Refining the region of branchio-oto-renal syndrome and defining the flanking markers on chromosome 8q by genetic mapping.

Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder associated with external-, middle-, and inner-ear malformations, branchial cleft sinuses, cervical fistulas, mixed hearing loss, and renal anomalies. The gene for BOR was mapped to the long arm of chromosome 8q. Several polymorphic dinucleotide repeat markers were investigated for linkage in two large BOR families, and the region of localization was refined. Two-point linkage analysis yielded the maximum lod scores of 7.44 at theta = .03 and 6.71 at theta = .04, with markers D8S279 and D8S260, respectively. A multipoint analysis was carried out to position the BOR gene with a defined region using markers D8S165, D8S285, PENK, D8S166, D8S260, D8S279, D8S164, D8S286, D8S84, D8S275, D8S167, D8S273, and D8S271. Haplotype analysis of recombination events at these polymorphic loci was also performed in multigeneration BOR kindreds. The linkage analysis and analysis of recombination events identified markers that clearly flank the BOR locus. The order was determined to be D8S260-BOR-D8S279 at odds > 10(3):1 over the other possible orders. This flanking markers provide a resource for high-resolution mapping toward cloning and characterizing the BOR gene.     

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.     

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.     

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.     

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.     

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.     

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.     

Johnson-McMillin syndrome: report of a new case with novel features

BACKGROUND: Johnson-McMillin syndrome (JMS) is a rare neuroectodermal disorder characterized by alopecia, ear malformations, conductive hearing loss, anosmia/hyposmia, and hypogonadotropic hypogonadism. It is inherited in an autosomal dominant manner; however, the causative gene has not yet been identified. CASE: Herein we report a patient with this condition who exhibits many of the features previously described, including alopecia, malformed auricles, conductive hearing loss, facial asymmetry, and developmental delays. Interestingly, she also has features that have not yet been reported, such as preauricular pits and tags, broad depressions at the lateral aspects of the eyes, and an abnormal left lower eyelid. CONCLUSIONS: In addition to demonstrating a pattern of anomalies consistent with JMS, this patient has several unique features. This phenotype supports the involvement of the branchial arches in the embryologic basis of this condition.     

Molecular Genetic Causes and Clinical Description of Branchio-Oto-renal Syndrome

Russian Journal of Genetics - Branchio-oto-renal (BOR) syndrome is an autosomal dominant disease characterized by a combination of hearing impairment with preauricular pits, cervical fistulas or...     

Whole-Exome Sequencing Identifies a Novel Genotype-Phenotype Correlation in the Entactin Domain of the Known Deafness Gene TECTA

Postlingual progressive hearing loss, affecting primarily the high frequencies, is the clinical finding in most cases of autosomal dominant nonsyndromic hearing loss (ADNSHL). The molecular genetic etiology of ADNSHL is extremely heterogeneous. We applied whole-exome sequencing to reveal the genetic etiology of high-frequency hearing loss in a mid-sized Korean family without any prior linkage data. Whole-exome sequencing of four family members (two affected and two unaffected), together with our filtering strategy based on comprehensive bioinformatics analyses, identified 21 potential pathogenic candidates. Sanger validation of an additional five family members excluded 20 variants, leaving only one novel variant, TECTA c.710C>T (p.T237I), as the strongest candidate. This variant resides in the entactin (ENT) domain and co-segregated perfectly with non-progressive high-frequency hearing loss in the family. It was absent among 700 ethnically matched control chromosomes, and the T237 residue is conserved among species, which supports its pathogenicity. Interestingly, this finding contrasted with a previously proposed genotype-phenotype correlation in which variants of the ENT domain of TECTA were associated with mid-frequency hearing loss. Based upon what we observed, we propose a novel “genotype to phenotype” correlation in the ENT domain of TECTA. Our results shed light on another important application of whole-exome sequencing: the establishment of a novel genotype-phenotype in the molecular genetic diagnosis of autosomal dominant hearing loss.     

Targeted massive parallel sequencing: the effective detection of novel causative mutations associated with hearing loss in small families

ABSTRACT: BACKGROUND: Hereditary hearing loss is one of the most common heterogeneous disorders, and genetic variants that can cause hearing loss have been identified in over fifty genes. Most of these hearing loss genes have been detected using classical genetic methods, typically starting with linkage analysis in large families with hereditary hearing loss. However, these classical strategies are not well suited for mutation analysis in smaller families who have insufficient genetic information. METHODS: Eighty known hearing loss genes were selected and simultaneously sequenced by targeted next-generation sequencing (NGS) in 8 Korean families with autosomal dominant non-syndromic sensorineural hearing loss. RESULTS: Five mutations in known hearing loss genes, including 1 nonsense and 4 missense mutations, were identified in 5 different genes (ACTG1, MYO1F, DIAPH1, POU4F3 and EYA4), and the genotypes for these mutations were consistent with the autosomal dominant inheritance pattern of hearing loss in each family. No mutational hot-spots were revealed in these Korean families. CONCLUSION: Targeted NGS allowed for the detection of pathogenic mutations in affected individuals who were not candidates for classical genetic studies. This report is the first documenting the effective use of an NGS technique to detect pathogenic mutations that underlie hearing loss in an East Asian population. Using this NGS technique to establish a database of common mutations in Korean patients with hearing loss and further data accumulation will contribute to the early diagnosis and fundamental therapies for hereditary hearing loss.     

