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Purpose: To analyze BRCA1 and BRCA2 genes using a cost-effective and rapid approach based on next generation sequencing (NGS) technology., Methods: A population of Spanish cancer patients with a personal or familial history of breast and/or ovarian cancer was analyzed for germline mutations in BRCA1 and BRCA2 genes. The methodology relies on a 5 multiplex PCR assay coupled to NGS., Results: Ten pathogenic mutations (four in BRCA1 and six in BRCA2 gene) were identified in a Spanish population. The deletion c.1792delA, in exon 10, and the duplication c.5869dupA, in exon 11 of BRCA2 gene were not previously reported and should be considered as pathogenic due to its frameshift nature., Conclusion: Two novel frameshift mutations in BRCA2 gene were detected using the multiplex PCR-based assay following by NGS.

We explored an approach to detect disease-causing sequence variants in 448 candidate genes from five index cases of autosomal dominant retinitis pigmentosa (adRP) by sequence DNA capture and next-generation DNA sequencing (NGS). Detection of sequence variants was carried out by sequence capture NimbleGen and NGS in a SOLiD platform. After filtering out variants previously reported in genomic databases, novel potential adRP-causing variants were validated by dideoxy capillary electrophoresis (Sanger) sequencing and co-segregation in the families. A total of 55 novel sequence variants in the coding or splicing regions of adRP candidate genes were detected, 49 of which were confirmed by Sanger sequencing. Segregation of these variants in the corresponding adRP families showed three variants present in all the RP-affected members of the family. A novel mutation, p.L270R in IMPDH1, was found to be disease causing in one family. In another family a variant, p.M96T in the NRL gene was detected; this variant was previously reported as probably causing adRP. However, the previously reported p.A76V mutation in NRL as a cause of RP was excluded by co-segregation in the family. We discuss the benefits and limitations of our approach in the context of mutation detection in adRP patients., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mutations in the gene encoding the transcription factor neural retina leucine zipper (NRL) are known to cause autosomal dominant (adRP) or recessive (arRP) retinitis pigmentosa (RP). In an adRP Spanish family, we detected a novel sequence variation (c.287T>C) in the NRL gene that results in the p.M96T protein change. A functional test of the ability of NRL, in conjunction with cone-rod homeobox (CRX), to transactivate a human rhodopsin (RHO) promoter was used to evaluate the pathogenic mechanisms of NRL. We found upregulation of the RHO promoter by p.M96T protein similar to that shown by other missense NRL mutations that cause adRP. Affected RP patients of the family carry the nucleotide change, although two other family members that also carry the c.287T>C variation remain asymptomatic. This result complicates the genetic counselling of the family. The pathogenic mechanisms associated with adRP NRL mutations appear to be caused by a gain of function. To suppress the negative effect of an NRL mutant, the suppression and replacement strategy seems to be the most suitable therapeutic approach capable of overcoming the mutational heterogeneity associated with NRL-linked adRP. Thus, we evaluated this methodology in the NRL gene for the first time., (© 2011 John Wiley & Sons A/S.)

Usher syndrome is defined by the association of sensorineural hearing loss, retinitis pigmentosa and variable vestibular dysfunction. Many disease-causative mutations have been identified in MYO7A and USH2A genes, which play a major role in Usher syndrome type I and type II, respectively. The pathogenic nature of mutations that lead to premature stop codons is not questioned; nevertheless, additional studies are needed to verify the pathogenicity of some changes such as those putatively involved in the splice process. Five putative splice-site variants were detected in our cohort of patients: c.2283-1G>T and c.5856G>A in MYO7A and c.1841-2A>G, c.2167+5G>A and c.5298+1G>C in the USH2A gene. In this study, we analyze these changes with bioinformatic tools and investigate the expression of MYO7A and USH2A transcripts through hybrid minigene assays. Our study showed that all five mutations abolished the consensus splice site producing the skipping of involved exons. In addition, for variant c.2167+5G>A, a new donor splice site was observed. Our data reveal the pathogenic nature of the analyzed variants. The fact that splicing mutations led to in-frame or out-of-frame alterations cannot explain phenotypic differences, thus, genotype-phenotype correlations cannot be inferred., (© 2010 John Wiley & Sons A/S.)

The nuclear receptor protein NR2E3 is postulated to play an important role in rod and cone photoreceptor development. NR2E3 gene mutational analyses were carried out in 103 unrelated subjects with different retinal diseases. A total of 14 different sequence variants were identified, including 3 mutations, 6 rare sequence variants and five polymorphisms. One of three mutations is novel (a frameshift mutation: c.1034_1038del5bp). Five of the six rare sequence variants and one of the polymorphisms identified are novel. Splice prediction programs and functional splicing assays were performed to study three of these variants. The c.119-2 A>C mutant allele construction produces, in addition to the normal one, an abnormal transcript of 180 bp resulting from an aberrant splicing with skipping of exon 2 and the generation of a premature stop codon in exon 3. These experimental data confirm the splice predictions made by the computer programs. The obtained results reinforce the idea that NR2E3 gene is involved in several retinal diseases without a clear genotype-phenotype correlation.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease, characterized phenotypically by mucocutaneous pigmentation and hamartomatous polyposis. Affected patients are at an increased risk of developing gastrointestinal and other malignancies. Mutations in the STK11/LKB1 (LKB1) gene, which encodes for a serine-threonine kinase, have been identified as a genetic cause of PJS. Molecular analysis of the LKB1 gene in a simplex case of PJS revealed a substitution of cytosine (C) for guanine (G) at codon 246 in exon 6, resulting in the Tyr246X mutation. The nucleotide substitution leads to a premature stop codon at the 246 residue, predicting a truncated protein and presumed loss of kinase activity. Analysis of DNA from both parents of the PJS patient did not show this mutation, which is therefore a de novo mutation. We isolated DNA from microdissected gastrointestinal hamartomatous polyps in the PJS patient and investigated the loss of heterozygosity (LOH) at the LKB1 locus by real-time fluorescence polymerase chain reaction genotyping using a fluorescent resonance energy transfer technique. The results suggest a different mechanism from LOH in the formation of hamartomatous polyps.

Objective: Genetic characterization of a series of patients with autosomal dominant retinitis pigmentosa (ADRP)., Methods: All patients underwent complete ophthalmological examination including computerized perimetry, electroretinography and occasionally fluorescein angiography. Blood samples were drawn for genetic analysis of candidate genes namely rhodopsin (RHO), peripherin-RDS, ROM-1, CRX, RP1 and NRL., Results: 148 ADRP index cases were examined at our hospital from June 1991 to September 2001. Genetic analysis detected the following mutations: 29 different families (19.5%) carried a RHO mutation among which the Pro-347-Leu was the most frequent one, five different RP-1 mutations (3.3%), 2 RDS mutations and one NRL mutation, which is the second reported in the world literature., Conclusions: RHO followed by RP1 are the most frequent ADRP-causing genes in our series as in other published ones, and RDS causes mainly macular dystrophies. Molecular characterization was possible in 37 families (25%) which is of great interest for visual prognosis and genetic counselling.