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.     

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.     

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.     

Exome Sequencing Identifies a Mutation in EYA4 as a Novel Cause of Autosomal Dominant Non-Syndromic Hearing Loss

Autosomal dominant non-syndromic hearing loss is highly heterogeneous, and eyes absent 4 (EYA4) is a disease-causing gene. Most EYA4 mutations founded in the Eya-homologous region, however, no deafness causative missense mutation in variable region of EYA4 have previously been found. In this study, we identified a pathogenic missense mutation located in the variable region of the EYA4 gene for the first time in a four-generation Chinese family with 57 members. Whole-exome sequencing (WES) was performed on samples from one unaffected and two affected individuals to systematically search for deafness susceptibility genes, and the candidate mutations and the co-segregation of the phenotype were verified by polymerase chain reaction amplification and by Sanger sequencing in all of the family members. Then, we identified a novel EYA4 mutation in exon 8, c.511G>C; p.G171R, which segregated with postlingual and progressive autosomal dominant sensorineural hearing loss (SNHL). This report is the first to describe a missense mutation in the variable region domain of the EYA4 gene, which is not highly conserved in many species, indicating that the potential unconserved role of 171G>R in human EYA4 function is extremely important.     

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.     

EYA1 mutations associated with the branchio‐oto‐renal syndrome result in defective otic development in Xenopus laevis

Background information. The BOR (branchio‐oto‐renal) syndrome is a dominant disorder most commonly caused by mutations in the EYA1 (Eyes Absent 1) gene. Symptoms commonly include deafness and renal anomalies. Results. We have used the embryos of the frog Xenopus laevis as an animal model for early ear development to examine the effects of different EYA1 mutations. Four eya1 mRNAs encoding proteins correlated with congenital anomalies in human were injected into early stage embryos. We show that the expression of mutations associated with BOR, even in the presence of normal levels of endogenous eya1 mRNA, leads to morphologically abnormal ear development as measured by overall otic vesicle size, establishment of sensory tissue and otic innervation. The molecular consequences of mutant eya1 expression were assessed by QPCR (quantitative PCR) analysis and in situ hybridization. Embryos expressing mutant eya1 showed altered levels of multiple genes (six1, dach, neuroD, ngnr‐1 and nt3) important for normal ear development. Conclusions. These studies lend support to the hypothesis that dominant‐negative effects of EYA1 mutations may have a role in the pathogenesis of BOR.     

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.     

Genomewide search and genetic localization of a second gene associated with autosomal dominant branchio-oto-renal syndrome: clinical and genetic implications

Branchio-oto-renal (BOR) syndrome is characterized by ear malformations, cervical fistulas, hearing loss, and renal anomalies. It is an autosomal dominant disorder with variable clinical manifestations. The most common features of BOR syndrome are branchial, hearing, and renal anomalies. However, many affected subjects have been observed with branchial-cleft anomalies and hearing loss but without renal anomalies, a condition called "branchio-otic" (BO) syndrome. It is logical to question whether the BOR and BO syndromes are allelic or whether they represent distinct genetic entities. We identified a very large extended family whose members had branchial and hearing anomalies associated with commissural lip pits that segregated in an autosomal dominant fashion. Using a genomewide search strategy, we identified genetic linkage, with a maximum LOD score of 4.81 at recombination fraction 0, between the BO phenotype and polymorphic marker D1S2757 in the genetic region of chromosome 1q31. This is the first report of linkage for a second gene associated with BOR syndrome. The findings have important clinical implications and will provide insight into the genetic basis of BOR syndrome.     

Two Novel Mutations on Exon 8 and Intron 65 of COL7A1 Gene in Two Chinese Brothers Result in Recessive Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa is an inherited bullous dermatosis caused by the COL7A1 gene mutation in autosomal dominant or recessive mode. COL7A1 gene encodes type VII collagen – the main component of the anchoring fibrils at the dermal–epidermal junction. Besides the 730 mutations reported, we identified two novel COL7A1 gene mutations in a Chinese family, which caused recessive dystrophic epidermolysis bullosa (RDEB). The diagnosis was established histopathologically and ultrastructurally. After genomic DNA extraction from the peripheral blood sample of all subjects (5 pedigree members and 136 unrelated control individuals), COL7A1 gene screening was performed by polymerase chain reaction amplification and direct DNA sequencing of the whole coding exons and flanking intronic regions. Genetic analysis of the COL7A1 gene in affected individuals revealed compound heterozygotes with identical novel mutations. The maternal mutation is a 2-bp deletion at exon 8 (c.1006_1007delCA), leading to a subsequent reading frame-shift and producing a premature termination codon located 48 amino acids downstream in exon 9 (p.Q336EfsX48), consequently resulting in the truncation of 2561 amino acids downstream. This was only present in two affected brothers, but not in the other unaffected family members. The paternal mutation is a 1-bp deletion occurring at the first base of intron 65 (c.IVS5568+1delG) that deductively changes the strongly conserved GT dinucleotide at the 5′ donor splice site, results in subsequent reading-through into intron 65, and creates a stop codon immediately following the amino acids encoded by exon 65 (GTAA→TAA). This is predicted to produce a truncated protein lacking of 1089 C-terminal amino acids downstream. The latter mutation was found in all family members except one of the two unaffected sisters. Both mutations were observed concurrently only in the two affected brothers. Neither mutation was discovered in 136 unrelated Chinese control individuals. This study reveals novel disease-causing mutations in the COL7A1 gene.     

