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

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

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

Biallelic mutation of Protocadherin-21 (PCDH21) causes retinal degeneration in humans

PurposeTo describe the clinical findings and mutations in affected members of two families with an autosomal recessive retinal dystrophy associated with mutations in the protocadherin-21 (PCDH21) gene.MethodsA full genome scan of members of two consanguineous families segregating an autosomal recessive retinal dystrophy was performed and regions identical by descent identified. Positional candidate genes were identified and sequenced. All patients had a detailed ophthalmic examination, including electroretinography and retinal imaging.ResultsAffected members of both families showed identical homozygosity for an overlapping region of chromosome 10q. Sequencing of a candidate gene, PCDH21, showed two separate homozygous single-base deletions, c.337delG (p.G113AfsX1) and c.1459delG (p.G487GfsX20), which were not detected in 282 control chromosomes. Affected members of the two families first reported nyctalopia in late teenage years and retained good central vision until their late 30s. No color vision was detected in any proband. The fundus appearance included the later development of characteristic circular patches of pigment epithelial atrophy at the macula and in the peripheral retina.ConclusionsBiallelic mutations in the photoreceptor-specific gene PCDH21 cause recessive retinal degeneration in humans

Prevalence and novelty of PRPF31 mutations in French autosomal dominant rod-cone dystrophy patients and a review of published reports

Background: Rod-cone dystrophies are heterogeneous group of inherited retinal disorders both clinically and genetically characterized by photoreceptor degeneration. The mode of inheritance can be autosomal dominant, autosomal recessive or X-linked. The purpose of this study was to identify mutations in one of the genes, PRPF31, in French patients with autosomal dominant RP, to perform genotype-phenotype correlations of those patients, to determine the prevalence of PRPF31 mutations in this cohort and to review previously identified PRPF31 mutations from other cohorts.Methods: Detailed phenotypic characterization was performed including precise family history, best corrected visual acuity using the ETDRS chart, slit lamp examination, kinetic and static perimetry, full field and multifocal ERG, fundus autofluorescence imaging and optic coherence tomography. For genetic diagnosis, genomic DNA of ninety families was isolated by standard methods. The coding exons and flanking intronic regions of PRPF31 were PCR amplified, purified and sequenced in the index patient.Results: We showed for the first time that 6.7% cases of a French adRP cohort have a PRPF31 mutation. We identified in total six mutations, which were all novel and not detected in ethnically matched controls. The mutation spectrum from our cohort comprises frameshift and splice site mutations. Co-segregation analysis in available family members revealed that each index patient and all affected family members showed a heterozygous mutation. In five families incomplete penetrance was observed. Most patients showed classical signs of RP with relatively preserved central vision and visual field.Conclusion: Our studies extended the mutation spectrum of PRPF31 and as previously reported in other populations, it is a major cause of adRP in France

Caracterización de PRPF31

Motivación: La retinitis pigmentosa es un grupo heterogéneo de distrofias retinales caracterizada por una progresiva degeneración de los fotorreceptores, que acaba produciendo deficiencia visual o ceguera. PRPF31 es uno de los genes cuya mutación causa este tipo de distrofia. Aunque la proteína Prpf31 es un factor de splicing presente en todo el organismo, su mutación sólo produce distrofia retinal. Actualmente no se conoce el mecanismo implicado en la enfermadad, por lo que intentaremos discernirlo con ayuda de este proyecto.Métodos: Utilizando la secuencia del gen PRPF31, planteamos clonarla en el vector p3XFLAG y transfectar con la construcción células de mamífero. La fusión con FLAG facilitará los estudios de localización subcelular y de las interacciones por inmunoprecipitación. Transfectaremos con la construcción las líneas celulare RPE1, derivada de epitelio pigmentario de la retina (RPE) humana y 293, para estudiar la localización y las interacciones de Prpf31 con otras proteínas, con el objetivo de estudiar la base molecular de la enfermedad y poder desarrollar terapias génicas, farmacológicas y/o celulares. Resultados: La clonación de la secuencia de PRPF31 aún está en proceso ya que no hemos obtenido la construcción diseñada a pesar de probar diversos métodos. Se han empezado a poner a punto las condiciones de transfección usando otra construcción similar, tanto con la línea 293 como con RPE1. Se ha estudiado la distribución de Prpf31 en retina y RPE de ratón por western blot, mostrando una mayor abundancia en RPE, lo que no es común a otros factores de splicing como el PRPF3 y PRPF8, con los que se ha comparado. Conclusiones: PRPF31 debe tener alguna función específica en RPE, aún por definir, por lo que la degeneración de los fotorreceptores será secundaria a una disfunción del RPE. Hay otros casos en los que un gen presenta una función específicamente diferente en la retina, como en el caso de ATR, que causa síndrome de Seckel. ATR es un controlador de la división celular, pero en la retina este gen es crítico para el desarrollo postnatal de los fotorreceptores, por lo que su mutación causa degeneraciónen la retina