常染色体显性遗传非综合征型耳聋致病基因定位研究

耳聋具有高度的遗传异质性, 迄今已定位了51个常染色体显性遗传非综合征型耳聋(autosomal dominant non-syndromic sensorineural hearing loss, DFNA)基因位点, 20个DFNA相关基因被克隆.文章收集了一个DFNA巨大家系, 家系中有血缘关系的家族成员共170人, 对73名家族成员进行了详细的病史调查、全身检查和耳科学检查, 提示39人有不同程度的迟发性感音神经性听力下降, 未见前庭及其他系统的异常.应用ABI公司382个常染色体微卫星多态标记进行全基因组扫描连锁分析, 将该家系致聋基因定位于14q12-13处D14S1021-D14S70之间约7.6 cM (3.18 Mb)的区域, 最大LOD值为6.69 (D14S1040), 与已知DFNA9位点有4.7 cM (2.57 Mb)的重叠区, DFNA9致病基因COCH位于重叠区域内.下一步拟进行COCH基因的突变筛查, 以揭示该家系耳聋的分子致病机制.     

常染色体显性遗传非综合征性耳聋致病基因定位研究

耳聋具有高度的遗传异质性, 迄今已定位了51个常染色体显性遗传非综合征型耳聋(autosomal dominant non-syndromic sensorineural hearing loss, DFNA)基因位点, 20个DFNA相关基因被克隆。文章收集了一个DFNA巨大家系, 家系中有血缘关系的家族成员共170人, 对73名家族成员进行了详细的病史调查、全身检查和耳科学检查, 提示39人有不同程度的迟发性感音神经性听力下降, 未见前庭及其他系统的异常。应用ABI公司382个常染色体微卫星多态标记进行全基因组扫描连锁分析, 将该家系致聋基因定位于14q12-13处D14S1021-D14S70之间约7.6 cM (3.18 Mb)的区域, 最大LOD值为6.69 (D14S1040), 与已知DFNA9位点有4.7 cM (2.57 Mb)的重叠区, DFNA9致病基因COCH位于重叠区域内。下一步拟进行COCH基因的突变筛查, 以揭示该家系耳聋的分子致病机制。     

Linkage of a gene causing familial focal segmental glomerulosclerosis to chromosome 11 and further evidence of genetic heterogeneity.

Focal segmental glomerulosclerosis (FSGS) is a pathological entity characterized by proteinuria, nephrotic syndrome, and the progressive loss of renal function. It is a common cause of end-stage renal disease (ESRD). Recently, familial forms of FSGS have been identified. Two families with autosomal dominant FSGS were evaluated for linkage using 351 genomic microsatellite markers. Linkage, multipoint analysis, and tests for heterogeneity were performed on the subsequent results. In addition, three small families were used for haplotype analysis. Evidence for linkage was found on chromosome 11q21-q22 for the largest family, with a maximum lod score of 9.89. The gene is currently localized to an 18-cM area between flanking markers D11S2002 and D11S1986. The disease in a second family was not linked to this locus or to a previously described locus on chromosome 19q13. There were no shared haplotypes among affected individuals in the three smaller families. Our findings demonstrate that genetic heterogeneity is prevalent in FSGS in that at least three genes cause the FSGS phenotype. Identification of the genes that cause familial FSGS will provide valuable insights into the molecular basis and pathophysiology of FSGS.     

Further delineation of renal-coloboma syndrome in patients with extreme variability of phenotype and identical PAX2 mutations.

Renal-coloboma syndrome is a recently described autosomal dominant syndrome of abnormal optic nerve and renal development. Two families have been reported with renal-coloboma syndrome and mutations of the PAX2 gene. The PAX2 gene, which encodes a DNA-binding protein, is expressed in the developing ear, CNS, eye, and urogenital tract. Ocular and/or renal abnormalities have been consistently noted in the five reports of patients with renal-coloboma syndrome, to date, but PAX2 expression patterns suggest that auditory and CNS abnormalities may be additional features of renal-coloboma syndrome. To determine whether additional clinical features are associated with PAX2 mutations, we have used PCR-SSCP to identify PAX2 gene mutations in patients. We report here four patients with mutations in exon 2, one of whom has severe ocular and renal disease, microcephaly, and retardation, and another who has ocular and renal disease with high-frequency hearing loss. Unexpectedly, extreme variability in clinical presentation was observed between a mother, her son, and an unrelated patient, all of whom had the same PAX2 mutation as previously described in two siblings with renal-coloboma syndrome. These results suggest that a sequence of seven Gs in PAX2 exon 2 may be particularly prone to mutation.     

Developmental defects in Gorlin syndrome related to a putative tumor suppressor gene on chromosome 9.

Gorlin syndrome is an autosomal dominant disorder that predisposes to basal cell carcinomas of the skin, ovarian fibromas, and medulloblastomas. Unlike other hereditary disorders associated with cancer, it features widespread developmental defects. To investigate the possibility that the syndrome is caused by mutation in a tumor suppressor gene, we searched for loss of heterozygosity in 16 sporadic basal cell carcinomas, 2 hereditary basal cell carcinomas, and 1 hereditary ovarian fibroma and performed genetic linkage studies in five Gorlin syndrome kindreds. Eleven sporadic basal cell carcinomas and all 3 hereditary tumors had allelic loss of chromosome 9q31, and all informative kindreds showed tight linkage between the Gorlin syndrome gene and a genetic marker in this region. Loss of heterozygosity at this chromosomal location, particularly in hereditary tumors, implies that the gene is homozygously inactivated and normally functions as a tumor suppressor. In contrast, hemizygous germline mutations lead to multiple congenital anomalies.