Cell death in retinitis pigmentosa: gap junctions and the 'bystander' effect

Retinitis pigmentosa (RP) constitutes a group of genetically mediated, degenerative retinal diseases that display a broad range of phenotypes. There is appreciable heterogeneity in the pathogenetic mechanisms that underlie the various forms of RP, but a substantial percentage of the known cases arise as a consequence of mutations in rhodopsin or other rod-specific proteins. However, despite the fact that the genetic defect is expressed solely in the rod photoreceptors, otherwise healthy cone photoreceptors invariably die, resulting in severe visual impairment. In this paper, the author proposes a mechanism that may be responsible, at least in part, for this unfortunate circumstance. The basic premise of the hypothesis is that the spread of the disease from dying rods to genetically normal cones is a form of 'bystander' effect, mediated by the gap junctions that exist between these photoreceptor subtypes. On this view, agents that trigger the apoptotic process permeate the intercellular gap-junctional channels to carry the disease from rods to neighboring cones. If permeation of noxious substances through gap junctions is a significant factor in the non-cell-autonomous spread of photoreceptor degeneration, blocking transmission through these channels may provide a means for therapeutic intervention. Many substances are known to block gap-junctional communication, but if the rod-cone channel is to be targeted, it will be essential to identify the connexins that form the gap junctions between the two types of photoreceptor, and to develop drugs that selectively affect their junctional properties. Clearly, passage of toxic agents through gap junctions may not be the only form of cell-cell interaction by which dying rods could cause cone cell death, and in this brief account, the author considers other avenues that are currently being explored to explain this phenomenon.     

视网膜色素变性家系的产前分子诊断

目的：对1例常染色体显性遗传视网膜色素变性家系患者所怀胎儿进行产前分子诊断。方法：应用聚合酶链反应（PCR）和直接测序技术法，对一常染色体显性遗传视网膜色素变性（ADRP）家系成员的所有现存人员的视紫红质基因的外显子进行测序分析。在采用STR位点分析方法排除母体基因组DNA污染后，应用DNA序列测定对胎儿-羊水基因组DNA进行分析。结果：该家系的25名成员中12名患者有视紫红质基因（rhodopsin，RHO）的512C〉T（P171L）突变，均呈杂合子，该错义突变使密码子171由CCA变成CTA。而未受累者的视紫红质基因表现为野生型。对该家系成员进行产前诊断，发现胎儿具有同种致病性突变。结论：视紫红质基因RHO的一种已知突变512C〉T（P171L）是该家系的病因，胎儿带P171L突变，产前诊断和早期干预能避免视网膜色素变性患儿的出生。     

Advantage of Whole Exome Sequencing over Allele-Specific and Targeted Segment Sequencing in Detection of Novel TULP1 Mutation in Leber Congenital Amaurosis

Background: Leber congenital amaurosis (LCA) is a severe form of retinal dystrophy with marked underlying genetic heterogeneity. Until recently, allele-specific assays and Sanger sequencing of targeted segments were the only available approaches for attempted genetic diagnosis in this condition. A broader next-generation sequencing (NGS) strategy, such as whole exome sequencing, provides an improved molecular genetic diagnostic capacity for patients with these conditions.

Materials and Methods: In a child with LCA, an allele-specific assay analyzing 135 known LCA-causing variations, followed by targeted segment sequencing of 61 regions in 14 causative genes was performed. Subsequently, exome sequencing was undertaken in the proband, unaffected consanguineous parents and two unaffected siblings. Bioinformatic analysis used two independent pipelines, BWA-GATK and SOAP, followed by Annovar and SnpEff to annotate the variants.

Results: No disease-causing variants were found using the allele-specific or targeted segment Sanger sequencing assays. Analysis of variants in the exome sequence data revealed a novel homozygous nonsense mutation (c.1081C?>?T, p.Arg361*) in TULP1, a gene with roles in photoreceptor function where mutations were previously shown to cause LCA and retinitis pigmentosa. The identified homozygous variant was the top candidate using both bioinformatic pipelines.

Conclusions: This study highlights the value of the broad sequencing strategy of exome sequencing for disease gene identification in LCA, over other existing methods. NGS is particularly beneficial in LCA where there are a large number of causative disease genes, few distinguishing clinical features for precise candidate disease gene selection, and few mutation hotspots in any of the known disease genes.     

Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases

Vision is the sense that we use to navigate the world around us. Thus it is not surprising that blindness is one of people's most feared maladies. Heritable diseases of the retina, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world, collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades, scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with retinal degeneration through drug therapy, gene augmentation or a cell-based transplantation approach. In this review we will discuss the use of the induced pluripotent stem cell technology to model and develop various treatment modalities for the treatment of inherited retinal degenerative disease. We will focus on the use of iPSCs for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell transplantation and replacement.     

