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.     

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.     

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.     

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.     

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.     

Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome.

Germ-line mutations of the tumor-suppressor gene p53 have been observed in some families with the Li-Fraumeni syndrome (LFS), a familial cancer syndrome in which affected relatives develop a diverse set of early-onset malignancies including breast carcinoma, sarcomas, and brain tumors. The analysis of the p53 gene in LFS families has been limited, in most studies to date, to the region between exon 5 and exon 9. In order to determine the frequency and distribution of germ-line p53 mutations in LFS, we sequenced the 10 coding exons of the p53 gene in lymphocytes and fibroblast cell lines derived from 15 families with the syndrome. Germ-line mutations were observed in eight families. Six mutations were missense mutations located between exons 5 and 8. One mutation was a nonsense mutation in exon 6, and one mutation was a splicing mutation in intron 4, generating aberrant shorter p53 RNA(s). In three families, a mutation of the p53 gene was observed in the fibroblast cell line derived from the proband. However, the mutation was not found in affected relatives in two families and in the blood from the one individual, indicating that the mutation probably occurred during cell culture in vitro. In four families, no mutation was observed. This study indicates that germ-line p53 mutations in LFS are mostly located between exons 5 and 8 and that approximately 50% of patients with LFS have no germ-line mutations in the coding region of the p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxanthine guanine phosphoribosyltransferase (HPRT) mutations in the Asian population

Mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior, or HPRT-related gout (Kelley-Seegmiller syndrome). The marked heterogeneity of HPRT deficiency is well known, with more than 300 mutations at the HPRT gene locus having been reported (deletions, insertions, duplications, abnormal splicing, and point mutations at different sites of the coding region from exons 1 to 9). We have identified mutations in Asian families with patients manifesting different clinical phenotypes, including rare cases of female subjects, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse-transcribed mRNA using the polymerase chain reaction technique coupled with direct sequencing. We developed suitable methods to detect the mutations identified from respective families with HPRT deficiency. Then, prenatal genetic diagnoses in HPRT-deficient families were carried out using both mRNA and genomic DNA from chorionic villi or amniotic fluid cells. As shown here in the heterogeneity of HPRT mutations, the spectrum of 70 mutations identified in the Asian population fits the four main conclusions that emerged previously from worldwide analysis.     

<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.     

Germline mutations in the Wilms' tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome.

Denys-Drash syndrome is a rare human condition in which severe urogenital aberrations result in renal failure, pseudohermaphroditism, and Wilms' tumor (nephroblastoma). To investigate its possible role, we have analyzed the coding exons of the Wilms' tumor suppressor gene (WT1) for germline mutations. In ten independent cases of Denys-Drash syndrome, point mutations in the zinc finger domains of one WT1 gene copy were found. Nine of these mutations are found within exon 9 (zinc finger III); the remaining mutation is in exon 8 (zinc finger II). These mutations directly affect DNA sequence recognition. In two families analyzed, the mutations were shown to arise de novo. Wilms' tumors from three individuals and one juvenile granulosa cell tumor demonstrate reduction to homozygosity for the mutated WT1 allele. Our results provide evidence of a direct role for WT1 in Denys-Drash syndrome and thus urogenital system development.     

Germ-line mutational analysis of the TSC2 gene in 90 tuberous-sclerosis patients.

Ninety patients with tuberous-sclerosis complex (TSC) were tested for subtle mutations in the TSC2 gene, by means of single-strand conformational analysis (SSCA) of genomic DNA. Patients included 56 sporadic cases and 34 familial probands. For all patients, SSCA was performed for each of the 41 exons of the TSC2 gene. We identified 32 SSCA changes, 22 disease-causing mutations, and 10 polymorphic variants. Interestingly, we detected mutations at a much higher frequency in the sporadic cases (32%) than in the multiplex families (9%). Among the eight families for which linkage to the TSC2 region had been determined, only one mutation was found. Mutations were distributed equally across the gene; they included 5 deletions, 3 insertions, 10 missense mutations, 2 nonsense mutations, and 2 tandem duplications. We did not detect an increase in mutations either in the GTPase-activating protein (GAP)-related domains of TSC2 or in the activating domains that have been identified in rat tuberin. We did not detect any mutations in the exons (25 and 31) that are spliced out in the isoforms. There was no evidence for correspondence between variability of phenotype and type of mutation (missense versus early termination). Diagnostic testing will be difficult because of the genetic heterogeneity of TSC (which has at least two causative genes: TSC1 and TSC2), the large size of the TSC2 gene, and the variety of mutations. More than half of the mutations that we identified (missense, small in-frame deletion, and tandem duplication) are not amenable to the mutation-detection methods, such as protein-truncation testing, that are commonly employed for genes that encode proteins with tumor-suppressor function.     

