Generation of a human iPS cell line from a patient with retinitis pigmentosa due to EYS mutation

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease. Mutations in EYS have been associated with autosomal recessive RP. The human iPS cell line, CABi002-A, derived from peripheral blood mononuclear cells from a patient carrying a heterozygous double mutation in EYS gene was generated by non-integrative reprogramming technology, using hOCT3/4, hSOX2, hc-MYC and hKLF4 reprogramming factors. Pluripotency and differentiation capacity were assessed by immunocytochemistry and RT-PCR. This iPSC line can be further differentiated towards the affected cells to understand the pathophysiology of the disease and test new therapeutic strategies.Cellex FoundationFundación Progreso y Salu

Disease-causing mutations in BEST1 gene are associated with altered sorting of bestrophin-1 protein

Doumanov, Jordan A. et al.Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery. © 2013 by the authors; licensee MDPI, Basel, Switzerland.This work was supported by Agence Nationale de la Recherche (Chaire d’Excellence to Shomi S. Bhattacharya), Fondation Voir et Entendre (Young Investigator Grants to Christina Zeitz and Emeline F. Nandrot), Centre National de la Recherche Scientifique (CNRS) and Fondation Bettencourt Schueller (to Emeline F. Nandrot), Université Pierre et Marie Curie-Paris6 (Bonus Qualité Recherche to Christina Zeitz), Foundation Fighting Blindness (grant number CD-CL-0808-0466-CHNO to Isabelle Audo), Centre d’Investigation Clinique 503 recognized as a Foundation Fighting Blindness Center (grant number C-CMM-0907-0428-INSERM04), Fundacion Progreso y Salud (to Shomi S. Bhattacharya, Maria Luz Bellido Diaz, Abhay Krishna and Paloma Dominguez Gimenez), Instituto de Salud Carlos III (grant number CM06/00183 to Maria Luz Bellido Diaz) and Bulgarian National Science Fund (grant number DDVU 02/10). Additionally, the Institut de la Vision is funded by Institut National de la Santé et de la Recherche Médicale, Université Pierre et Marie Curie-Paris 6, Centre National de la Recherche Scientifique and Départment de Paris.Peer Reviewe

Human iPSC derived disease model of MERTK-associated retinitis pigmentosa

Retinitis pigmentosa (RP) represents a genetically heterogeneous group of retinal dystrophies affecting mainly the rod photoreceptors and in some instances also the retinal pigment epithelium (RPE) cells of the retina. Clinical symptoms and disease progression leading to moderate to severe loss of vision are well established and despite significant progress in the identification of causative genes, the disease pathology remains unclear. Lack of this understanding has so far hindered development of effective therapies. Here we report successful generation of human induced pluripotent stem cells (iPSC) from skin fibroblasts of a patient harboring a novel Ser331Cysfs*5 mutation in the MERTK gene. The patient was diagnosed with an early onset and severe form of autosomal recessive RP (arRP). Upon differentiation of these iPSC towards RPE, patient-specific RPE cells exhibited defective phagocytosis, a characteristic phenotype of MERTK deficiency observed in human patients and animal models. Thus we have created a faithful cellular model of arRP incorporating the human genetic background which will allow us to investigate in detail the disease mechanism, explore screening of a variety of therapeutic compounds/reagents and design either combined cell and gene- based therapies or independent approaches.This work was supported by Andalusian Health Council (PI-0324-2013), Instituto de Salud Carlos III (PI13/01331), Spanish Ministry of Economy and Competitiveness-FEDER BFU2012-36845, Instituto de Salud Carlos III RETICS RD12/0034/0010 and Academy of Finland (218050; 272808)

A novel 1-bp deletion in PITX3 causing congenital posterior polar cataract

Purpose: Cataracts are the most common cause of blindness worldwide. Inherited cataract is a clinically and genetically heterogeneous disease. Here we report a novel mutation in the paired-like homeodomain 3 (PITX3) gene segregating in a four generation English family with an isolated autosomal dominant posterior polar cataract.Methods: A genome-wide linkage was performed by means of single nucleotide polymorphism (SNP) and microsatellite markers. Linkage analyses were performed with the GeneHunter and MLINK programs. Direct sequencing of PCR products was performed to detect mutation in the gene, using the BigDye version 3.1 and analyzed using Sequence analysis version 5.2.Results: Genome-wide linkage analysis with SNP markers, identified a disease-haplotype interval on chromosome 10q. Two point positive logarithm of odds (LOD) scores was obtained with markers D10S205 (Z=3.10 at theta=0.00), flanked by markers D10S1709 and D10S543, which harbors the homeobox gene PITX3. Sequence analysis of PITX3 revealed a 1-bp deletion that cosegregated with all the affected members of this family which resulted in a frameshift in codon 181 and likely to produce an aberrant protein consisting of 127 additional residues.Conclusions: The 542delC is a novel mutation in PITX3 causing an isolated posterior polar cataract

