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.     

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.     

A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration (‘retinal disease-specific''); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one ‘retinal disease-specific'' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype–phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.     

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.     

Panel-based next generation sequencing as a reliable and efficient technique to detect mutations in unselected patients with retinal dystrophies

Hereditary retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different forms of RD can be caused by mutations in >100 genes, including >1600 exons. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. So far, NGS is not routinely used in gene diagnostics. We developed a diagnostic NGS pipeline to identify mutations in 170 genetically and clinically unselected RD patients. NGS was applied to 105 RD-associated genes. Underrepresented regions were examined by Sanger sequencing. The NGS approach was successfully established using cases with known sequence alterations. Depending on the initial clinical diagnosis, we identified likely causative mutations in 55% of retinitis pigmentosa and 80% of Bardet–Biedl or Usher syndrome cases. Seventy-one novel mutations in 40 genes were newly associated with RD. The genes USH2A, EYS, ABCA4, and RHO were more frequently affected than others. Occasionally, cases carried mutations in more than one RD-associated gene. In addition, we found possible dominant de-novo mutations in cases with sporadic RD, which implies consequences for counseling of patients and families. NGS-based mutation analyses are reliable and cost-efficient approaches in gene diagnostics of genetically heterogeneous diseases like RD.     

An USH2A founder mutation is the major cause of Usher syndrome type 2 in Canadians of French origin and confirms common roots of Quebecois and Acadians

Congenital hearing loss affects approximately one child in 1000. About 10% of the deaf population have Usher syndrome (USH). In USH, hearing loss is complicated by retinal degeneration with onset in the first (USH1) or second (USH2) decade. In most populations, diagnostic testing is hampered by a multitude of mutations in nine genes. We have recently shown that in French Canadians from Quebec, USH1 largely results from a single USH1C founder mutation, c.216G>A ('Acadian allele'). The genetic basis of USH2 in Canadians of French descent, however, has remained elusive. Here, we have investigated nine USH2 families from Quebec and New Brunswick (the former Acadia) by haplotype analyses of the USH2A locus and sequencing of the three known USH2 genes. Seven USH2A mutations were identified in eight patients. One of them, c.4338_4339delCT, accounts for 10 out of 18 disease alleles (55.6%). This mutation has previously been reported in an Acadian USH2 family, and it was found in homozygous state in the three Acadians of our sample. As in the case of c.216G>A (USH1C), a common haplotype is associated with c.4338_4339delCT. With a limited number of molecular tests, it will now be possible in these populations to estimate whether children with congenital hearing impairment of different degrees will develop retinal disease - with important clinical and therapeutic implications. USH2 is the second example that reveals a significant genetic overlap between Quebecois and Acadians: in contrast to current understanding, other genetic disorders present in both populations are likely based on common founder mutations as well.     

Genesis of two most prevalent PROP1 gene variants causing combined pituitary hormone deficiency in 21 populations

Two variants (c.[301_302delAG][301_302delAG] and c.[150delA][150delA]) in the PROP1 gene are the most common genetic causes of recessively inherited combined pituitary hormones deficiency (CPHD). Our objective was to analyze in detail the origin of the two most prevalent variants. In the multicentric study were included 237 patients with CPHD and their 15 relatives carrying c.[301_302delAG][301_302delAG] or c.[150delA][150delA] or c.[301_302delAG][ 150delA]. They originated from 21 different countries worldwide. We genotyped 21 single-nucleotide variant markers flanking the 9.6-Mb region around the PROP1 gene that are not in mutual linkage disequilibrium in the general populations – a finding of a common haplotype would be indicative of ancestral origin of the variant. Haplotypes were reconstructed by Phase and Haploview software, and the variant age was estimated using an allelic association method. We demonstrated the ancestral origin of both variants – c.[301_302delAG] was carried on 0.2 Mb-long haplotype in a majority of European patients arising ~101 generations ago (confidence interval 90.1–116.4). Patients from the Iberian Peninsula displayed a different haplotype, which was estimated to have emerged 23.3 (20.1–29.1) generations ago. Subsequently, the data indicated that both the haplotypes were transmitted to Latin American patients ~13.8 (12.2–17.0) and 16.4 (14.4–20.1) generations ago, respectively. The c.[150delA] variant that was carried on a haplotype spanning about 0.3 Mb was estimated to appear 43.7 (38.4–52.7) generations ago. We present strong evidence that the most frequent variants in the PROP1 gene are not a consequence of variant hot spots as previously assumed, but are founder variants.     

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.     

