New mutations found by Next-Generation Sequencing screening of Spanish patients with Nemaline Myopathy.

Nemaline Myopathy (NM) is a rare genetic disorder that encompasses a large spectrum of myopathies characterized by hypotonia and generalized muscle weakness. To date, mutations in thirteen different genes have been associated with NM. The most frequently responsible genes are NEB (50% of cases) and ACTA1 (15-25% of cases). In this report all known NM related genes were screened by Next Generation Sequencing in five Spanish patients in order to genetically confirm the clinical and histological diagnosis of NM. Four mutations in NEB (c.17779_17780delTA, c.11086A>C, c.21076C>T and c.2310+5G>A) and one mutation in ACTA1 (c.871A>T) were found in four patients. Three of the four mutations in NEB were novel. A cDNA sequencing assay of the novel variants c.17779_17780delTA, c.11086A>C and c.2310+5G>A revealed that the intronic variant c.2310+5G>A affected the splicing process. Mutations reported here could help clinicians and geneticists in NM diagnosis.

Targeted next generation sequencing for molecular diagnosis of Usher syndrome.

BACKGROUND: Usher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and time-consuming. Consequently, the aim of the present study was to develop a molecular diagnostics method for Usher syndrome, based on targeted next generation sequencing. METHODS: A custom HaloPlex panel for Illumina platforms was designed to capture all exons of the 10 known causative Usher syndrome genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31 and CLRN1), the two Usher syndrome-related genes (HARS and PDZD7) and the two candidate genes VEZT and MYO15A. A cohort of 44 patients suffering from Usher syndrome was selected for this study. This cohort was divided into two groups: a test group of 11 patients with known mutations and another group of 33 patients with unknown mutations. RESULTS: Forty USH patients were successfully sequenced, 8 USH patients from the test group and 32 patients from the group composed of USH patients without genetic diagnosis. We were able to detect biallelic mutations in one USH gene in 22 out of 32 USH patients (68.75%) and to identify 79.7% of the expected mutated alleles. Fifty-three different mutations were detected. These mutations included 21 missense, 8 nonsense, 9 frameshifts, 9 intronic mutations and 6 large rearrangements. CONCLUSIONS: Targeted next generation sequencing allowed us to detect both point mutations and large rearrangements in a single experiment, minimizing the economic cost of the study, increasing the detection ratio of the genetic cause of the disease and improving the genetic diagnosis of Usher syndrome patients.

Novel deletions involving the USH2A gene in patients with Usher syndrome and retinitis pigmentosa.

PURPOSE: The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa. METHODS: The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analysis was applied to 40 unrelated patients previously screened for point mutations in the USH2A gene in which none or only one pathologic mutation was identified. RESULTS: We detected six large deletions involving USH2A in six out of the 40 cases studied. Three of the patients were homozygous for the deletion, and the remaining three were compound heterozygous with a previously identified USH2A point mutation. In five of these cases, the patients displayed Usher type 2, and the remaining case displayed nonsyndromic retinitis pigmentosa. The exact breakpoint junctions of the deletions found in USH2A in four of these cases were characterized. CONCLUSIONS: Our study highlights the need to develop improved efficient strategies of mutation screening based upon next generation sequencing (NGS) that reduce cost, time, and complexity and allow simultaneous identification of all types of disease-causing mutations in diagnostic procedures.

Two novel disease-causing mutations in the CLRN1 gene in patients with Usher syndrome type 3.

PURPOSE: To identify the genetic defect in Spanish families with Usher syndrome (USH) and probable involvement of the CLRN1 gene. METHODS: DNA samples of the affected members of our cohort of USH families were tested using an USH genotyping array, and/or genotyped with polymorphic markers specific for the USH3A locus. Based on these previous analyses and clinical findings, CLRN1 was directly sequenced in 17 patients susceptible to carrying mutations in this gene. RESULTS: Microarray analysis revealed the previously reported mutation p.Y63X in two unrelated patients, one of them homozygous for the mutation. After CLRN1 sequencing, we found two novel mutations, p.R207X and p.I168N. Both novel mutations segregated with the phenotype. CONCLUSIONS: To date, 18 mutations in CLRN1 have been reported. In this work, we report two novel mutations and a third one previously identified in the Spanish USH sample. The prevalence of CLRN1 among our patients with USH is low.

Mutational screening of the USH2A gene in Spanish USH patients reveals 23 novel pathogenic mutations.

BACKGROUND: Usher Syndrome type II (USH2) is an autosomal recessive disorder, characterized by moderate to severe hearing impairment and retinitis pigmentosa (RP). Among the three genes implicated, mutations in the USH2A gene account for 74-90% of the USH2 cases. METHODS: To identify the genetic cause of the disease and determine the frequency of USH2A mutations in a cohort of 88 unrelated USH Spanish patients, we carried out a mutation screening of the 72 coding exons of this gene by direct sequencing. Moreover, we performed functional minigene studies for those changes that were predicted to affect splicing. RESULTS: As a result, a total of 144 DNA sequence variants were identified. Based upon previous studies, allele frequencies, segregation analysis, bioinformatics' predictions and in vitro experiments, 37 variants (23 of them novel) were classified as pathogenic mutations. CONCLUSIONS: This report provide a wide spectrum of USH2A mutations and clinical features, including atypical Usher syndrome phenotypes resembling Usher syndrome type I. Considering only the patients clearly diagnosed with Usher syndrome type II, and results obtained in this and previous studies, we can state that mutations in USH2A are responsible for 76.1% of USH2 disease in patients of Spanish origin.

Microarray-based mutation analysis of 183 Spanish families with Usher syndrome.

PURPOSE: The purpose of this study was to test the ability of the genotyping microarray for Usher syndrome (USH) to identify the mutations responsible for the disease in a cohort of 183 patients with USH. METHODS: DNA from 183 patients with Usher syndrome from the Spanish population was analyzed using a genotyping microarray containing 429 previously identified disease-associated variants in eight USH genes. Mutations detected by the array were confirmed by direct sequencing. Haplotype analysis was also performed in families carrying common Spanish mutations. RESULTS: The genotyping microarray identified 43 different variants, divided into 32 disease causative and 11 probably nonpathologic. Mutations were detected in 62 patients with USH (33.9%). According to the clinical classification of patients, pathologic variants were detected in 31.4% patients with USH1, 39.4% of with USH2, 22.2% with USH3 and 15.8% with unclassified Usher syndrome. Ninety-seven pathologic alleles were detected, corresponding to 26.5% of expected alleles. The USH2A mutations p.C3267R and p.T3571M were revealed as common in the Spanish population, and two major haplotypes linked to these mutations were observed. CONCLUSIONS: The genotyping microarray is a robust, low-cost, rapid technique that is effective for the genetic study of patients with USH. However, it also indicates variants of unclear pathologic nature and detection failures have also been observed. Results must be confirmed by direct sequencing to avoid misdiagnosis, and continuous updates of the microarray should be performed to increase the efficiency and rate of detection of mutations.