视网膜色素变性的分子遗传学研究进展

视网膜色素变性（RP）是常见的遗传性眼病,具有高度的遗传异质性,患者常有进行性夜盲和视野缺损。对于常染色体显性遗传RP已经发现有12个基因座与之有关,其中6个致病基因已被克隆,对于这些致病基因的结构、突变及其功能目前已有了新的研究进展。 Abstract:Retinitis pigmentosa (RP) describes a genetically and clinically heterogeneous group of disorders that are characterized by gradual degeneration of photoreceptor cells.Common clinical features include a progressive loss of night vision,leading to night blindness and peripheral-visual-field loss.At least 12 loci have been mapped to chromosomes,and mutations in an ever increasing number of genes have been found to cause autosomal dominant retinitis pigmentosa (ADRP).Six of the 12 genes known to cause ADRP have been cloned.New progress has been made on the studies of structure,mutation and function of these genes.     

A Novel Missense SNRNP200 Mutation Associated with Autosomal Dominant Retinitis Pigmentosa in a Chinese Family

The SNRNP200 gene encodes hBrr2, a helicase essential for pre-mRNA splicing. Six mutations in SNRNP200 have recently been discovered to be associated with autosomal dominant retinitis pigmentosa (adRP). In this work, we analyzed a Chinese family with adRP and identified a novel missense mutation in SNRNP200. To identify the genetic defect in this family, exome of the proband was captured and sequencing analysis was performed to exclude known genetic defects and find possible pathogenic mutations. Subsequently, candidate mutations were validated in affected family members using Sanger sequencing. A novel missense mutation, c.2653C>G transition (p.Q885E), in exon 20 of SNRNP200 was identified. The mutation co-segregated with the disease phenotype over four generations and was absent in 100 normal unaffected individuals. This mutation occurs at highly conserved position in hBrr2 and is predicted to have a functional impact, suggesting that hBrr2-dependent small nuclear riboproteins (snRNPs) unwinding and spliceosome activation is important in the pathogenesis of some variants of RP.     

A Novel PRPF31 Mutation in a Large Chinese Family with Autosomal Dominant Retinitis Pigmentosa and Macular Degeneration

Purpose

This study was intended to identify the disease causing genes in a large Chinese family with autosomal dominant retinitis pigmentosa and macular degeneration.

Methods

A genome scan analysis was conducted in this family for disease gene preliminary mapping. Snapshot analysis of selected SNPs for two-point LOD score analysis for candidate gene filter. Candidate gene PRPF31 whole exons'' sequencing was executed to identify mutations.

Results

A novel nonsense mutation caused by an insertion was found in PRPF31 gene. All the 19 RP patients in 1085 family are carrying this heterozygous nonsense mutation. The nonsense mutation is in PRPF31 gene exon9 at chr19:54629961-54629961, inserting nucleotide “A” that generates the coding protein frame shift from p.307 and early termination at p.322 in the snoRNA binding domain (NOP domain).

Conclusion

This report is the first to associate PRPF31 gene''s nonsense mutation and adRP and JMD. Our findings revealed that PRPF31 can lead to different clinical phenotypes in the same family, resulting either in adRP or syndrome of adRP and JMD. We believe our identification of the novel “A” insertion mutation in exon9 at chr19:54629961-54629961 in PRPF31 can provide further genetic evidence for clinical test for adRP and JMD.     

The Severe Autosomal Dominant Retinitis Pigmentosa Rhodopsin Mutant Ter349Glu Mislocalizes and Induces Rapid Rod Cell Death

Mutations in the rhodopsin gene cause approximately one-tenth of retinitis pigmentosa cases worldwide, and most result in endoplasmic reticulum retention and apoptosis. Other rhodopsin mutations cause receptor mislocalization, diminished/constitutive activity, or faulty protein-protein interactions. The purpose of this study was to test for mechanisms by which the autosomal dominant rhodopsin mutation Ter349Glu causes an early, rapid retinal degeneration in patients. The mutation adds an additional 51 amino acids to the C terminus of the protein. Folding and ligand interaction of Ter349Glu rhodopsin were tested by ultraviolet-visible (UV-visible) spectrophotometry. The ability of the mutant to initiate phototransduction was tested using a radioactive filter binding assay. Photoreceptor localization was assessed both in vitro and in vivo utilizing fluorescent immunochemistry on transfected cells, transgenic Xenopus laevis, and knock-in mice. Photoreceptor ultrastructure was observed by transmission electron microscopy. Spectrally, Ter349Glu rhodopsin behaves similarly to wild-type rhodopsin, absorbing maximally at 500 nm. The mutant protein also displays in vitro G protein activation similar to that of WT. In cultured cells, mislocalization was observed at high expression levels whereas ciliary localization occurred at low expression levels. Similarly, transgenic X. laevis expressing Ter349Glu rhodopsin exhibited partial mislocalization. Analysis of the Ter349Glu rhodopsin knock-in mouse showed a rapid, early onset degeneration in homozygotes with a loss of proper rod outer segment development and improper disc formation. Together, the data show that both mislocalization and rod outer segment morphogenesis are likely associated with the human phenotype.     

