Identification of a novel EYA1 splice-site mutation in a Danish branchio-oto-renal syndrome family

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by variable clinical manifestations including branchial fistulae, preauricular pits, ear malformations, hearing impairment, and renal anomalies. BOR is caused by mutations in the genes EYA1 and SIX1. A Danish BOR family with five affected individuals in three generations was analyzed for mutations in all 17 exons of EYA1 using direct sequencing of polymerase chain reaction (PCR) amplified genomic DNA. A novel splice-site mutation (IVS9+1 G>C) was detected in all affected family members but not in unaffected family members or in 96 controls. We conclude that this mutation is causing BOR in the family, most likely as a result of haploinsufficiency or an abnormal protein product caused by aberrant splicing of EYA1 mRNA.     

Mutational Analysis of EYA1, SIX1 and SIX5 Genes and Strategies for Management of Hearing Loss in Patients with BOR/BO Syndrome

Background

Branchio-oto-renal (BOR) or branchio-otic (BO) syndrome is one of the most common forms of autosomal dominant syndromic hearing loss. Mutations in EYA1, SIX1 and SIX5 genes have been associated with BOR syndrome. In this study, clinical and genetic analyses were performed in patients with BOR/BO syndrome focusing on auditory manifestations and rehabilitation.

Methods

The audiologic manifestations were reviewed in 10 patients with BOR/BO syndrome. The operative findings and hearing outcome were analyzed in patients who underwent middle ear surgeries. The modality and outcome of auditory rehabilitation were evaluated. Genetic analysis was performed for EYA1, SIX1, and SIX5 genes.

Results

All patients presented with mixed hearing loss. Five patients underwent middle ear surgeries without successful hearing gain. Cochlear implantation performed in two patients resulted in significant hearing improvement. Genetic analysis revealed four novel EYA1 mutations and a large deletion encompassing the EYA1 gene.

Conclusions

Auditory rehabilitation in BOR/BO syndrome should be individually tailored keeping in mind the high failure rate after middle ear surgeries. Successful outcome can be expected with cochlear implantations in patients with BOR/BO syndrome who cannot benefit from hearing aids. The novel EYA1 mutations may add to the genotypic and phenotypic spectrum of BOR syndrome in the East Asian population.     

Identification of a novel nonsynonymous mutation of EYA1 disrupting splice site in a Korean patient with BOR syndrome

The EYA1 gene is known as the causative gene of BOR (Branchio-oto-renal) syndrome which is a genetic disorder associated with branchial cleft cysts of fistulae, hearing loss, ear malformation, and renal anomalies. Although approximately 40 % of patients with BOR syndrome have mutations in the EYA1 gene and over 130 disease-causing mutations in EYA1 have been reported in various populations, only a few mutations have been reported in Korean families. In this study, genetic analysis of the EYA1 gene was performed in a Korean patient diagnosed with BOR syndrome and his parents. A de novo novel missense mutation, c.418G>A, located at the end of exon 6, changed glycine to serine at amino acid position 140 (p.G140S) and was suspected to affect normal splicing. Our in vitro splicing assay demonstrated that this mutation causes exon 6 skipping leading to frameshift and truncation of the protein to result in the loss of eyaHR. To the best of our knowledge, this is the first report revealing that a missense mutation in the exon disturbs normal splicing as a result of a substitution of the last nucleotide of an exon in EYA1.     

Genome-wide copy number variation analysis of a Branchio-oto-renal syndrome cohort identifies a recombination hotspot and implicates new candidate genes

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial arch anomalies, hearing loss and renal dysmorphology. Although haploinsufficiency of EYA1 and SIX1 are known to cause BOR, copy number variation analysis has only been performed on a limited number of BOR patients. In this study, we used high-resolution array-based comparative genomic hybridization on 32 BOR probands negative for coding-sequence and splice-site mutations in known BOR-causing genes to identify potential disease-causing genomic rearrangements. Of the >1,000 rare and novel copy number variants we identified, four were heterozygous deletions of EYA1 and several downstream genes that had nearly identical breakpoints associated with retroviral sequence blocks, suggesting that non-allelic homologous recombination seeded by this recombination hotspot is important in the pathogenesis of BOR. A different heterozygous deletion removing the last exon of EYA1 was identified in an additional proband. Thus, in total five probands (14 %) had deletions of all or part of EYA1. Using a novel disease-gene prioritization strategy that includes network analysis of genes associated with other deletions suggests that SHARPIN (Sipl1), FGF3 and the HOXA gene cluster may contribute to the pathogenesis of BOR.     

