidic sequence were associated with BO/BOR phenotypes. 3.5. Literature review A total of 323 articles were including in our analysis, 30 of which best matched our inclusion criteria. Novel mutations that we found in the literature (not yet reported at http://www.healthcare.uiowa.edu/labs/pendredandbor/, last update December 1st, 2013) are shown in Table 6 [1,13–22].
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Fig. 3. Case 2. Left (A) and right (B) ear of the second patient. Note that external ear malformations are not directly related with middle and inner malformation entity and are also not directly predictable for hearing impairment. (C) Coronal CT scan showing ossicular chain defects; (D) axial CT scan of the left ear showing bulbous inner ear canal and hypoplastic semi-circular canals; (E) family tree of the proband and (F) tonal and speech audiometry.
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4. Discussion and conclusions In the present study, we present two cases of EYA1-related branchiotorenal spectrum disorders. The first case, a young boy affected by mild bilateral conductive hearing loss (not due to otitis), preauricular pits and mild deformities of the external auditory canal, presented conditions that may be sufficient to require additional genetic and imaging investigations to achieve an accurate diagnosis. Using PCR and sequencing, we identified one novel sporadic mutation in the EYA1 gene: c.1597G > A, causing the G533R substitution in the isoform 1 (592 aa). This mutation appears to display a dominant inheritance pattern. Clinical findings and in silico analysis supported the pathogenic role of the new variant.
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In the second clinical case, we found the c. 1777T > A mutation, causing the p.X593QextX6 substitution, a C-terminal variant which is known to be involved in branchio-otorenal spectrum disorders. Therefore, mutations in the EYA1 gene are responsible for similar disorders with variable phenotypes: BOR syndrome (subtypes 1), BO syndrome (subtype 1) and OFC syndrome. These conditions can be referred to as EYA1-related disorders and should be included in branchiotorenal spectrum disorders. The present results and previously reported mutations in the EYA1 gene demonstrate a great variety, including nonsense mutations, deletions and insertions, resulting in frameshifts, aberrant splicing and deletions. It is thought that these mutation types have unpredictable phenotypes (Table 6). Moreover, available data about genotype-phenotype
Fig. 4. EYA1-related disorders: diagnostic flow chart.
Please cite this article in press as: A. Castiglione, et al., EYA1-related disorders: Two clinical cases and a literature review, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.032
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correlations in clinical studies are limited, especially those concerning the most common symptom: hearing loss. It has been reported that hypoacusis may be either conductive (30% of cases), sensorineural (20% of cases) or mixed (50% of cases), ranging from mild to profound in degree. Imaging evaluations should include renal function tests, kidney/urinary tract ultrasound and voiding cystourethrogram (with contrast) to exclude vesicoureteral reflux. Generally, this exam is not routinely performed due to the X-ray exposure and contrast use, particularly in young patients. In such cases, as well in case of contraindication to the contrast use, additional urine/serum analyses may be useful. The following conditions must be investigated: (1) renal agenesis, hypoplasia or dysplasia; (2) uretero-pelvic junction (UPJ) obstruction; (3) calyceal cyst/diverticulum; and (4) calyectasis, pelviectasis, hydronephrosis and vesicoureteral reflux. Even if the renal involvement is not always present (13–67% of affected), when a renal dysfunction of any degree is documented, it must be monitored and eventually treated. This is because it could be responsible for severe renal failure, leading to renal transplantation. A temporal bone CT scan without contrast is mandatory to study the middle and internal ear structures. A temporal bone MRI (without contrast) may be necessary to complete the imaging assessment. Interestingly, the entity of external ear malformations does not relate to inner and middle ear malformations, and the entity of hearing loss is not directly proportional to the neuroradiological findings. In summary, an effective clinical management effort should consider: (1) surgery to treat branchial defects (cleft cysts, cleft palate, fistulae or sinuses) and, when possible, hearing loss (by canaloplasty, ossiculoplasty or cochlear implantation); (2) an appropriate auditory habilitation/rehabilitation; (3) medical and surgical treatment for renal abnormalities; (4) neuroimaging; (5) genetic investigation; (6) monitoring (semiannual/annual examinations especially to assess stability of hearing loss and renal function); (7) screening of relatives for hearing loss and renal involvement; and, finally, (8) nephrotoxic and ototoxic agents should be avoided. Regardless of the potential necessity of further studies to establish prognostic conditions or genotype/phenotype correlations, the syndrome subtypes are generally associated with different clinical features; an accurate differential diagnosis may contribute to defining the evolution or progression of symptoms. Furthermore, it is important to note that in our cases, the neuroimaging pictures were not directly proportional to the audiological pictures. Similar conditions are expected to have severe/profound hearing impairment. In conclusion, these results add to the spectrum of EYA1-related disorders and indicate that the BO/BOR/BOU/OFC/BOF phenotypes are an indication for additional molecular studies. Nevertheless, a comprehensive multidisciplinary approach is mandatory to obtain an accurate differential diagnosis (Fig. 4). 5. Consent Written informed consent was obtained from the patients and other family members according to the current national rules and laws for the publication of these cases and any accompanying images. Competing interests The authors declare that they have no competing interests.