Genomic rearrangements of the APC tumor-suppressor gene in familial adenomatous polyposis

Germline mutations of the adenomatous polyposis coli (APC) tumor-suppressor gene result in the hereditary colorectal cancer syndrome familial adenomatous polyposis (FAP). Almost all APC mutations that have been identified are single-nucleotide alterations, small insertions, or small deletions that would truncate the protein product of the gene. No well-characterized intragenic rearrangement of APC has been described, and the prevalence of this type of mutation in FAP patients is not clear. We screened 49 potential FAP families and identified 26 different germline APC mutations in 30 families. Four of these mutations were genomic rearrangements resulting from homologous and nonhomologous recombinations mediated by Alu elements. Two of these four rearrangements were complex, involving deletion and insertion of nucleotides. Of these four rearrangements, one resulted in the deletion of exons 11 and 12 and two others resulted in either complete or partial deletion of exon 14. The fourth rearrangement grossly altered the sequence within intron 14. Although this rearrangement did not affect any coding sequence of APC at the genomic DNA level, it caused inappropriate splicing of exon 14. These rearrangements were initially revealed by analyzing cDNAs and could not have been identified by using mutation detection methods that screened each exon individually. The identification of a rearrangement that did not alter any coding exons yet affected the splicing further underscores the importance of using cDNA for mutation analysis. The identification of four genomic rearrangements among 30 mutations suggests that genomic rearrangements are frequent germline APC mutations.     

Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome

Craniofrontonasal syndrome (CFNS) is an X-linked craniofacial disorder with an unusual manifestation pattern, in which affected females show multiple skeletal malformations, whereas the genetic defect causes no or only mild abnormalities in male carriers. Recently, we have mapped a gene for CFNS in the pericentromeric region of the X chromosome that contains the EFNB1 gene, which encodes the ephrin-B1 ligand for Eph receptors. Since Efnb1 mutant mice display a spectrum of malformations and an unusual inheritance reminiscent of CFNS, we analyzed the EFNB1 gene in three families with CFNS. In one family, a deletion of exons 2-5 was identified in an obligate carrier male, his mildly affected brother, and in the affected females. In the two other families, missense mutations in EFNB1 were detected that lead to amino acid exchanges P54L and T111I. Both mutations are located in multimerization and receptor-interaction motifs found within the ephrin-B1 extracellular domain. In all cases, mutations were found consistently in obligate male carriers, clinically affected males, and affected heterozygous females. We conclude that mutations in EFNB1 cause CFNS.     

Mutations in NDUFB11, Encoding a Complex I Component of the Mitochondrial Respiratory Chain,Cause Microphthalmia with Linear Skin Defects Syndrome

Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject’s mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.     

LDL-R and Apo-B-100 gene mutations in Polish familial hypercholesterolemias

A group of 30 Polish families with clinical signs of familial hypercholesterolemia was studied for the presence of germ-line mutations in the LDL-R and ApoB-100 genes. Screening of the LDL-R gene was performed at the genomic DNA level by single-strand conformation polymorphism analysis of all 18 exons and extended by sequencing of polymerase chain reaction (PCR) products showing abnormalities. The occurrence of large LDL-R gene alterations was evaluated by analysis of restriction enzyme patterns on Southern blots and using the long-PCR technique. The ApoB-100 gene was studied by combined allele-specific and asymmetric PCR for the occurrence of the common B-3500 missense mutation G to A at nucleotide position 10,708. Germ-line mutations were found in 17 families. In 12 of them LDL-R gene mutations were detected. Three of 11 different mutations had previously been described in other populations (3-bp deletion of codon 197; Ser156Leu; Gly571Glu). Of the mutations not previously recognized and identified in Polish families, there were three small deletions (2-bp deletion AG at codon 291; 4-bp deletion CCCT at codons 661–662; 1-bp deletion A at codon 830), and four point mutations (Arg239Stop, Cys331Stop, Asn543Ser, Gln665Stop). Additionally, one large (∼1-kb) LDL-R gene deletion between exons 6 and 9 was identified. In five families, the B-3500 mutation within the ApoB-100 gene was revealed. Received: 15 September 1997 / Accepted: 10 February 1998     

Diverse growth hormone receptor gene mutations in Laron syndrome.

To better understand the molecular genetic basis and genetic epidemiology of Laron syndrome (growth-hormone insensitivity syndrome), we analyzed the growth-hormone receptor (GHR) genes of seven unrelated affected individuals from the United States, South America, Europe, and Africa. We amplified all nine GHR gene exons and splice junctions from these individuals by PCR and screened the products for mutations by using denaturing gradient gel electrophoresis (DGGE). We identified a single GHR gene fragment with abnormal DGGE results for each affected individual, sequenced this fragment, and, in each case, identified a mutation likely to cause Laron syndrome, including two nonsense mutations (R43X and R217X), two splice-junction mutations, (189-1 G to T and 71 + 1 G to A), and two frameshift mutations (46 del TT and 230 del TA or AT). Only one of these mutations, R43X, has been previously reported. Using haplotype analysis, we determined that this mutation, which involves a CpG dinucleotide hot spot, likely arose as a separate event in this case, relative to the two prior reports of R43X. Aside from R43X, the mutations we identified are unique to patients from particular geographic regions. Ten GHR gene mutations have now been described in this disorder. We conclude that Laron syndrome is caused by diverse GHR gene mutations, including deletions, RNA processing defects, translational stop codons, and missense codons. All the identified mutations involve the extracellular domain of the receptor, and most are unique to particular families or geographic areas.