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

Retinal pigment epithelium degeneration caused by aggregation of PRPF31 and the role of HSP70 family of proteins

Background Mutations in pre-mRNA splicing factor PRPF31 can lead to retinitis pigmentosa (RP). Although the exact disease mechanism remains unknown, it has been hypothesized that haploinsufficiency might be involved in the pathophysiology of the disease. Methods In this study, we have analyzed a mouse model containing the p.A216P mutation in Prpf31 gene. Results We found that mutant Prpf31 protein produces cytoplasmic aggregates in the retinal pigment epithelium and decreasing the protein levels of this splicing factor in the nucleus. Additionally, normal protein was recruited in insoluble aggregates when the mutant protein was overexpressed in vitro. In response to protein aggregation, Hspa4l is overexpressed. This member of the HSP70 family of chaperones might contribute to the correct folding and solubilization of the mutant protein, allowing its translocation to the nucleus. Conclusions Our data suggests that a mechanism haploinsufficiency and dominant-negative is involved in retinal degeneration due to mutations in PRPF31. HSP70 over-expression might be a new therapeutic target for the treatment of retinal degeneration due to PRPF31 mutations.This project has been financed through a) The ISCIII (Miguel Servet-I, 2015), co-financed by the European Regional Development Fund (ERDF), No CP15/00071. b) The European Union’s Horizon 2020 research and innovation program, under grant agreement No 634479. c) Regional Ministry of Economy, Innovation and Science of the Junta de Andalucía, No P09-CTS-04967.info:eu-repo/semantics/publishedVersio

Biallelic Variants in TTLL5, Encoding a Tubulin Glutamylase, Cause Retinal Dystrophy

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with “cone-first” retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs∗2];[Glu529Valfs∗2], and c.[401delT(;)3354G>A], p.[Leu134Argfs∗45(;)Trp1118∗]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543∗) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families

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

Conducta suicida no letal en Castellón: un estudio descriptivo y comparativo.

Treball Final de Grau en Medicina. Codi: MD1158. Curs acadèmic: 2018/2019.Background: Due to the suicidal behavior variability regarding the sociocultural values of each territory, we conducted this study to define the sociodemographic and clinical features of the patients from Castellon. The objective is to obtain deeper knowledge that facilitate the detection and implementation of effective interventions. In addition, we carried out a comparative analysis between non-lethal suicide cases (our sample) and completed suicide cases. Methods: Our sample includes every person assisted for non-lethal suicidal behavior in the Psychiatric Emergency room at Castellon’s Provincial Hospital from January 19th to February 15th, performing an 8 year follow-up. Our completed suicide sample was taken from a previous study conducted in 2018.2 Results: Our sample showed an average age of 39, with a majority of women (56.1%) and psychiatric pathology diagnosis (74.2%, mainly anxiety-depressive disorders). Most of them were being followed by a health service (81.2%, generally their GP). The most frequent suicide method was drug overdose (94.4%). After the suicide episode, only 27 patients (40.9%) attended the healthcare service they were referred to, what may explain the persistance of the suicidal behavior during the follow-up (7.7% suicidal threats and 34.5% attempts) Conclusions: Non-lethal suicidal behavior in Castellon is more frequent among young, women, and those with psychiatric disorders, using as main suicidal method drug overdose. Most of them don’t attend the health care service responsible for their follow-up. Moreover, it was found that the completed suicide profile differs from the non-lethal; it is therefore required to develop different therapeutical and preventive strategies for each one.Introducción: Dada la variabilidad en la conducta suicida según los valores socioculturales de cada territorio, realizamos este estudio para definir las características sociodemográficas y clínicas de los pacientes con comportamiento suicida no letal de nuestra provincia. Pretendemos ampliar nuestro conocimiento para facilitar la detección de individuos en riesgo e implantar intervenciones efectivas. Además, realizamos una comparativa entre las características de nuestra muestra con las de suicidio consumado. Material y métodos: La muestra incluye toda conducta suicida no letal registrada en el Hospital Provincial de Castellón entre el 19 de enero y 15 de febrero de 2009, realizándose un seguimiento hasta febrero de 2017. La muestra de suicidio consumado es tomada de un proyecto previo de 2018. Resultados: La muestra la integran mayormente individuos jóvenes (media de 39 años), de sexo femenino (56.1%), con patología psiquiátrica (74.2%, principalmente trastornos ansioso-depresivos) y en seguimiento sanitario (81.2%, predominantemente MAP (77.3%)). El método de suicidio más frecuente fue la sobreingesta medicamentosa (94.4%). Sólo 27 pacientes (40.9%) acudieron tras el episodio a los servicios de salud remitidos, facilitándose así la persistencia de la conducta durante el seguimiento (7.7% amenazas, 34.5% tentativas). Conclusiones: La conducta suicida no letal de Castellón predomina entre adultos jóvenes, de sexo femenino, con trastornos psiquiátricos, que recurren como método a la sobredosis medicamentosa e incumplen el seguimiento a posteriori. Además, se comprobó que el perfil del comportamiento suicida letal difiere de los rasgos de la conducta suicida no letal, evidenciando la necesidad de desarrollar estrategias preventivas y terapéuticas distintas entre ellos

A common allele in RPGRIP1L is a modifier of retinal degeneration in ciliopathies