The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line

To understand the molecular basis of sensory organ development and disease, we have cloned and characterized the zebrafish mutation dog-eared (dog) that is defective in formation of the inner ear and lateral line sensory systems. The dog locus encodes the eyes absent-1 (eya1) gene and single point mutations were found in three independent dog alleles, each prematurely truncating the expressed protein within the Eya domain. Moreover, morpholino-mediated knockdown of eya1 gene function phenocopies the dog-eared mutation. In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis but Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts. Increased levels of apoptosis occur in the migrating primordia of the posterior lateral line in dog embryos and as well as in regions of the developing otocyst that are mainly fated to give rise to sensory cells of the cristae. Importantly, mutation of the EYA1 or EYA4 gene causes hereditary syndromic deafness in humans. Determination of eya gene function during zebrafish organogenesis will facilitate understanding the molecular etiology of human vestibular and hearing disorders.     

<Emphasis Type="Italic">CHX10</Emphasis> mutations cause non-syndromic microphthalmia/anophthalmia in Arab and Jewish kindreds

Microphthalmia/anophthalmia is a clinically heterogeneous disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. The genetic defect underlying isolated autosomal recessive microphthalmia/anophthalmia is yet unclear. We studied four families (two of Arab origin, one of Bedouin origin, and one of Persian-Jewish origin) with autosomal recessive microphthalmia/anophthalmia and no associated eye anomalies, and one Syrian–Jewish family with associated colobomas. Assuming a founder effect in each of the families, we performed homozygosity mapping using polymorphic markers adjacent to human homologues of genes known to be associated with eye absence in various species, namely EYA1, EYA2, EYA3, SIX4, SIX6, PAX6 and CHX10. No association was found with EYA1, EYA2, EYA3, SIX6 or PAX6. In two families, linkage analysis was consistent with possible association with SIX4, but no mutations were found in the coding region of the gene or its flanking intron sequences. In three of the five families, linkage analysis followed by sequencing demonstrated that affected individuals in each family were homozygous for a different CHX10 aberration: a mutation in the CVC domain and a deletion of the homeobox domain were found in two Arab families, and a mutation in the donor-acceptor site in the first intron in the Syrian-Jewish family. There was phenotypic variation between families having different mutations, but no significant phenotypic variation within each family. It has been previously shown that mutations in a particular nucleotide in CHX10 are associated with an autosomal recessive syndrome of microphthalmia/anophthalmia with iris colobomas and cataracts in two families. We now show that different mutations in other domains of the same gene underlie isolated microphthalmia/anophthalmia.     

Oto-facio-cervical (OFC) syndrome is a contiguous gene deletion syndrome involving EYA1: molecular analysis confirms allelism with BOR syndrome and further narrows the Duane syndrome critical region to 1 cM

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder involving hearing loss, branchial defects, ear pits and renal abnormalities. Oto-facio-cervical (OFC) syndrome is clinically similar to BOR syndrome, with clinical features in addition to those of BOR syndrome. Mutations in the EYA1 gene (localised to 8q13.3) account for nearly 70% of BOR syndrome cases exhibiting at least three of the major features. Small intragenic deletions of the 3' region of the gene have also been reported in patients with BOR syndrome. We have developed a fluorescent quantitative multiplex polymerase chain reaction for three 3' exons (7, 9 and 13) of the EYA1 gene. This dosage assay, combined with microsatellite marker analysis, has identified de novo deletions of the EYA1 gene and surrounding region in two patients with complex phenotypes involving features of BOR syndrome. One patient with OFC syndrome carried a large deletion of the EYA1 gene region, confirming that OFC syndrome is allelic with BOR syndrome. Microsatellite analysis has shown that comparison of the boundaries of this large deletion with other reported rearrangements of the region reduces the critical region for Duane syndrome (an eye movement disorder) to between markers D8S553 and D8S1797, a genetic distance of approximately 1 cM.     