Focal Choroidal Excavation in Retinal Dystrophies

Aim: To investigate the presence of focal choroidal excavation (FCE) in patients with retinitis pigmentosa (RP), Stargardt’s disease (STGD), and Best disease in the Indian population. Methods: This retrospective consecutive case series included 309 eyes of 157 patients with RP (183 eyes), STGD (93 eyes), and Best disease (33 eyes) with good-quality, enhanced-depth spectral domain optical coherence tomography scans. Comprehensive ophthalmic examination data were collected. Characteristics of FCE, including location of FCE, type (conforming and non-conforming), maximal width, and depth, were noted. Results: FCE was found in 2 out of 33 (6%) eyes with Best disease and no FCE was found in eyes with RP or STGD. The location of the FCE was extrafoveal in both cases. The first case had non-conforming FCE while the second case had the conforming type and the FCE occurred in association with choroidal neovascularization in the second case. The first case maintained good visual acuity of 20/20 over the entire period of follow-up (14 months), while the second case had a visual acuity of 20/200 at the last follow-up (three years) due to scarred choroidal neovascular membranes. The FCE showed no change in both eyes over the entire duration of follow-up. Conclusion: Focal choroidal excavation was found in 6% of eyes with Best disease, which remained stable throughout follow up. Eyes with RP and STGD did not have any FCE. Further studies are required to determine the role of vitelliform material in FCE development in Best disease.     

Targeted next-generation sequencing reveals that a compound heterozygous mutation in phosphodiesterase 6a gene leads to retinitis pigmentosa in a Chinese family

Purpose: Retinitis pigmentosa (RP) is a genetically heterogeneous disease with over 70 causative genes identified to date. However, approximately 40% of RP cases remain genetically unsolved, suggesting that many novel disease-causing mutations are yet to be identified. The purpose of this study is to identify the causative mutations of a Chinese RP family.

Methods: Targeted next-generation sequencing (NGS) for a total of 163 genes which involved in inherited retinal disorders were used to screen the possible causative mutations. Sanger sequencing was used to verify the mutations.

Results: As results, we identified two heterozygous mutations: a splicing site mutation c.1407 + 1G>C and a nonsense mutation c. 1957C>T (p.R653X) in phosphodiesterase 6A (PDE6A) gene in the RP patient. These two mutations are inherited from his father and mother, respectively. Furthermore, these mutations are unique in our in-house database and are rare in human genome databases, implicating that these two mutations are pathological.

Conclusion: By using targeted NGS method, we identified a compound heterozygous mutation in PDE6A gene that is associated with RP in a Chinese family.     

Slowly progressive retinitis pigmentosa caused by two novel mutations in the MAK gene

Background: The growing number of clinical trials currently underway for inherited retinal diseases has highlighted the importance of achieving a molecular diagnosis for all new cases presenting to hospital eye services. The male germ cell-associated kinase (MAK) gene encodes a cilium-associated protein selectively expressed in the retina and testis, and has recently been implicated in autosomal recessive retinitis pigmentosa (RP). Whole exome sequencing has previously identified a homozygous Alu insertion in probands with recessive RP and nonsense and missense mutations have also been reported.

Materials and methods: Here we describe two novel mutations in different alleles of the MAK gene in a 75-year-old British female, who had a clinical diagnosis of RP () with onset in the fourth decade and no relevant family history. The mutations were established through next generation sequencing of a panel of 111 genes associated with RP and RP-like phenotypes.

Results: Two novel null mutations were identified within the MAK gene. The first c.1195_1196delAC p.(Thr399fs), was a two base-pair deletion creating a frame-shift in exon 9 predicted to result in nonsense-mediated decay. The second, c.279-2A>G, involved the splice acceptor consensus site upstream of exon 4, predicted to lead to aberrant splicing.

Conclusions: The natural history of this individual’s RP is consistent with previously described MAK mutations, being significantly milder than that associated with other photoreceptor ciliopathies. We suggest inclusion of MAK as part of wider genetic testing in all individuals presenting with RP.     

视网膜色素变性Rd1小鼠眼球中增殖细胞核抗原（PCNA）的表达

目的　探讨增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）在Rd1小鼠视网膜上的表达情况。方法　取出生后14 d、21 d、28 d的Rd1小鼠和C57BL/6J小鼠各5只,摘出眼球进行HE染色,观察视网膜形态学结构变化;免疫组织化学染色与实时荧光定量PCR检测PCNA蛋白和基因在不同鼠龄小鼠中的表达。结果　HE染色结果显示:PCNA蛋白均在两种小鼠视网膜神经节细胞中表达;C57BL/6J小鼠视网膜结构完整;与C57BL/6J小鼠相比,Rd1小鼠视网膜发育随着鼠龄的增加,发生进行性退化。免疫组织化学染色结果显示:PCNA蛋白表达仅局限于视网膜神经节细胞,免疫阳性信号出现在第14天和第21天,而在第28天未检测到。PCR 实验结果显示:与C57BL/6J小鼠相比较,鼠龄21 d时Rd1小鼠PCNA基因表达水平增高,是C57BL/6J小鼠的2.23倍;鼠龄14 d和28 d时 Rd1小鼠PCNA基因表达均低于C57BL/6J小鼠（均为P<0.05）。结论　PCNA基因参与了Rd1小鼠视网膜发育退化过程。