USH1C: a rare cause of USH1 in a non-Acadian population and a founder effect of the Acadian allele

Usher syndrome (USH) is characterized by the associated findings of hearing loss and retinitis pigmentosa (RP), leading to progressive loss of vision. Three forms of USH can be distinguished clinically. In the most severe form, USH1, profound congenital deafness is associated with vestibular dysfunction and RP. To determine the frequency of USH1C mutations as a cause for USH1, 128 probands with Usher syndrome type 1 including seven from Acadian and 121 from non-Acadian populations were systematically screened for mutations in USH1C using a combined single-strand conformational polymorphisms (SSCP)/heteroduplex and sequencing method. All seven Acadian USH1 patients were found to be homozygous for both the 216G>A mutation and the 9-repeat VNTR which characterizes the Acadian allele, confirming previous evidence for a founder effect by haplotype analysis. However, USH1C mutations were identified in only two non-Acadian USH1 probands (1.65%) including one from Pakistan who was homozygous for a 238-239insC mutation and one from Canada was also homozygous for the Acadian allele. The low prevalence of USH1C mutations in the present study suggests that the high prevalence of the 238-239insC in Germany may reflect a founder effect. Comparison of the affected haplotypes in the Canadian patient with the Acadian USH1 patients yielded evidence for a founder effect. Our data suggest that USH1C is a relatively rare form of USH1 in non-Acadian populations and that in addition to the 216G>A Acadian mutation, the 238-239insC mutation appears to be common in some populations.     

Mutation profile of the MYO7A gene in Spanish patients with Usher syndrome type I

Usher syndrome type I is the most severe form of Usher syndrome. It is an autosomal recessive disorder characterized by profound congenital sensorineural deafness, retinitis pigmentosa, and vestibular abnormalities. Mutations in the myosin VIIA gene (MYO7A) are responsible for Usher syndrome type 1B (USH1B). This gene is thought to bear greatest responsibility for USH1 and, depending on the study, has been reported to account for between 24% and 59% of USH1 cases. In this report a mutation screening of the MYO7A gene was carried out in a series of 48 unrelated USH1 families using single strand conformation polymorphism analysis (SSCP) and direct sequencing of those fragments showed an abnormal electrophoretic pattern. Twenty-five mutations were identified in 23 out of the 48 families studied (47.9%). Twelve of these mutations were novel, including five missense mutations, three premature stop codons, three frameshift, and one putative splice-site mutation. Based on our results we can conclude there is an absence of hot spot mutations in the MYO7A gene and that this gene plays a major role in Usher syndrome.     

The contribution of USH1C mutations to syndromic and non-syndromic deafness in the UK

Denaturing high-performance liquid chromatography (DHPLC) was used to screen 14 UK patients with Usher syndrome type 1, in order to assess the contribution of mutations in USH1C to type 1 Usher. In addition, 16 Caucasian sib pairs and two small consanguineous families with non-syndromic deafness, who were concordant for haplotypes around DFNB18, were also screened for mutations in the USH1C gene. Two Usher type 1 patients were found to have the 238-239insC mutation reported previously; one of Greek Cypriot origin was homozygous for the mutation and another Caucasian was heterozygous. This indicates that mutations in the USH1C gene make a greater contribution to Usher syndrome type 1 than originally thought, which has implications for the genetic testing of families with Usher syndrome in the UK. Analysis using intragenic single nucleotide polymorphisms (SNPs) revealed that the haplotypic background bearing this common mutation was not consistent across the gene in two families, and that there are either two haplotypes on which the mutation has arisen or that there has been a recombination on a single haplotype. We found no evidence of mutations in USH1C in the patients with non-syndromic deafness, suggesting that the gene is not a major contributor to autosomal-recessive non-syndromic deafness in the UK.     