Despite rapid advances in the identification of genes involved in disease, the predictive power of the genotype remains limited, in part owing to poorly understood effects of second-site modifiers. Here we demonstrate that a polymorphic coding variant of RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein-1 like), a ciliary gene mutated in Meckel-Gruber (MKS) and Joubert (JBTS) syndromes, is associated with the development of retinal degeneration in individuals with ciliopathies caused by mutations in other genes. As part of our resequencing efforts of the ciliary proteome, we identified several putative loss-of-function RPGRIP1L mutations, including one common variant, A229T. Multiple genetic lines of evidence showed this allele to be associated with photoreceptor loss in ciliopathies. Moreover, we show that RPGRIP1L interacts biochemically with RPGR, loss of which causes retinal degeneration, and that the Thr229-encoded protein significantly compromises this interaction. Our data represent an example of modification of a discrete phenotype of syndromic disease and highlight the importance of a multifaceted approach for the discovery of modifier alleles of intermediate frequency and effect.This work was supported by grants R01EY007961 from the National Eye Institute (H.K. and A.S.), R01HD04260 from the National Institute of Child Health and Development (N.K.), R01DK072301, R01DK075972 (N.K.), R01DK068306, R01DK064614, R01DK069274 (F.H.), NRSA fellowship F32 DK079541 (E.E.D.) from the National Institute of Diabetes, Digestive and Kidney disorders, Intramural program of NEI (A.S.), the Macular Vision Research Foundation (N.K.), the Foundation for Fighting Blindness (H.K., S.S.B., A.S. and N.K.), the Foundation for Fighting Blindness Canada (R.K.K.), Le Fonds de la recherche en sante du Québec (FRSQ) (R.K.K.), Research to Prevent Blindness (A.S.), Harold Falls Collegiate Professorship (A.S.), the Midwest Eye Banks and Transplantation Center (H.K.), the Searle Scholars Program (M.A.B.), the Deutsche Forschungsgemeinschaft (DFG grant BE 3910/4-1; C.B.) the UK Medical Research Council (grant number G0700073; C.A.J.), NIHR Biomedical Research Centre for Ophthalmology (S.S.B.) and EU-GENORET Grant LSHG-CT-2005-512036 (S.S.B.). F.H. is an investigator of the Howard Hughes Medical Institute (HHMI) and a Doris Duke Distinguished Clinical Scientist (DDCF)

Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy

A novel locus for autosomal dominant cone-rod dystrophy maps to chromosome 10q

Kamenarova, Kunka et al.Here we report recruitment of a three-generation Romani (Gypsy) family with autosomal dominant cone-rod dystrophy (adCORD). Involvement of known adCORD genes was excluded by microsatellite (STR) genotyping and linkage analysis. Subsequently, two independent total-genome scans using STR markers and single-nucleotide polymorphisms (SNPs) were performed. Haplotype analysis revealed a single 6.7-Mb novel locus between markers D10S1757 and D10S1782 linked to the disease phenotype on chromosome 10q26. Linkage analysis gave a maximum LOD score of 3.31 for five fully informative STR markers within the linked interval corresponding to the expected maximum in the family. Multipoint linkage analysis of SNP genotypes yielded a maximum parametric linkage score of 2.71 with markers located in the same chromosomal interval. There is no previously mapped CORD locus in this interval, and therefore the data reported here is novel and likely to identify a new gene that may eventually contribute to new knowledge on the pathogenesis of this condition. Sequencing of several candidate genes within the mapped interval led to negative findings in terms of the underlying molecular pathogenesis of the disease in the family. Analysis by comparative genomic hybridization excluded large chromosomal aberrations as causative of adCORD in the pedigree.This work was supported by grants from: Fundación Progresso y Salud (Project No: 113.GI02.0.0000), Spain; Foundation Fighting Blindness (USA); RP Fighting Blindness (UK); National Science Fund, Bulgarian Ministry of Education, Youth and Science (Contract G-3/2004). The Molecular Medicine Center was supported by infrastructure grants from: National Science Fund, Bulgarian Ministry of Education, Youth and Science (DUNK01-2/2009) and the Science Fund, Medical University – Sofia (8I/2009).Peer Reviewe

TOPORS, a dual E3 ubiquitin and Sumo1 ligase, interacts with 26 S protease regulatory subunit 4, encoded by the PSMC1 gene

The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS.The work was funded by the following: 1. BRITISH EYE RESEARCH FOUNDATION (Fight for Sight; charity number 1111438): www.fightforsight.org.uk (PhD Studentship 1816 grant to BC and SSB); 2. THE TERESA ROSENBAUM GOLDEN CHARITABLE TRUST (Rosetrees Trust; charity number 298582): www.rosetreestrust.co.uk (Grant number M233-CD1 to BC AZS GA SSB); 3. THE SPECIAL TRUSTEES OF MOORFIELDS EYE HOSPITAL GENERAL FUND (Charity number 228064): www.moorfields.nhs.uk (Personal award to SSB for infrastructures); 4. MOORFIELDS EYE CHARITY (Moorfields Eye Hospital NHS Foundation Trust; charity number 1140679): www.moorfields.nhs.uk (Personal award to SSB for infrastructures); and 5. National Institute for Health Research: http://www.nihr.ac.uk (Personal award to SSB for infrastructures).Peer Reviewe