Web resources The URLs for data presented herein are as follows: Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm. ih.gov/Omim/, http://www.healthcare.uiowa.edu/labs/pendredandbor/, http://www.genecards.org/ and http://www.uniprot.org/. References [1] D.J. Orten, S.M. Fischer, J.L. Sorensen, U. Radhakrishna, C.W. Cremers, H.A. Marres, et al., Branchio-oto-renal syndrome (BOR): novel mutations in the EYA1 gene, and a review of the mutational genetics of BOR, Hum. Mutat. 29 (4) (2008) 537–544. [2] S. Abdelhak, V. Kalatzis, R. Heilig, S. Compain, D. Samson, C. Vincent, et al., A human homologue of the Drosophila eyes absent gene underlies branchiooto-renal (BOR) syndrome and identifies a novel gene family, Nat. Genet. 15 (2) (1997) 157–164. [3] M.A. Mosrati, B. Hammami, I.B. Rebeh, L. Ayadi, L. Dhouib, K. Ben Mahfoudh, et al., A novel dominant mutation in SIX1, affecting a highly conserved residue, result in only auditory defects in humans, Eur. J. Med. Genet. 54 (5) (2011) e484–e488. [4] S.H. Wang, C.C. Wu, Y.C. Lu, Y.H. Lin, Y.N. Su, W.L. Hwu, et al., Mutation screening of the EYA1, SIX1, and SIX5 genes in an East Asian cohort with branchio-oto-renal syndrome, Laryngoscope 122 (5) (2012) 1130–1136. [5] A.A. Salam, F.M. Hafner, T.E. Linder, T. Spillmann, A.A. Schinzel, S.M. Leal, A novel locus (DFNA23) for prelingual autosomal dominant nonsyndromic hearing loss maps to 14q21-q22 in a Swiss German kindred, Am. J. Hum. Genet. 66 (6) (2000) 1984–1988. [6] T. Trummer, D. Muller, A. Schulze, W. Vogel, W. Just, Branchio-oculo-facial syndrome and branchio-otic/branchio-oto-renal syndromes are distinct entities, J. Med. Genet. 39 (1) (2002) 71–73. [7] A. Castiglione, M. Busi, A. Martini, Syndromic hearing loss: an update, Hearing, Balance Commun. 11 (3) (2013) 146–159. [8] B.E. Hoskins, C.H. Cramer 2nd, V. Tasic, E.O. Kehinde, S. Ashraf, R. Bogdanovic, et al., Missense mutations in EYA1 and TCF2 are a rare cause of urinary tract malformations, Nephrol. Dial. Transplant.: Off. Publ. Eur. Dial. Transplant Assoc. – Eur. Ren. Assoc. 23 (2) (2008) 777–779. [9] H. Ohto, S. Kamada, K. Tago, S.I. Tominaga, H. Ozaki, S. Sato, et al., Cooperation of six and eya in activation of their target genes through nuclear translocation of Eya, Mol. Cell. Biol. 19 (10) (1999) 6815–6824. [10] E.H. Chang, M. Menezes, N.C. Meyer, R.A. Cucci, V.S. Vervoort, C.E. Schwartz, et al., Branchio-oto-renal syndrome: the mutation spectrum in EYA1 and its phenotypic consequences, Hum. Mutat. 23 (6) (2004) 582–589. [11] S. Ceruti, C. Stinckens, C.W. Cremers, J.W. Casselman, Temporal bone anomalies in the branchio-oto-renal syndrome: detailed computed tomographic and magnetic resonance imaging findings, Otol. Neurotol. 