A novel mutation in GRK1 causes Oguchi disease in a consanguineous Pakistani family

Contains fulltext : 76070.pdf (publisher's version ) (Open Access)PURPOSE: The purpose of this study was to identify the underlying molecular genetic defect in a large consanguineous Pakistani family with Oguchi disease who had been given a diagnosis of autosomal recessive retinitis pigmentosa. METHODS: The family was genotyped with the Affymetrix 10K single nucleotide polymorphism array. Fine-mapping of a common homozygous region on chromosome 13q was performed using fluorescent microsatellite markers. Mutation analysis was done by direct sequencing of the candidate gene GRK1 located in the region. The segregation of a novel mutation in the family and the frequency of the identified mutation in the Pakistani population were determined by StuI RFLP analysis. RESULTS: Genetic mapping supported the diagnosis of typical Oguchi disease in a Pakistani family and also resulted in the identification of a novel nonsense mutation (c.614C>A; p.S205X) in exon 1 of GRK1. This mutation is predicted to result in premature termination of the protein product, thereby affecting the phototransduction cascade. A clinical reappraisal of the family revealed that all patients homozygous for this variant had Oguchi disease. CONCLUSIONS: This is the first report to describe a mutation causing typical Oguchi disease in a large consanguineous Pakistani family. This mutation segregated in eight affected members.6 p

Identification of recurrent and novel mutations in TULP1 in Pakistani families with early-onset retinitis pigmentosa

Contains fulltext : 108208.pdf (publisher's version ) (Open Access)PURPOSE: To identify the genetic defects underlying retinitis pigmentosa (RP) in Pakistani families. METHODS: Genome-wide high-density single-nucleotide-polymorphism microarray analysis was performed using the DNA of nine affected individuals from two large families with multiple consanguineous marriages. Data were analyzed to identify homozygous regions that are shared by affected sibs in each family. Sanger sequencing was performed for genes previously implicated in autosomal recessive RP and allied retinal dystrophies that resided in the identified homozygous regions. Probands from both families underwent fundus examination and electroretinogram measurements. RESULTS: The tubby-like protein 1 gene (TULP1) was present in the largest homozygous region in both families. Sequence analysis identified a previously reported mutation (c.1138A>G; p.Thr380Ala) in one family and a novel pathogenic variant (c.1445G>A; p.Arg482Gln) in the other family. Both variants were found to be present in a homozygous state in all affected individuals, were heterozygous present in the unaffected parents, and heterozygous present or absent in normal individuals. Affected individuals of both families showed an early-onset form of RP. CONCLUSIONS: Homozygosity mapping, combined with candidate-gene analysis, successfully identified genetic defects in TULP1 in two large Pakistani families with early-onset retinitis pigmentosa

Novel CNGA3 and CNGB3 mutations in two Pakistani families with achromatopsia

PURPOSE: To identify the genetic defect in two Pakistani families with autosomal recessive achromatopsia. METHODS: Two families (RP26 and RP44) were originally diagnosed with retinal dystrophy based upon their medical history. To localize the causative genes in these families, homozygosity mapping was performed using Affymetrix 10K single nucleotide polymorphism (SNP) arrays. Sequence analysis was used to find the mutations in candidate genes cyclic nucleotide-gated channel alpha-3 (CNGA3; family RP26) and cyclic nucleotide-gated channel beta-3 (CNGB3; family RP44). Control individuals were analyzed by allele-specific PCR for the CNGA3 mutation and BstXI restriction analysis for the CNGB3 mutation. After genetic analysis, clinical diagnosis was re-evaluated by electroretinography and color vision testing. During the course of this study, selected affected members of family RP26 were given pink glasses as supportive therapy. RESULTS: Sequence analysis of the positional candidate genes identified a novel missense mutation in CNGA3 (c.822G>T; p.R274S) in family RP26, and a novel CNGB3 frameshift mutation (c.1825delG; p.V609WfsX9) in family RP44. Clinical re-evaluation after genetic analysis revealed that both families have segregating autosomal recessive achromatopsia. CONCLUSIONS: Genetic analysis of two Pakistani families with retinal disease enabled the establishment of the correct diagnosis of achromatopsia. Two novel mutations were identified in CNGA3 and CNGB3 that are both specifically expressed in cone photoreceptors. Re-evaluation of the clinical status revealed that both families had achromatopsia. The use of pink glasses in patients was helpful in reducing photophobia and enabled rod-mediated vision

A homozygous p.Glu150Lys mutation in the opsin gene of two Pakistani families with autosomal recessive retinitis pigmentosa