Mutations in myosin VIIA (MYO7A) and usherin (USH2A) in Spanish patients with Usher syndrome types I and II,respectively

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing impairment and retinitis pigmentosa. Three clinical types are known (USH1, USH2 and USH3), and there is an extensive genetic heterogeneity, with at least ten genes implicated. The most frequently mutated genes are MYO7A, which causes USH1B, and usherin, which causes USH2A. We carried out a mutation analysis of these two genes in the Spanish population. Analysis of the MYO7A gene in patients from 30 USH1 families and sporadic cases identified 32% of disease alleles, with mutation Q821X being the most frequent. Most of the remaining variants are private mutations. With regard to USH2, mutation 2299delG was detected in 25% of the Spanish patients. Altogether the mutations detected in USH2A families account for 23% of the disease alleles.     

Twelve novel myosin VIIA mutations in 34 patients with Usher syndrome type I: confirmation of genetic heterogeneity

Usher syndrome is a heterogeneous autosomal recessive trait and the most common cause of hereditary deaf-blindness. Usher syndrome type I (USH1) is characterised by profound congenital sensorineural hearing loss, vestibular dysfunction, and prepubertal onset of retinitis pigmentosa. Of the at least six different loci for USH1, USH1B maps on chromosome 11q13, and the MYO7A gene has been shown to be defective in USH1B. MYO7A encodes myosin VIIA, an unconventional myosin, and it consists of 48 coding exons. In this study, MYO7A was analysed in 34 unrelated Usher type I patients by single-strand conformation polymorphism analysis and direct sequencing. We identified a total of 12 novel and unique mutations, all single base changes. In addition, we found a previously reported nonsense mutation (C31X) on nine alleles of a total of six patients from Denmark.     

Identification and in vitro expression of novel CDH23 mutations of patients with Usher syndrome type 1D

Usher syndrome (USH) is a group of autosomal recessive sensory disorders characterized by progressive retinitis pigmentosa (RP) and sensorineural hearing impairment. Usher syndrome type 1 (USH1), with additional vestibular dysfunction, represents the most severe form and shows extensive allelic and non-allelic heterogeneity. At least six USH1 loci exist (USH1A-F), and four of the underlying genes have been identified. Recently, a novel gene, cadherin 23 (CDH23), was shown to be mutated in USH1D. We performed mutation screening by single strand conformation polymorphism (SSCP) analysis and direct sequencing on 33 USH1 patients previously excluded for USH1B and USH1C. On eight disease alleles of four patients, four different mutations were identified, three of them novel (c.6933delT, c.5712G-->A, and IVS45-9G-->A). Exon trapping experiments were performed with two mutations. In the case of a c.5712G-->A transition of the last base of exon 42, that is an apparently synonymous mutation, skipping of exon 42 was observed. By the mutation IVS45-9G-->A, a novel splice acceptor site was created and the insertion of 7 intronic bp was observed. Two mutations, IVS45-9G-->A and the previously described IVS51+5G-->A, were each found in more than one patient. Haplotype analysis by SNPs within CDH23 suggests common ancestors for each of the mutations. Among the total of 52 USH1 cases studied by us, CDH23 mutations account for about 10% of all disease alleles. Our results further suggest that in patients with a typical USH1D phenotype, a significant portion of CDH23 mutations leads to premature termination of translation or loss of numerous amino acid residues, with a high frequency of changes causing aberrant splicing of CDH23 mRNA.     

UMD-USHbases: a comprehensive set of databases to record and analyse pathogenic mutations and unclassified variants in seven Usher syndrome causing genes

Using the Universal Mutation Database (UMD) software, we have constructed "UMD-USHbases", a set of relational databases of nucleotide variations for seven genes involved in Usher syndrome (MYO7A, CDH23, PCDH15, USH1C, USH1G, USH3A and USH2A). Mutations in the Usher syndrome type I causing genes are also recorded in non-syndromic hearing loss cases and mutations in USH2A in non-syndromic retinitis pigmentosa. Usher syndrome provides a particular challenge for molecular diagnostics because of the clinical and molecular heterogeneity. As many mutations are missense changes, and all the genes also contain apparently non-pathogenic polymorphisms, well-curated databases are crucial for accurate interpretation of pathogenicity. Tools are provided to assess the pathogenicity of mutations, including conservation of amino acids and analysis of splice-sites. Reference amino acid alignments are provided. Apparently non-pathogenic variants in patients with Usher syndrome, at both the nucleotide and amino acid level, are included. The UMD-USHbases currently contain more than 2,830 entries including disease causing mutations, unclassified variants or non-pathogenic polymorphisms identified in over 938 patients. In addition to data collected from 89 publications, 15 novel mutations identified in our laboratory are recorded in MYO7A (6), CDH23 (8), or PCDH15 (1) genes. Information is given on the relative involvement of the seven genes, the number and distribution of variants in each gene. UMD-USHbases give access to a software package that provides specific routines and optimized multicriteria research and sorting tools. These databases should assist clinicians and geneticists seeking information about mutations responsible for Usher syndrome.     