Structure-Function Modeling of Optical Coherence Tomography and Standard Automated Perimetry in the Retina of Patients with Autosomal Dominant Retinitis Pigmentosa

Purpose

To assess relationships between structural and functional biomarkers, including new topographic measures of visual field sensitivity, in patients with autosomal dominant retinitis pigmentosa.

Methods

Spectral domain optical coherence tomography line scans and hill of vision (HOV) sensitivity surfaces from full-field standard automated perimetry were semi-automatically aligned for 60 eyes of 35 patients. Structural biomarkers were extracted from outer retina b-scans along horizontal and vertical midlines. Functional biomarkers were extracted from local sensitivity profiles along the b-scans and from the full visual field. These included topographic measures of functional transition such as the contour of most rapid sensitivity decline around the HOV, herein called HOV slope for convenience. Biomarker relationships were assessed pairwise by coefficients of determination (R²) from mixed-effects analysis with automatic model selection.

Results

Structure-function relationships were accurately modeled (conditional R²>0.8 in most cases). The best-fit relationship models and correlation patterns for horizontally oriented biomarkers were different than vertically oriented ones. The structural biomarker with the largest number of significant functional correlates was the ellipsoid zone (EZ) width, followed by the total photoreceptor layer thickness. The strongest correlation observed was between EZ width and HOV slope distance (marginal R² = 0.85, p<10⁻¹⁰). The mean sensitivity defect at the EZ edge was 7.6 dB. Among all functional biomarkers, the HOV slope mean value, HOV slope mean distance, and maximum sensitivity along the b-scan had the largest number of significant structural correlates.

Conclusions

Topographic slope metrics show promise as functional biomarkers relevant to the transition zone. EZ width is strongly associated with the location of most rapid HOV decline.     

Mutations in ARL2BP,Encoding ADP-Ribosylation-Factor-Like 2 Binding Protein,Cause Autosomal-Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal degeneration characterized by photoreceptor death, which results in visual failure. Here, we used a combination of homozygosity mapping and exome sequencing to identify mutations in ARL2BP, which encodes an effector protein of the small GTPases ARL2 and ARL3, as causative for autosomal-recessive RP (RP66). In a family affected by RP and situs inversus, a homozygous, splice-acceptor mutation, c.101−1G>C, which alters pre-mRNA splicing of ARLBP2 in blood RNA, was identified. In another family, a homozygous c.134T>G (p.Met45Arg) mutation was identified. In the mouse retina, ARL2BP localized to the basal body and cilium-associated centriole of photoreceptors and the periciliary extension of the inner segment. Depletion of ARL2BP caused cilia shortening. Moreover, depletion of ARL2, but not ARL3, caused displacement of ARL2BP from the basal body, suggesting that ARL2 is vital for recruiting or anchoring ARL2BP at the base of the cilium. This hypothesis is supported by the finding that the p.Met45Arg amino acid substitution reduced binding to ARL2 and caused the loss of ARL2BP localization at the basal body in ciliated nasal epithelial cells. These data demonstrate a role for ARL2BP and ARL2 in primary cilia function and that this role is essential for normal photoreceptor maintenance and function.     

Whole Exome Analysis Identifies Frequent CNGA1 Mutations in Japanese Population with Autosomal Recessive Retinitis Pigmentosa

Objective

The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population.

Methods

In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed.

Results

Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation.

Conclusions

This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.     