Developmental expression profile of the optic atrophy gene product: OPA1 is not localized exclusively in the mammalian retinal ganglion cell layer

PURPOSE: Autosomal dominant optic atrophy (ADOA) is characterized by primary degeneration of retinal ganglion cells and atrophy of the optic nerve. The OPA1 gene encodes a 960-amino-acid protein. In the current study the temporal and spatial localization of OPA1 were examined in developing and adult murine ocular tissues and the adult human eye. Because the Bst/+ mouse has been postulated as a model of ADOA, the mOPA1 expression in the Bst/+ retina was also examined. METHODS: A polyclonal antibody generated against a C-terminal peptide of OPA1 was used to assess by immunohistochemistry the expression of mOPA1 in the wild-type embryonic and postnatal mouse ocular tissues and the Bst/+ retina. Western blot analyses of total proteins from a panel of adult human tissues were used to examine the expression of human OPA1, and spatial localization was assessed by immunohistochemistry. RESULTS: The ocular expression of mOPA1 begins at E15 in the inner retina in a location corresponding to that of the subsequently developing ganglion cell layer (GCL) and peaks between postnatal day (P)0 and P1 in the retina and the optic nerve. There is a sharp decline in mOPA1 expression after P2, but it is expressed at a basal level until at least P12 in the GCL, inner plexiform layer (IPL), and inner nuclear layer (INL) of the retina as well as in the optic nerve. In the adult Bst/+ retina, mOPA1 is strongly expressed in the GCL and IPL and weakly in the INL. In the adult human eye, OPA1 is expressed in the GCL, IPL, INL, and outer plexiform layer (OPL) of the retina and in the optic nerve, where it is observed only in the myelinated region. CONCLUSIONS: OPA1 is not restricted to the GCL of the mammalian retina, and its expression extends into the IPL, INL, and OPL. OPA1 is distinctly expressed in the myelinated region beyond the lamina cribrosa in the human optic nerve, whereas its expression is weaker in the mouse optic nerve. In the Bst/+ mouse retina, despite the structural defects, mOPA1 expression is comparable to that observed in the wild-type adult mouse retina. These observations suggest a wider role for OPA1 than previously anticipated

NMNAT1 Mutations Cause Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss. Two-thirds of LCA cases are caused by mutations in 17 known disease genes (RetNet Retinal Information Network). Using exome sequencing, we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 as likely disease-causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in their kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide $(NAD^+)$ biosynthesis. Functional studies showed the p.Val9Met mutation decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated LCA families identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease and indicate that NMNAT1 mutations cause LCA

Disease mechanism for retinitis pigmentosa (RP11) caused by missense mutations in the splicing factor gene PRPF31

Purpose: Missense mutations in the splicing factor gene PRPF31 cause a dominant form of retinitis pigmentosa (RP11) with reduced penetrance. Missense mutations in PRPF31 have previously been shown to cause reduced protein solubility, suggesting insufficiency of functional protein as the disease mechanism. Here we examine in further detail the effect of the A216P mutation on splicing function. Methods: Splicing activity was assayed using an in vivo assay in transfected mammalian cells with rhodopsin (RHO) and transducin (GNAT1) splicing templates. Pull-down assays were used to study the interaction between PRPF31 and one of its cognate partners in the spliceosome, PRPF6. Results: Splicing of RHO intron 3 and GNAT1 introns 3-5 mini-gene templates was inefficient with both spliced and unspliced products clearly detected. Assays using the RHO minigene template revealed a direct negative effect on splicing efficiency of the mutant. However, no effect of the mutation on splicing efficiency could be detected using the longer GNAT1 minigene template or using a full-length RHO transcript, splicing of which had an efficiency of 100%. No unspliced RHO transcripts could be detected in RNA from human retina. Pull-down assays between PRPF31 and PRPF6 proteins showed a stronger interaction for the mutant than wild type, suggesting a mechanism for the negative effect. Conclusions: Splicing of full-length RHO is more efficient than splicing of the minigene, and assays using a full-length template more accurately mimic splicing in photoreceptors. The RP11 missense mutations exert their pathology mainly via a mechanism based on protein insufficiency due to protein insolubility, but there is also a minor direct negative effect on function