Usher syndrome type 1 due to missense mutations on both CDH23 alleles: investigation of mRNA splicing

Usher syndrome (USH) is an autosomal recessive condition characterized by sensorineural hearing loss, vestibular dysfunction, and visual impairment due to retinitis pigmentosa. Truncating mutations in the cadherin-23 gene (CDH23) result in Usher syndrome type 1D (USH1D), whereas missense mutations affecting strongly conserved motifs of the CDH23 protein cause non-syndromic deafness (DFNB12). Four missense mutations constitute an exception from this genotype-phenotype correlation: they have been described in USH1 patients in homozygous state. Using a minigene assay, we have investigated these changes (c.1450G>C, p.A484P; c.3625A>G, p.T1209A; c.4520G>A, p.R1507Q; and c.5237G>A, p.R1746Q) for a possible impact on mRNA splicing which could explain the syndromic phenotype. While in silico analysis suggested impairment of splicing in all four cases, we found aberrant splicing for only one mutation, p.R1746Q. However, splicing was normal in case of p.A484P, p.T1209A and p.R1507Q. These three latter CDH23 missense mutations could interfere with functions of both, the auditory and the visual system. Alternatively, they could represent rare non-pathogenic polymorphisms.     

Map refinement of the Usher syndrome type 1B gene, MYO7A, relative to 11q13.5 microsatellite markers

Usher syndrome (US) is clinically and genetically a heterogeneous group of disorders characterized by the association of deafness with retinitis pigmentosa. So far, eight genes responsible for US have been mapped, of which only the gene responsible for the most common form, USH1B, has been identified. The USH1B is a large gene containing 49 exons and encoding for an unconventional myosin-VIIA (MYO7A). Mutation analysis within the MYO7A gene showed a wide variety of mutations dispersed all over the gene. The present report refines the location of the MYO7A gene relative to microsatellite markers mapped to this region, thereby allowing a reliable and efficient carrier detection by linkage analysis.     

Novel mutations of the RPGR gene in RP3 families

X-linked retinitis pigmentosa is a severe retinal degeneration characterized by night blindness and visual field constriction, leading to complete blindness within the third decade of life. Mutations in the RPGR gene (retinitis pigmentosa GTPase regulator), located on Xp21.1 in the RP3 region, have been associated with an RP phenotype. Further to our previous mutation screening of RPGR in families segregating with the RP3 locus, we have expanded this study to include other 8 RP3 pedigrees. Here we report the results of this expanded study and the identification of five mutations in RPGR, four of which are novel (IVS6+5 G>A, 950-951delAA, 963 T>C, EX8del) and one of which occurs in the donor splice site of intron 1 (IVS1+1 G>A). These findings bring the proportion of "RP3 genotypes" with a mutation in this gene to 27% (10/37). Hum Mutat 15:386, 2000.     

Identification of 14 novel mutations in the long isoform of USH2A in Spanish patients with Usher syndrome type II

Mutations in USH2A gene have been shown to be responsible for Usher syndrome type II, an autosomal recessive disorder characterised by hearing loss and retinitis pigmentosa. USH2A was firstly described as consisting of 21 exons, but 52 novel exons at the 3' end of the gene were recently identified. In this report, a mutation analysis of the new 52 exons of USH2A gene was carried out in 32 unrelated patients in which both disease-causing mutations could not be found after the screening of the first 21 exons of the USH2A gene. On analysing the new 52 exons, fourteen novel mutations were identified in 14 out of the 32 cases studied, including 7 missense, 5 frameshift, 1 duplication and a putative splice-site mutation.     