A Likelihood-Based Framework for Variant Calling and De Novo Mutation Detection in Families

Family samples, which can be enriched for rare causal variants by focusing on families with multiple extreme individuals and which facilitate detection of de novo mutation events, provide an attractive resource for next-generation sequencing studies. Here, we describe, implement, and evaluate a likelihood-based framework for analysis of next generation sequence data in family samples. Our framework is able to identify variant sites accurately and to assign individual genotypes, and can handle de novo mutation events, increasing the sensitivity and specificity of variant calling and de novo mutation detection. Through simulations we show explicit modeling of family relationships is especially useful for analyses of low-frequency variants and that genotype accuracy increases with the number of individuals sequenced per family. Compared with the standard approach of ignoring relatedness, our methods identify and accurately genotype more variants, and have high specificity for detecting de novo mutation events. The improvement in accuracy using our methods over the standard approach is particularly pronounced for low-frequency variants. Furthermore the family-aware calling framework dramatically reduces Mendelian inconsistencies and is beneficial for family-based analysis. We hope our framework and software will facilitate continuing efforts to identify genetic factors underlying human diseases.     

Dominant Retinitis Pigmentosa,p.Gly56Arg Mutation in NR2E3: Phenotype in a Large Cohort of 24 Cases

Importance

This research is the single largest NR2E3 genotype-phenotype correlation study performed to date in autosomal dominant Retinitis Pigmentosa.

Objective

The aim of this study is to analyse the frequency of the p.Gly56Arg mutation in NR2E3 for the largest cohort of autosomal dominant Retinitis Pigmentosa patients to date and its associated phenotype.

Patients and Methods

A cohort of 201 unrelated Spanish families affected by autosomal dominant Retinitis Pigmentosa. The p.Gly56Arg mutation in the NR2E3 (NM_014249.2) gene was analysed in 201 families. In the 24 cases where the mutation had been detected, a haplotype analysis linked to the p.Gly56Arg families was performed, using four extragenic polymorphic markers D15S967, D15S1050, D15S204 and D15S188. Phenotype study included presence and age of onset of night blindness, visual field loss and cataracts; and an ophthalmoscopic examination after pupillary dilation and electroretinogram for the 24 cases.

Results

Seven of the 201 analyzed families were positive for the p.Gly56Arg, leading to a prevalence of 3.5%. Clinical data were available for 24 subjects. Night blindness was the first noticeable symptom (mean 15.9 years). Visual field loss onset was variable (23.3 ± 11.9 years). Loss of visual acuity appeared late in the disease´s evolution. Most of the patients with cataracts (50%) presented it from the third decade of life. Fundus changes showed inter and intrafamiliar variability, but most of the patients showed typical RP changes and it was common to find macular affectation (47.4%). Electroretinogram was impaired from the beginning of the disease. Two families shared a common haplotype. Additionally, all patients shared a 104Kb region between D15S1050 and the NR2E3 gene.

Conclusions

This study highlights the importance of p.Gly56Arg in the NR2E3 gene as a common mutation associated with adRP, and provides new clues to its phenotype, which can allow for a better clinical management and genetic counselling of patients and their families.     

Optimal Control in the Treatment of Retinitis Pigmentosa

Numerous therapies have been implemented in an effort to minimize the debilitating effects of the degenerative eye disease Retinitis Pigmentosa (RP), yet none have provided satisfactory long-term solution. To date there is no treatment that can halt the degeneration of photoreceptors. The recent discovery of the RdCVF protein has provided researchers with a potential therapy that could slow the secondary wave of cone death. In this work, we build on an existing mathematical model of photoreceptor interactions in the presence of RP and incorporate various treatment regiments via RdCVF. Our results show that an optimal control exists for the administration of RdCVF. In addition, our numerical solutions show the experimentally observed rescue effect that the RdCVF has on the cones.     

Autosomal Recessive Transmission of a Rare KRT74 Variant Causes Hair and Nail Ectodermal Dysplasia: Allelism with Dominant Woolly Hair/Hypotrichosis

Pure hair and nail ectodermal dysplasia (PHNED) comprises a heterogeneous group of rare heritable disorders characterized by brittle hair, hypotrichosis, onychodystrophy and micronychia. Autosomal recessive (AR) PHNED has previously been associated with mutations in either KRT85 or HOXC13 on chromosome 12p11.1-q14.3. We investigated a consanguineous Pakistani family with AR PHNED linked to the keratin gene cluster on 12p11.1 but without detectable mutations in KRT85 and HOXC13. Whole exome sequencing of affected individuals revealed homozygosity for a rare c.821T>C variant (p.Phe274Ser) in the KRT74 gene that segregates AR PHNED in the family. The transition alters the highly conserved Phe274 residue in the coil 1B domain required for long-range dimerization of keratins, suggesting that the mutation compromises the stability of intermediate filaments. Immunohistochemical (IHC) analyses confirmed a strong keratin-74 expression in the nail matrix, the nail bed and the hyponychium of mouse distal digits, as well as in normal human hair follicles. Furthermore, hair follicles and epidermis of an affected family member stained negative for Keratin-74 suggesting a loss of function mechanism mediated by the Phe274Ser substitution. Our observations show for the first time that homozygosity for a KRT74 missense variant may be associated with AR PHNED. Heterozygous KRT74 mutations have previously been associated with autosomal dominant woolly hair/hypotrichosis simplex (ADWH). Thus, our findings expand the phenotypic spectrum associated with KRT74 mutations and imply that a subtype of AR PHNED is allelic with ADWH.     