PURPOSE: To identify the gene mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in Pakistani families. METHODS: A cohort of consanguineous families with typical RP phenotype in patients was screened by homozygosity mapping using microsatellite markers that mapped close to 21 known arRP genes and five arRP loci. Mutation analysis was performed by direct sequencing of the candidate gene. RESULTS: In two families, RP21 and RP53, homozygosity mapping suggested RHO, the gene encoding rhodopsin, as a candidate disease gene on chromosome 3q21. In six out of seven affected members from the two families, direct sequencing of RHO identified a homozygous c.448G>A mutation resulting in the p.Glu150Lys amino acid change. This variant was first reported in PMK197, an Indian arRP family. Single nucleotide polymorphism analysis in RP21, RP53, and PMK197 showed a common disease-associated haplotype in the three families. CONCLUSIONS: In two consanguineous Pakistani families with typical arRP phenotype in the patients, we identified a disease-causing mutation (p.Glu150Lys) in the RHO gene. Single nucleotide polymorphism analysis suggests that the previously reported Indian family (PMK197) and the two Pakistani families studied here share the RHO p.Glu150Lys mutation due to a common ancestry

Whole-exome sequencing reveals ZNF408 as a new gene associated with autosomal recessive retinitis pigmentosa with vitreal alterations

Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies that cause visual impairment as a result of photoreceptor cell death. RP is heterogeneous, both clinically and genetically making difficult to establish precise genotype–phenotype correlations. In a Spanish family with autosomal recessive RP (arRP), homozygosity mapping and whole-exome sequencing led to the identification of a homozygous mutation (c.358_359delGT; p.Ala122Leufs*2) in the ZNF408 gene. A screening performed in 217 additional unrelated families revealed another homozygous mutation (c.1621C>T; p.Arg541Cys) in an isolated RP case. ZNF408 encodes a transcription factor that harbors 10 predicted C2H2-type fingers thought to be implicated in DNA binding. To elucidate the ZNF408 role in the retina and the pathogenesis of these mutations we have performed different functional studies. By immunohistochemical analysis in healthy human retina, we identified that ZNF408 is expressed in both cone and rod photoreceptors, in a specific type of amacrine and ganglion cells, and in retinal blood vessels. ZNF408 revealed a cytoplasmic localization and a nuclear distribution in areas corresponding with the euchromatin fraction. Immunolocalization studies showed a partial mislocalization of the p.Arg541Cys mutant protein retaining part of the WT protein in the cytoplasm. Our study demonstrates that ZNF408, previously associated with Familial Exudative Vitreoretinopathy (FEVR), is a new gene causing arRP with vitreous condensations supporting the evidence that this protein plays additional functions into the human retina.This work is supported by CIBERER (06/07/0036), FIS (PI013/00226), Ministry of Economy and Competitiveness-FEDER (BFU2012-36845), RETICS (RD12/0034/0010), Fundación ONCE, Fundaluce and grants BIO2011-27069 from the Spanish Ministry of Economy and Competitiveness, and PROMETEOII/2014/025 from the Conselleria de Educacio of the Valencia Community. PC is supported by Fundación Conchita Rábago (FCR), MC by Miguel Servet ISCIII (CP/03256) and dS by CAPES Foundation, Ministry of Education of Brazil