Mutational spectrum in Usher syndrome type II

Usher syndrome type II is an autosomal recessive disorder characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa (RP). We carried out a mutation screening of the USH2A gene in 88 probands with Usher syndrome type II to determine the frequency of USH2A mutations as a cause for USH2. Six mutations, including 2299delG, 921-922insCAGC, R334W, N346H, R626X, and N357T were identified, with 2299delG mutation being the most frequent (16.5% of alleles), accounting for 77.5% of the pathologic alleles. Thirty-five percent (31/88) of the probands had a USH2A mutation. Nine of them carried two pathogenic mutations: six cases were homozygotes and three were compound heterozygotes. Twenty-two probands (25%) were found to carry only single USH2A mutations. One new missense mutation (N357T) occuring within the laminin N-terminal (type VI) domain of usherin was identified. Eight polymorphisms were found, five of which are novel. Our data support the view that the 2299delG is the most common mutation in USH2A.     

Identification of 11 novel mutations in USH2A among Japanese patients with Usher syndrome type 2

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A , which accounts for 74–90% of USH2 cases. This is the first study reporting the results of scanning for USH2A mutations in Japanese patients with USH2. In 8 of 10 unrelated patients, we identified 14 different mutations. Of these mutations, 11 were novel. Although the mutation spectrum that we identified differed from that for Caucasians, the incidence of mutations in USH2A was 80% for all patients tested, which is consistent with previous findings. Further, c.8559-2A>G was identified in four patients and accounted for 26.7% of mutated alleles; it is thus a frequent mutation in Japanese patients. Hence, mutation screening for c.8559-2A>G in USH2A may prove very effective for the early diagnosis of USH2.     

Identification of novel USH2A mutations: implications for the structure of USH2A protein

Usher syndrome type II is an autosomal recessive disorder, characterised by stable hearing impairment from childhood and progressive retinitis pigmentosa from the late teens. Mutations in the USH2A gene, located on 1q41, were recently shown to be responsible for Usher syndrome type IIa. We have investigated the molecular pathology of Usher type II by screening the USH2A gene for mutations in 31 unrelated patients from Denmark and Norway. Besides the frequent 2299delG mutation, which accounted for 44% of the disease alleles, a heterogeneous spectrum of mutations was identified. Sixteen new, putative disease-causing mutations were detected, of which 12 were private and four were shared by unrelated patients. The disease-causing mutations were scattered throughout the gene and included six nonsense and seven missense mutations, two deletions and one small insertion. In addition, six non-pathogenic polymorphisms were identified. All missense mutations resulted in major amino acid side-chain alterations. Four missense mutations affected the N-terminal part of USH2A, whereas three missense mutations affected the laminin-type epidermal growth factor-like (LE) domain. The structural consequences of the mutations affecting the LE domain are discussed in relation to the three-dimensional structure of a LE-module of the mouse laminin gamma1 chain.     

Novel USH2A mutations in Japanese Usher syndrome type 2 patients: marked differences in the mutation spectrum between the Japanese and other populations

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A. In a recent mutation screening of USH2A in Japanese USH2 patients, we identified 11 novel mutations in 10 patients and found the possible frequent mutation c.8559-2A>G in 4 of 10 patients. To obtain a more precise mutation spectrum, we analyzed further nine Japanese patients in this study. We identified nine mutations, of which eight were novel. This result indicates that the mutation spectrum for USH2A among Japanese patients largely differs from Caucasian, Jewish and Palestinian patients. Meanwhile, we did not find the c.8559-2A>G in this study. Haplotype analysis of the c.8559-2G (mutated) alleles using 23 single nucleotide polymorphisms surrounding the mutation revealed an identical haplotype pattern of at least 635?kb in length, strongly suggesting that the mutation originated from a common ancestor. The fact that all patients carrying c.8559-2A>G came from western Japan suggests that the mutation is mainly distributed in that area; indeed, most of the patients involved in this study came from eastern Japan, which contributed to the absence of c.8559-2A>G.     

