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Sanchez-Alcudia R.,Fundacion Jimenez Diaz University Hospital | Sanchez-Alcudia R.,Center for Biomedical Network Research on Rare Diseases | Corton M.,Fundacion Jimenez Diaz University Hospital | Corton M.,Center for Biomedical Network Research on Rare Diseases | And 26 more authors.
Investigative Ophthalmology and Visual Science

Purpose. The aim of this study was to deepen our knowledge on the basis of intrafamilial genetic heterogeneity of inherited retinal dystrophies (RD) to further discern the contribution of individual alleles to the pathology.Methods. Families with intrafamilial locus and/or allelic heterogeneity were selected from a cohort of 873 characterized of 2468 unrelated RD families. Clinical examination included visual field assessments, electrophysiology, fundus examination, and audiogram. Molecular characterization was performed using a combination of different methods: genotyping microarray, single strand conformational polymorphism (SSCP), denaturing high pressure liquid chromatography (dHPLC), high resolution melt (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, whole-genome homozygosity mapping, and next-generation sequencing (NGS).Results. Overall, intrafamilial genetic heterogeneity was encountered in a total of 8 pedigrees. There were 5 of 873 families (~0.6%) with causative mutations in more than one gene (locus heterogeneity), involving the genes: (1) USH2A, RDH12, and TULP1; (2) PDE6B and a new candidate gene; (3) CERKL and CRB1; (4) BBS1 and C2orf71; and (5) ABCA4 and CRB1. Typically, in these cases, each mutated gene was associated with different phenotypes. In the 3 other families (~0.35%), different mutations in the same gene (allelic heterogeneity) were found, including the frequent RD genes ABCA4 and CRB1.Conclusions. This systematic research estimates that the frequency of overall mutation load promoting RD intrafamilial heterogeneity in our cohort of Spanish families is almost 1%. The identification of the genetic mechanisms underlying RD locus and allelic heterogeneity is essential to discriminate the real contribution of the monoallelic mutations to the disease, especially in the NGS era. Moreover, it is decisive to provide an accurate genetic counseling and in disease treatment. © 2014 The Association for Research in Vision and Ophthalmology, Inc. Source

Cebrian-Torrejon G.,University Paris - Sud | Mackiewicz N.,University Paris - Sud | Vazquez-Manrique R.P.,Institute Investigacion Sanitaria IIS La Fe | Fournet A.,University Paris - Sud | And 3 more authors.
European Journal of Organic Chemistry

Based on the biosynthetic line of canthin-6-one alkaloids from their simple precursors such as tryptamine, the present work is focused on the study of alternative protocol of the Bischler-Napieralski reaction and has led to a full coverage of the different biosynthetic intermediates. Nanoparticles were also prepared as mimics of biosynthetic assembly lines and some interesting biological results in term of chemical ecology are also reported. Canthin-6-one, a particular representative β-carboline alkaloid, was targeted for synthesis keeping in mind its biosynthetic origin. In fact, several biosynthetic intermediates were synthesized and nanoparticular mimicry of key steps was also achieved permitting further evaluation of biological properties for this class of alkaloids. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Vache C.,Montpellier University Hospital Center | Besnard T.,Montpellier University Hospital Center | Besnard T.,French Institute of Health and Medical Research | Besnard T.,Montpellier University | And 17 more authors.
Human Mutation

USH2A sequencing in three affected members of a large family, referred for the recessive USH2 syndrome, identified a single pathogenic alteration in one of them and a different mutation in the two affected nieces. As the patients carried a common USH2A haplotype, they likely shared a mutation not found by standard sequencing techniques. Analysis of RNA from nasal cells in one affected individual identified an additional pseudoexon (PE) resulting from a deep intronic mutation. This was confirmed by minigene assay. This is the first example in Usher syndrome (USH) with a mutation causing activation of a PE. The finding of this alteration in eight other individuals of mixed European origin emphasizes the importance of including RNA analysis in a comprehensive diagnostic service. Finally, this mutation, which would not have been found by whole-exome sequencing, could offer, for the first time in USH, the possibility of therapeutic correction by antisense oligonucleotides (AONs). © 2011 Wiley Periodicals, Inc. Source