X 性连锁视网膜色素变性中的RP G R 基因的研究进展

视网膜色素变性是一组常见的遗传性致盲眼病,患病率约为1／3500。X染色体连锁遗传RP作为其中的一种类型,具有发病早,损害最为严重等特点。而在XLRP的相关基因中RPGR有着重要的意义。本文就RPGR的定位克隆、结构、功能及其突变谱予以综述,并对该基因的突变研究的临床意义作出了相关阐述。     

Clinical and Rehabilitative Management of Retinitis Pigmentosa: Up-to-Date

The term retinitis pigmentosa (RP) indicates a heterogeneous group of genetic rare ocular diseases in which either rods or cones are prevalently damaged. RP represents the most common hereditary cause of blindness in people from 20 to 60 years old. In general, the different RP forms consist of progressive photo-receptorial neuro-degenerations, which are characterized by variable visual disabilities and considerable socio-sanitary burden. Sometimes, RP patients do not become visually impaired or legally blind until their 40-50 years of age and/or maintain a quite acceptable sight for all their life. Other individuals with RP become completely blind very early or in middle childhood. Although there is no treatment that can effectively cure RP, in some case-series the disease's progression seems to be reducible by specific preventive approaches. In the most part of RP patients, the quality of vision can be considerably increased by means of nanometer-controlled filters. In the present review, the main aspects of the routine clinical and rehabilitative managements for RP patients are described, particularly focusing on the importance of specific referral Centers to practice a real multidisciplinary governance of these dramatic diseases.     

Electrostatic Compensation Restores Trafficking of the Autosomal Recessive Retinitis Pigmentosa E150K Opsin Mutant to the Plasma Membrane

Rhodopsin is the rod photoreceptor G protein-coupled receptor responsible for capturing light. Mutations in the gene encoding this protein can lead to a blinding disease called retinitis pigmentosa, which is inherited frequently in an autosomal dominant manner. The E150K opsin mutant associated with rarely occurring autosomal recessive retinitis pigmentosa localizes to trans-Golgi network membranes rather than to plasma membranes of rod photoreceptor cells. We investigated the molecular mechanisms underlying opsin retention in the Golgi apparatus. Electrostatic calculations reveal that the E150K mutant features an overall accumulation of positive charges between helices H-IV and H-II. Human E150K and several other closely related opsin mutants were then expressed in HEK-293 cells. Spectral characteristics and functional biochemistry of each mutant were analyzed after reconstitution with the cis-retinoid chromophore. UV-visible spectra and rhodopsin/transducin activation assays revealed only minor differences between the purified wild type control and rhodopsin mutants. However, partial restoration of the surface electrostatic charge in the compensatory R69E/E150K double mutant rescues the plasma membrane localization of opsin. These findings emphasize the fundamental importance of electrostatic interactions for appropriate membrane trafficking of opsin and advance our understanding of the pathophysiology of autosomal recessive retinitis pigmentosa due to the E150K mutation.     

Genetic Analysis of a Kindred With X-linked Mental Handicap and Retinitis Pigmentosa

A kindred is described in which X-linked nonspecific mental handicap segregates together with retinitis pigmentosa. Carrier females are mentally normal but may show signs of the X-linked retinitis pigmentosa carrier state and become symptomatic in their later years. Analysis of polymorphic DNA markers at nine loci on the short arm of the X chromosome shows that no crossing-over occurs between the disease and Xp11 markers DXS255, TIMP, DXS426, MAOA, and DXS228. The 90% confidence limits show that the locus is in the Xp21-q21 region. Haplotype analysis is consistent with the causal gene being located proximal to the Xp21 loci DXS538 and 5'-dystrophin on the short arm of the X chromosome. The posterior probability of linkage to the RP2 region of the X chromosome short arm (Xp11.4-p11.23) is .727, suggesting the possibility of a contiguous-gene-deletion syndrome. No cytogenetic abnormality has been identified.