Delivery of oligonucleotide-based therapeutics : challenges and opportunities

Funding Information: This work was supported by funding from Cooperation of Science and Technology (COST) Action CA17103 (networking grant to V.A-G). V.A-G holds a Miguel Servet Fellowship from the ISCIII [grant reference CPII17/00004] that is part-funded by the European Regional Development Fund (ERDF/FEDER) and also acknowledges funding from Ikerbasque (Basque Foundation for Science). S.M.H is funded by the Medical Research Council and Muscular Dystrophy UK. A.A-R receives funding from amongst others the Duchenne Parent Project, Spieren voor Spieren, the Prinses Beatrix Spierfonds, Duchenne UK and through Horizon2020 project BIND. A.G and R.W.J.C are supported by several foundations including the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Stichting Blinden-Penning, Landelijke Stichting voor Blinden en Slechtzienden, Stichting Oogfonds Nederland, Stichting Macula Degeneratie Fonds, and Stichting Retina Nederland Fonds (who contributed through UitZicht 2015-31 and 2018-21), together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, Stichting voor Ooglijders, and Stichting Dowilvo; as well as the Foundation Fighting Blindness USA, grant no. PPA-0517-0717-RAD. R.A.M.B is supported by Hersenstichting Nederland Grant DR-2018-00253. G.G. is supported by Ministry of Research and Innovation in Romania/National Program 31N/2016/PN 16.22.02.05. S.A is supported by Project PTDC/BBB-BMD/6301/2014 (Funda??o para a Ci?ncia e a Tecnologia?MCTES, Portugal). L.R.D. is supported by Fundaci?n Ram?n Areces Grant XVII CN and Spanish Ministry of Science and Innovation (MICINN, grant PID2019-105344RB-I00). T.L is supported by Estonian Research Council grant PSG226. S.K is supported by the Friedrich-Baur-Stiftung. C.F is funded by The Danish Council for Independent Research, Technology and Production Sciences (grant number DFF-4184-00422). W.vRM is supported by ZonMw Programme Translational Research 2 [Project number 446002002], Campaign Team Huntington and AFM Telethon [Project number 20577]. S.E.B is supported by the H2020 projects B-SMART, Grant number 721058, and REFINE, Grant number 761104. A.T.G is supported by the Institut National de la sant? et la recherche m?dicale (INSERM) and the Association Monegasque contre les myopathies (AMM). L.E. is founded by the Association Monegasque contre les myopathies (AMM). Publisher Copyright: © 2021 The Authors. Published under the terms of the CC BY 4.0 licenseNucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid-based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid-based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide-based therapeutics.publishersversionPeer reviewe

Genetic landscape of 6089 inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications

Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes were PRPH2, ABCA4 and RS1 in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; RHO, USH2A and RPGR in AD, AR and XL for non-syndromic RP; and USH2A and MYO7A in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in ABCA4 and c.2276G > T (p.Cys759Phe) in USH2A were the most frequent variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis, counselling and new therapeutic strategies to both the Spanish population and other related populations.This work was supported by the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Health (FIS; PI16/00425 and PI19/00321), Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER, 06/07/0036), IIS-FJD BioBank (PT13/0010/0012), Comunidad de Madrid (CAM, RAREGenomics Project, B2017/BMD-3721), European Regional Development Fund (FEDER), the Organización Nacional de Ciegos Españoles (ONCE), Fundación Ramón Areces, Fundación Conchita Rábago and the University Chair UAM-IIS-FJD of Genomic Medicine. Irene Perea-Romero is supported by a PhD fellowship from the predoctoral Program from ISCIII (FI17/00192). Ionut F. Iancu is supported by a grant from the Comunidad de Madrid (CAM, PEJ-2017-AI/BMD7256). Marta del Pozo-Valero is supported by a PhD grant from the Fundación Conchita Rábago. Berta Almoguera is supported by a Juan Rodes program from ISCIII (JR17/00020). Pablo Minguez is supported by a Miguel Servet program from ISCIII (CP16/00116). Marta Corton is supported by a Miguel Servet program from ISCIII (CPII17/00006). The funders played no role in study design, data collection, data analysis, manuscript preparation and/or publication decisions

Generation of three isogenic control lines from patient-derived iPSCs carrying bi-allelic ABCA4 variants underlying Stargardt disease

Stargardt disease, a progressive retinal disorder, is associated with bi-allelic variants in ABCA4. Employing the CRISPR/Cas9 approach, we generated isogenic control lines (RMCGENi005-A-1, RMCGENi018-A-1, RMCGENi017-A-1) for each of three induced pluripotent stem cell lines (RMCGENi005-A, RMCGENi018-A, RMCGENi017-A) derived from Stargardt patients carrying compound heterozygous ABCA4 variants. All of the generated lines showed pluripotent characteristics, no chromosomal aberrations and no indication of off-targets. The availability of these isogenic control lines will allow us to have a fair comparison between health and disease state within our studies on Stargardt disease

Generation of iPSC lines from three Stargardt patients carrying bi-allelic ABCA4 variants

Stargardt disease, a progressive retinal disorder, is associated with bi-allelic variants in ABCA4, a protein that is expressed in the retina. Induced pluripotent stem cell lines (RMCGENi005-A, SCTCi018-A, SCTCi017-A) were generated by lentivirus reprogramming of fibroblasts derived from Stargardt patients carrying different bi-allelic ABCA4 variants. All the generated lines showed pluripotent characteristics and no chromosomal aberrations. The availability of these lines will allow us to generate patient-derived photoreceptor precursor cells and retinal organoids to further study ABCA4 and thereby, Stargardt disease in relevant model systems carrying the patient’s genetic background