Garcia-Garcia G.,French Institute of Health and Medical Research | Garcia-Garcia G.,Institute Investigacion Sanitaria IIS La Fe | Besnard T.,French Institute of Health and Medical Research | Besnard T.,Montpellier University | And 9 more authors.
Molecular Vision

Background: Usher syndrome type 2 (USH2) is an autosomal recessive disease characterized by moderate to severe hearing loss and retinitis pigmentosa. To date, three disease-causing genes have been identified, USH2A, GPR98, and DFN B31, of which USH2A is clearly the major contributor. The aim of this work was to determine the contribution of GPR98 and DF N B31 genes in a Spanish cohort of USH2A negative patients using exhaustive molecular analysis, including sequencing, dosage, and splicing analysis. Methods: Linkage analysis was performed to prioritize the gene to study, followed by sequencing of exons and intronexon boundaries of the selected gene, GPR98 (90 exons) or DF N B31 (12 exons). Functional splicing analyses and comparative genomic hybridization array to detect large rearrangements were performed when appropriate. Results: We confirmed that mutations in GPR98 contribute a significant but minor role to Usher syndrome type 2. In a group of patients referred for molecular diagnosis, 43 had been found to be positive for USH2A mutations, the remaining 19 without USH2A alterations were screened, and seven different mutations were identified in the GPR98 gene in seven patients (five in the homozygous state), of which six were novel. All detected mutations result in a truncated protein; deleterious missense mutations were not found. No pathological mutations were identified in the DF N B31 gene. Conclusions: In Spain, USH2A and GPR98 are responsible for 95.8% and 5.2% of USH2 mutated cases, respectively. DFNB31 plays a minor role in the Spanish population. There was a group of patients in whom no mutation was found. These fndings confrm the importance of including at least GPR98 analysis for comprehensive USH2 molecular diagnosis. © 2013 Molecular Vision. Source

Aparisi M.J.,Institute Investigacion Sanitaria IIS La Fe | Garcia-Garcia G.,Institute Investigacion Sanitaria IIS La Fe | Jaijo T.,Institute Investigacion Sanitaria IIS La Fe | Jaijo T.,CIBER ISCIII | And 14 more authors.
Molecular Vision

Purpose: Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by severe-profound sensorineural hearing loss, retinitis pigmentosa, and vestibular areflexia. To date, five USH1 genes have been identified. One of these genes is Usher syndrome 1C (USH1C), which encodes a protein, harmonin, containing PDZ domains. The aim of the present work was the mutation screening of the USH1C gene in a cohort of 33 Usher syndrome patients, to identify the genetic cause of the disease and to determine the relative involvement of this gene in USH1 pathogenesis in the Spanish population. Methods: Thirty-three patients were screened for mutations in the USH1C gene by direct sequencing. Some had already been screened for mutations in the other known USH1 genes (myosin VIIA [MYO7A], cadherin-related 23 [CDH23], protocadherin-related 15 [PCDH15], and Usher syndrome 1G [USH1G]), but no mutation was found. Results: Two novel mutations were found in the USH1C gene: a non-sense mutation (p.C224X) and a frame-shift mutation (p.D124TfsX7). These mutations were found in a homozygous state in two unrelated USH1 patients. Conclusions: In the present study, we detected two novel pathogenic mutations in the USH1C gene. Our results suggest that mutations in USH1C are responsible for 1.5% of USH1 disease in patients of Spanish origin (considering the total cohort of 65 Spanish USH1 patients since 2005), indicating that USH1C is a rare form of USH in this population. © 2010 Molecular Vision. Source

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