23 (2) (2002) 200–207. [12] E. Senel, B.N. Gulleroglu, S. Senel, Additional temporal bone findings on computed tomography imaging in branchio-oto-renal syndrome, Laryngoscope 119 (4) (2009) 832. [13] P. Krug, V. Moriniere, S. Marlin, V. Koubi, H.D. Gabriel, E. Colin, et al., Mutation screening of the EYA1, SIX1, and SIX5 genes in a large cohort of patients harboring branchio-oto-renal syndrome calls into question the pathogenic role of SIX5 mutations, Hum. Mutat. 32 (2) (2011) 183–190. [14] J.D. Lee, S.C. Kim, Y.W. Koh, H.J. Lee, S.Y. Choi, U.K. Kim, A novel frameshift mutation in the EYA1 gene in a Korean family with branchio-oto-renal syndrome, Ann. Clin. Lab. Sci. 39 (3) (2009) 303–306. [15] T.L. Stockley, R. Mendoza-Londono, E.J. Propst, S. Sodhi, L. Dupuis, B.C. Papsin, A recurrent EYA1 mutation causing alternative RNA splicing in branchio-oto-renal syndrome: implications for molecular diagnostics and disease mechanism, Am. J. Med. Genet. Part A 149 (3) (2009) 322–327. [16] E. Nardi, A. Palermo, P. Cusimano, G. Mule, G. Cerasola, Young woman with branchio-oto-renal syndrome and a novel mutation in the EYA-1 gene, Clin. Nephrol. 76 (4) (2011) 330–333. [17] M.J. Kwon, S.H. Boo, H.J. Kim, Y.S. Cho, W.H. Chung, S.H. Hong, A novel splice site mutation in the EYA1 gene in a Korean family with branchio-oto (BO) syndrome, Acta Otolaryngol. 129 (6) (2009) 688–693. [18] T. Matsunaga, M. Okada, S. Usami, T. Okuyama, Phenotypic consequences in a Japanese family having branchio-oto-renal syndrome with a novel frameshift mutation in the gene EYA1, Acta Otolaryngol. 127 (1) (2007) 98–104. [19] M. Gigante, M. d’Altilia, E. Montemurno, S. Diella, F. Bruno, G.S. Netti, et al., Branchio-Oto-Renal Syndrome (BOR) associated with focal glomerulosclerosis in a patient with a novel EYA1 splice site mutation, BMC Nephrol. 14 (2013) 60. [20] K.M. Sanggaard, N.D. Rendtorff, K.W. Kjaer, H. Eiberg, T. Johnsen, S. Gimsing, et al., Branchio-oto-renal 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.: EJHG 15 (11) (2007) 1121–1131. [21] M.H. Song, T.J. Kwon, H.R. Kim, J.H. Jeon, J.I. Baek, W.S. Lee, et al., Mutational analysis of EYA1, SIX1 and SIX5 genes and strategies for management of hearing loss in patients with BOR/BO syndrome, PloS ONE 8 (6) (2013) e67236. [22] H.R. Kim, M.H. Song, M.A. Kim, Y.R. Kim, K.Y. Lee, J.K. Sonn, et al., Identification of a novel nonsynonymous mutation of EYA1 disrupting splice site in a Korean patient with BOR syndrome, Mol. Biol. Rep. (2014).
Please cite this article in press as: A. Castiglione, et al., EYA1-related disorders: Two clinical cases and a literature review, Int. J. Pediatr. Otorhinolaryngol. (2014), http://dx.doi.org/10.1016/j.ijporl.2014.03.032