Erratum: analysis of DNA elements that modulate myosin VIIa expression in humans

Usher syndromeIb (USH1B), an autosomal recessive disorder caused by mutations in myosin VIIa (MYO7A), is characterized by congenital profound hearing loss, vestibular abnormalities and retinitis pigmentosa. Promoter elements in the 5 kb upstream of the translation start were identified using adult retinal pigment epithelium cells (ARPE-19) as a model system. A 160 bp minimal promoter within the first intron was active in ARPE-19 cells, but not in HeLa cells that do not express MYO7A. A 100 bp sequence, 5' of the first exon, and repeated with 90% homology within the first intron, appeared to modulate expression in both cell lines. Segments containing these elements were screened by heteroduplex analysis. No heteroduplexes were detected in the minimal promoter, suggesting that this sequence is conserved. A -2568 A>T transversion in the 5' 100 bp repeat, eliminating a CCAAT element, was found only in USH1B patients. However, in all 5 families, -2568 A>T was in cis with the same missense mutation in the myosin VIIa tail (Arg1240Gln), and 4 of the 5 families were Dutch. These observations suggest either 1) linkage disequilibrium or 2)that a combination of a promoter mutation with a less active myosin VIIa protein results in USH1B.     

Survey of the frequency of USH1 gene mutations in a cohort of Usher patients shows the importance of cadherin 23 and protocadherin 15 genes and establishes a detection rate of above 90%

Background

Usher syndrome, a devastating recessive disorder which combines hearing loss with retinitis pigmentosa, is clinically and genetically heterogeneous. Usher syndrome type 1 (USH1) is the most severe form, characterised by profound congenital hearing loss and vestibular dysfunction.

Objective

To describe an efficient protocol which has identified the mutated gene in more than 90% of a cohort of patients currently living in France.

Results

The five genes currently known to cause USH1 (MYO7A, USH1C, CDH23, PCDH15, and USH1G) were tested for. Disease causing mutations were identified in 31 of the 34 families referred: 17 in MYO7A, 6 in CDH23, 6 in PCDH15, and 2 in USH1C. As mutations in genes other than myosin VIIA form nearly 50% of the total, this shows that a comprehensive approach to sequencing is required. Twenty nine of the 46 identified mutations were novel. In view of the complexity of the genes involved, and to minimise sequencing, a protocol for efficient testing of samples was developed. This includes a preliminary linkage and haplotype analysis to indicate which genes to target. It proved very useful and demonstrated consanguinity in several unsuspected cases. In contrast to CDH23 and PCDH15, where most of the changes are truncating mutations, myosin VIIA has both nonsense and missense mutations. Methods for deciding whether a missense mutation is pathogenic are discussed.

Conclusions

Diagnostic testing for USH1 is feasible with a high rate of detection and can be made more efficient by selecting a candidate gene by preliminary linkage and haplotype analysis.     

Genetic analysis of 2299delG and C759F mutations (USH2A) in patients with visual and/or auditory impairments

The most common mutation in the USH2A gene (Usherin), 2299delG, causes both typical Usher (USH) syndrome type II and atypical USH syndrome, two autosomal recessive disorders, characterised by moderate to severe sensorineural hearing loss and retinitis pigmentosa (RP). Furthermore, the C759F mutation in the USH2A gene has been described in 4.5% of patients with nonsyndromic recessive RP. We have investigated the presence of the 2299delG and/or the C759F mutations in 191 unrelated Spanish patients with different syndromic and nonsyndromic retinal diseases, or with nonsyndromic hearing impairment. The 2299delG mutation was observed in patients with clinical signs of USHII or of atypical USH syndrome, whereas the C759F mutation, regardless of being associated with the 2299delG mutation or not, was identified in cases with nonsyndromic RP, as well as in patients with RP associated with a variability of hearing impairment. The comparative analysis of both phenotypic and genotypic data supports the hypothesis that sensorineural hearing loss in patients with RP may depend on the nature and on the association of the USH2A allele variants present.