Profound, prelingual nonsyndromic deafness maps to chromosome 10q21 and is caused by a novel missense mutation in the Usher syndrome type IF gene PCDH15

We studied a consanguineous family (Family A) from the island of Newfoundland with an autosomal recessive form of prelingual, profound, nonsyndromic sensorineural hearing loss. A genome-wide scan mapped the deafness trait to 10q21-22 (max LOD score of 4.0; D10S196) and fine mapping revealed a 16 Mb ancestral haplotype in deaf relatives. The PCDH15 gene was mapped within the critical region and was an interesting candidate because truncating mutations cause Usher syndrome type IF (USH1F) and two missense mutations have been previously associated with isolated deafness (DFNB23). Sequencing of the PCDH15 gene revealed 33 sequencing variants. Three of these variants were homozygous exclusively in deaf siblings but only one of them was not seen in ethnically matched controls. This novel c.1583 T>A transversion predicts an amino-acid substitution of a valine with an aspartic acid at codon 528 (V528D). Like the two DFNB23 mutations, the V528D mutation in Family A occurs in a highly conserved extracellular cadherin (EC) domain of PCDH15 and is predicted to be more deleterious than the previously identified DFNB23 missense mutations (R134G and G262D). Physical assessment, vestibular and visual function testing in deaf adults ruled out syndromic deafness because of Usher syndrome. This study validates the DFNB23 designation and supports the hypothesis that missense mutations in conserved motifs of PCDH15 cause nonsyndromic hearing loss. This emerging genotype–phenotype correlation in USH1F is similar to that in several other USH1 genes and cautions against a prognosis of a dual sensory loss in deaf children found to be homozygous for hypomorphic mutations at the USH1F locus.     

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.     

Haplotype analysis of the 185delAG BRCA1 mutation in ethnically diverse populations

The 185delAG* BRCA1 mutation is encountered primarily in Jewish Ashkenazi and Iraqi individuals, and sporadically in non-Jews. Previous studies estimated that this is a founder mutation in Jewish mutation carriers that arose before the dispersion of Jews in the Diaspora ∼2500 years ago. The aim of this study was to assess the haplotype in ethnically diverse 185delAG* BRCA1 mutation carriers, and to estimate the age at which the mutation arose. Ethnically diverse Jewish and non-Jewish 185delAG*BRCA1 mutation carriers and their relatives were genotyped using 15 microsatellite markers and three SNPs spanning 12.5 MB, encompassing the BRCA1 gene locus. Estimation of mutation age was based on a subset of 11 markers spanning a region of ∼5 MB, using a previously developed algorithm applying the maximum likelihood method. Overall, 188 participants (154 carriers and 34 noncarriers) from 115 families were included: Ashkenazi, Iraq, Kuchin-Indians, Syria, Turkey, Iran, Tunisia, Bulgaria, non-Jewish English, non-Jewish Malaysian, and Hispanics. Haplotype analysis indicated that the 185delAG mutation arose 750–1500 years ago. In Ashkenazim, it is a founder mutation that arose 61 generations ago, and with a small group of founder mutations was introduced into the Hispanic population (conversos) ∼650 years ago, and into the Iraqi–Jewish community ∼450 years ago. The 185delAG mutation in the non-Jewish populations in Malaysia and the UK arose at least twice independently. We conclude that the 185delAG* BRCA1 mutation resides on a common haplotype among Ashkenazi Jews, and arose about 61 generations ago and arose independently at least twice in non-Jews.     

Further refinement of the Usher 2A locus at 1q41.

Usher syndrome (USH) is characterised by congenital sensorineural hearing loss and progressive pigmentary retinopathy. All three subtypes (USH1, USH2, and USH3) are inherited as recessive traits. People with Usher type 2 (USH2) have normal vestibular responses and moderate to severe hearing loss. These syndromes have been found to be genetically heterogeneous, with a single locus for USH2 at 1q41 (USH2A), six loci for USH1, and one for USH3. Some USH2 families have been excluded from the 1q41 locus suggesting that a second, as yet unidentified, locus (USH2B) must exist. Linkage studies suggest that around 90% of USH2 families are USH2A. Four USH2 families were analysed for linkage to markers flanking the USH2A locus. In one of these families a recombination event was observed in an affected subject which excludes the USH2A gene from proximal to the marker AFM143XF10 and defines this as the new centromeric flanking marker for the USH2A locus. A further recombination event in another patient from this family confirmed AFM144XF2 as the telomeric flanking marker. The interval between these polymorphic markers is estimated to be 400 kb. This region is completely contained in each of three YACs from the CEPH library: 867g9, 919h3, and 848b9. This refinement more than halves the critical genetic interval and will greatly facilitate positional cloning of the USH2A gene.     