<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.     

Characterization of the threonine-phosphatase of mouse eyes absent 3

Eyes absent (EYA) has tyrosine- and threonine-phosphatase activities in their C-terminal and N-terminal regions, respectively. Using various mutants of mouse EYA3, we showed that the 68-amino acid domain between positions 53 and 120 was necessary and sufficient for its threonine-phosphatase activity. Point mutations were then introduced, and residues Cys-56, Tyr-77, His-79, and Tyr-90 were essential for the EYA3s threonine-phosphatase. The 68-amino acid domain is not well conserved among the four mouse EYA members, but is evolutionally highly conserved in the orthologous EYA members of different species, suggesting that the threonine-phosphatase of EYA3 has a function distinct from that of the other EYAs.     

No genetic differences between affected and unaffected members of a German family with Leber's hereditary optic neuropathy (LHON) with respect to ten mtDNA point mutations associated with LHON.

In order to investigate possible synergistic influences of different mtDNA mutations on penetrance and severity of Leber's hereditary optic neuropathy (LHON), a large German LHON pedigree is characterized with respect to 10 different mutations associated with LHON. All members of the family carry three different mtDNA mutations (at nucleotide 4,216, 11,778 and 13,708) in a homoplasmic form, regardless of whether or not they are clinically affected. Testing for another 7 mutations reveals negative results in all family members. Hence, the variable disease expression of the family members cannot be explained by varying combinations of LHON-associated mtDNA mutations.     

Spondyloepiphyseal dysplasia congenita: genetic linkage to type II collagen (COL2AI).

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominantly inherited chondrodysplasia characterized by disproportionate short stature (short trunk), abnormal epiphyses, and flattened vertebral bodies. Manifestations are present at birth. We ascertained a 4-generation family exhibiting the clinical manifestations of the disorder. Previous evidence suggesting defects of type II collagen associated with the SEDC phenotype led us to genotype the family for various COL2A1 gene-associated RFLPs. A total of 17 affected and unaffected members of this family were studied. The family was informative for a recently discovered HinfI RFLP. No recombinants between the marker and the phenotype were found in eight informative meioses. A maximum LOD score of 3.01 was obtained at a recombination fraction of .00. Our results indicate that the SEDC phenotype in this family is caused by mutations in or very close to the COL2A1 locus.     

Identification and functional analysis of three distinct mutations in the human galactose-1-phosphate uridyltransferase gene associated with galactosemia in a single family.

We have identified three mutations associated with transferase-deficiency galactosemia in a three-generation family including affected members in two generations and have modeled all three mutations in a yeast-expression system. A sequence of pedigree, biochemical, and molecular analyses of the galactose-1-phosphate uridyltransferase (GALT) enzyme and genetic locus in both affected and carrier individuals revealed three distinct base substitutions in this family, two (Q188R and S135L) that had been reported previously and one (V151A) that was novel. Biochemical analyses of red-blood-cell lysates from the relevant family members suggested that each of these mutations was associated with dramatic impairment of GALT activity in these cells. While this observation was consistent with our previous findings concerning the Q188R mutation expressed both in humans and in a yeast-model system, it was at odds with a report by Reichardt and colleagues, indicating that in their COS cell-expression system the S135L substitution behaved as a neural polymorphism. To address this apparent paradox, as well as to investigate the functional significance of the newly identified V151A substitution, all three mutations were recreated by site-directed mutagenesis of the otherwise wild-type human GALT sequence and were expressed both individually and in the appropriate allelic combinations in a GALT-deficient strain of the yeast Saccharomyces cerevisiae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations of CDKL5 cause a severe neurodevelopmental disorder with infantile spasms and mental retardation

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused, in most classic cases, by mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2). A large degree of phenotypic variation has been observed in patients with RTT, both those with and without MECP2 mutations. We describe a family consisting of a proband with a phenotype that showed considerable overlap with that of RTT, her identical twin sister with autistic disorder and mild-to-moderate intellectual disability, and a brother with profound intellectual disability and seizures. No pathogenic MECP2 mutations were found in this family, and the Xq28 region that contains the MECP2 gene was not shared by the affected siblings. Three other candidate regions were identified by microsatellite mapping, including 10.3 Mb at Xp22.31-pter between Xpter and DXS1135, 19.7 Mb at Xp22.12-p22.11 between DXS1135 and DXS1214, and 16.4 Mb at Xq21.33 between DXS1196 and DXS1191. The ARX and CDKL5 genes, both of which are located within the Xp22 region, were sequenced in the affected family members, and a deletion of nucleotide 183 of the coding sequence (c.183delT) was identified in CDKL5 in the affected family members. In a screen of 44 RTT cases, a single splice-site mutation, IVS13-1G-->A, was identified in a girl with a severe phenotype overlapping RTT. In the mouse brain, Cdkl5 expression overlaps--but is not identical to--that of Mecp2, and its expression is unaffected by the loss of Mecp2. These findings confirm CDKL5 as another locus associated with epilepsy and X-linked mental retardation. These results also suggest that mutations in CDKL5 can lead to a clinical phenotype that overlaps RTT. However, it remains to be determined whether CDKL5 mutations are more prevalent in specific clinical subgroups of RTT or in other clinical presentations.