Contractile activity is required for Z‐disc sarcomere maturation in vivo

Sarcomere structure underpins structural integrity, signaling, and force transmission in the muscle. In embryos of the frog Xenopus tropicalis, muscle contraction begins even while sarcomerogenesis is ongoing. To determine whether contractile activity plays a role in sarcomere formation in vivo, chemical tools were used to block acto‐myosin contraction in embryos of the frog X. tropicalis, and Z‐disc assembly was characterized in the paralyzed dicky ticker mutant. Confocal and ultrastructure analysis of paralyzed embryos showed delayed Z‐disc formation and defects in thick filament organization. These results suggest a previously undescribed role for contractility in sarcomere maturation in vivo. genesis 53:299–307, 2015. © 2015 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Human NDUFB9 gene: genomic organization and a possible candidate gene associated with deafness disorder mapped to chromosome 8q13

Human NADH dehydrogenase (ubiquinone) 1beta-subcomplex, 9 (NDUFB9) is a nuclear encoded mitochondrial protein with the respiratory electron transport chain. It has been physically mapped to a 1-Mb deletion at chromosome 8q13 which also contains the gene for branchio-oto-renal (BOR) syndrome. BOR syndrome is characterized by branchial and renal abnormalities with hearing impairment. Since several hereditary deafness disorders have been associated with mitochondrial mutations, NDUFB9 was considered a candidate gene for BOR syndrome. Recently, EYA1 gene has been identified in the region which underlies the BOR syndrome but majority of BOR families did not show mutations in the EYA1 gene. Here we have determined the genomic structure of the NDUFB9 gene, including the nucleotide sequence, organization and the boundaries of the four coding exons. PCR primers were designed from the adjacent intron sequences that allow amplification of the four exons that encode the complete open reading frame. To identify whether mutations in NDUFB9 are involved in causing the BOR syndrome, we screened 9 BOR families which did not show mutations in the EYA1 gene by heteroduplex analysis; however, no mutations were found.     

Hearing dysfunction in heterozygous MitfMi‐wh/+ mice,a model for Waardenburg syndrome type 2 and Tietz syndrome

The human deafness‐pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia‐White (Mitf^Mi‐wh/+) mice were studied and hearing function of these mice characterized. Mitf^Mi‐wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf^Mi‐wh/+ embryos have fewer melanoblasts during embryonic development than their wild‐type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf^Mi‐wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness‐pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.     

Defective calmodulin‐dependent rapid apical endocytosis in zebrafish sensory hair cell mutants

Vertebrate mechanosensory hair cells contain a narrow “pericuticular” zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1‐43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 424–434, 1999     

Novel three‐way complex rearrangement of TRPM1‐PUM1‐LCK in a case of agminated Spitz nevi arising in a giant congenital hyperpigmented macule

The genetic anomalies associated with the agminated variant of Spitz nevus have so far been limited to HRAS G13R mutations, especially when arising within a nevus spilus. A previous report exposed the case of a man with a giant pigmented macule involving his upper right limb and trunk. Since childhood, Spitz nevi have been periodically arising, within the pigmented area. The histopathology of several lesions displayed the usual criteria of junctional, compound, or intradermal Spitz nevi with a diversity of cytomorphological and architectural features. Some lesions spontaneously regressed. Genetic studies confirmed in three lesions an identical translocation involving TRPM1, PUM1, and LCK. No mutations in HRAS, NRAS, BRAF, or other known fusion genes linked to Spitz nevus were detected. LCK break‐apart fluorescence in situ hybridization confirmed the rearrangement was present not only in the melanocytic proliferation but also in the surrounding non‐spitzoid melanocytes. This report expands the list of genetic alterations involved both in giant congenital macules and in agminated Spitz nevi, and also extends the concept of mosaicism in melanocytes to gene translocations.