Partial USH2A deletions contribute to Usher syndrome in Denmark

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing impairment, progressive visual loss owing to retinitis pigmentosa and in some cases vestibular dysfunction. Usher syndrome is divided into three subtypes, USH1, USH2 and USH3. Twelve loci and eleven genes have so far been identified. Duplications and deletions in PCDH15 and USH2A that lead to USH1 and USH2, respectively, have previously been identified in patients from United Kingdom, Spain and Italy. In this study, we investigate the proportion of exon deletions and duplications in PCDH15 and USH2A in 20 USH1 and 30 USH2 patients from Denmark using multiplex ligation-dependent probe amplification (MLPA). Two heterozygous deletions were identified in USH2A, but no deletions or duplications were identified in PCDH15. Next-generation mate-pair sequencing was used to identify the exact breakpoints of the two deletions identified in USH2A. Our results suggest that USH2 is caused by USH2A exon deletions in a small fraction of the patients, whereas deletions or duplications in PCDH15 might be rare in Danish Usher patients.     

High-resolution population-specific recombination rates and their effect on phasing and genotype imputation

Previous research has shown that using population-specific reference panels has a significant effect on downstream population genomic analyses like haplotype phasing, genotype imputation, and association, especially in the context of population isolates. Here, we developed a high-resolution recombination rate mapping at 10 and 50 kb scale using high-coverage (20–30×) whole-genome sequenced data of 55 family trios from Finland and compared it to recombination rates of non-Finnish Europeans (NFE). We tested the downstream effects of the population-specific recombination rates in statistical phasing and genotype imputation in Finns as compared to the same analyses performed by using the NFE-based recombination rates. We found that Finnish recombination rates have a moderately high correlation (Spearman’s ρ = 0.67–0.79) with NFE, although on average (across all autosomal chromosomes), Finnish rates (2.268 ± 0.4209 cM/Mb) are 12–14% lower than NFE (2.641 ± 0.5032 cM/Mb). Finnish recombination map was found to have no significant effect in haplotype phasing accuracy (switch error rates ~2%) and average imputation concordance rates (97–98% for common, 92–96% for low frequency and 78–90% for rare variants). Our results suggest that haplotype phasing and genotype imputation mostly depend on population-specific contexts like appropriate reference panels and their sample size, but not on population-specific recombination maps. Even though recombination rate estimates had some differences between the Finnish and NFE populations, haplotyping and imputation had not been noticeably affected by the recombination map used. Therefore, the currently available HapMap recombination maps seem robust for population-specific phasing and imputation pipelines, even in the context of relatively isolated populations like Finland.Subject terms: Genetic variation, Haplotypes     

Spectrum of USH2A mutations in Scandinavian patients with Usher syndrome type II

Usher syndrome type II (USH2) is an autosomal recessive disorder, characterised by moderate to severe high-frequency hearing impairment, normal balance function and progressive visual impairment due to retinitis pigmentosa. Usher syndrome type IIa, the most common subtype, is defined by mutations in the USH2A gene encoding a short and a recently discovered long usherin isoform comprising 21 and 73 exons, respectively. More than 120 different disease-causing mutations have been reported, however, most of the previous reports concern mutations restricted to exons 1-21 of the USH2A gene. To explore the spectrum of USH2A disease-causing mutations among Scandinavian USH2 cases, patients from 118 unrelated families of which 27 previously had been found to carry mutations in exons 1-21 were subjected to extensive DNA sequence analysis of the full size USH2A gene. Altogether, 122 USH2A DNA sequence alterations were identified of which 57 were predicted to be disease-causing, 7 were considered to be of uncertain pathogenicity and 58 were predicted to be benign variants. Of 36 novel pathogenic USH2A mutations 31 were located in exons 22-73, specific to the long isoform. USH2A mutations were identified in 89/118 (75.4%) families. In 79/89 (88.8%) of these families two pathogenic mutations were identified whereas in 10/89 (11.2%) families the second mutation remained unidentified. In 5/118 (4.2%) families the USH phenotype could be explained by mutations in the USH3A gene. The results presented here provide a comprehensive picture of the genetic aetiology of Usher syndrome type IIA in Scandinavia as it is known to date.     

Homozygosity mapping to the USH2A locus in two isolated populations

Usher syndrome is a group of autosomal recessive disorders characterised by progressive visual loss from retinitis pigmentosa and moderate to severe sensorineural hearing loss. Usher syndrome is estimated to account for 6-10% of all congenital sensorineural hearing loss. A gene locus in Usher type II (USH2) families has been assigned to a small region on chromosome 1q41 called the UHS2A locus. We have investigated two families with Usher syndrome from different isolated populations. One family is a Norwegian Saami family and the second family is from the Cayman Islands. They both come from relatively isolated populations and are inbred families suitable for linkage analysis. A lod score of 3.09 and 7.65 at zero recombination was reached respectively in the two families with two point linkage analysis to the USH2A locus on 1q41. Additional homozygosity mapping of the affected subjects concluded with a candidate region of 6.1 Mb. This region spans the previously published candidate region in USH2A. Our study emphasises that the mapped gene for USH2 is also involved in patients from other populations and will have implications for future mutation analysis once the USH2A gene is cloned.     

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.