Autosomal Recessive Retinitis Pigmentosa Research Articles

Long-read sequencing improves the genetic diagnosis of retinitis pigmentosa by identifying an Alu retrotransposon insertion in the EYS gene

BackgroundBiallelic variants in EYS are the major cause of autosomal recessive retinitis pigmentosa (arRP) in certain populations, a clinically and genetically heterogeneous disease that may lead to legal blindness. EYS is one of the largest genes (~ 2 Mb) expressed in the retina, in which structural variants (SVs) represent a common cause of disease. However, their identification using short-read sequencing (SRS) is not always feasible. Here, we conducted targeted long-read sequencing (T-LRS) using adaptive sampling of EYS on the MinION sequencing platform (Oxford Nanopore Technologies) to definitively diagnose an arRP family, whose affected individuals (n = 3) carried the heterozygous pathogenic deletion of exons 32–33 in the EYS gene. As this was a recurrent variant identified in three additional families in our cohort, we also aimed to characterize the known deletion at the nucleotide level to assess a possible founder effect.ResultsT-LRS in family A unveiled a heterozygous AluYa5 insertion in the coding exon 43 of EYS (chr6(GRCh37):g.64430524_64430525ins352), which segregated with the disease in compound heterozygosity with the previously identified deletion. Visual inspection of previous SRS alignments using IGV revealed several reads containing soft-clipped bases, accompanied by a slight drop in coverage at the Alu insertion site. This prompted us to develop a simplified program using grep command to investigate the recurrence of this variant in our cohort from SRS data. Moreover, LRS also allowed the characterization of the CNV as a ~ 56.4kb deletion spanning exons 32–33 of EYS (chr6(GRCh37):g.64764235_64820592del). The results of further characterization by Sanger sequencing and linkage analysis in the four families were consistent with a founder variant.ConclusionsTo our knowledge, this is the first report of a mobile element insertion into the coding sequence of EYS, as a likely cause of arRP in a family. Our study highlights the value of LRS technology in characterizing and identifying hidden pathogenic SVs, such as retrotransposon insertions, whose contribution to the etiopathogenesis of rare diseases may be underestimated.

Mechanisms of cone sensitivity loss in retinitis pigmentosa.

To explore the mechanisms of cone sensitivity loss in retinitis pigmentosa by combining two-colour perimetry with threshold versus intensity (tvi) testing. Seven subjects with autosomal recessive retinitis pigmentosa and 10 normal subjects were recruited and underwent perimetric testing of one eye using 480- and 640-nm Goldman size V targets presented under scotopic conditions (no background illumination) and against a white background ranging in luminance from -1.5 to 2 log cd m-2 in 0.5 log cd m-2 steps. Data were fitted with tvi functions of the form logT = logT0 + log ((A + A0)/A0)n, where T is the threshold, T0 is the absolute threshold, A is the background intensity, A0 is the 'dark-light' constant and n is a gain constant. Reliable tvi functions could not be obtained within the region of the visual field corresponding to loss of the ellipsoid zone on optical coherence tomography. At fixation, changes in both T0 and A0 were observed, consistent with a d1 mechanism loss, which resulted in an upwards and rightwards shift of the tvi function. Losses at [±3°, ±3°] demonstrated changes in T0, consistent with a d3 mechanism loss, resulting in an upwards translation of the tvi curve. Although the absolute cone threshold was elevated at each location, shifts in the tvi function (so-called d1 mechanism loss) at fixation minimise threshold elevation in the presence of white adapting backgrounds, such as those typically employed in standard two-colour perimetry. At more peripheral testing locations, changes in threshold occurred independent of background luminance (so-called d3 mechanism loss). These findings suggest that backgrounds which selectively adapt rods while maintaining cones at, or near, absolute threshold may be preferable to conventional two-colour perimetry for assessing loss of cone sensitivity, especially at the point of fixation.

The temporal progression of retinal degeneration and early-stage idebenone treatment in the Pde6brd1/rd1 mouse model of retinal dystrophy

Photoreceptor cell death, primarily through apoptosis, related to retinal disorders like retinitis pigmentosa (RP), would result in vision loss. The pathological processes and crucial mutant conditions preceding photoreceptor cell demise are not well understood. This study aims to conduct an in-depth examination of early-stage changes in the widely utilized Pde6brd1/rd1 (rd1) mouse model, which has Pde6b gene mutations representing autosomal recessive RP disorder. We investigated the morphology and ultrastructure of retinal cells, including second-order neurons, during the initial phase of disease progression. Our findings revealed that mitochondrial alterations in rod photoreceptors were present as a predeath mutant state as early as postnatal day 3 (P3). The bipolar and horizontal cells from the rd1 mouse retina exhibited significant morphological changes in response to loss of photoreceptor cells, indicating that second-order neurons rely on these cells for their structures. Subsequent oral administration of idebenone, a mitochondria-protective agent, enhanced retinal function and promoted both photoreceptor cell survival and inner retinal second-order synaptogenesis in rd1 mice at P14. Our findings offer a mechanistic framework, suggesting that mitochondrial damage acts as an early driver for photoreceptor cell death in retinal degeneration.

Functional Vision in Patients With Biallelic USH2A Variants

To describe functional vision (FV) and investigate the relationship between FV, visual acuity (VA), and hill of vision (VTOT) at baseline in patients with biallelic USH2A variants. Multicenter, international, cross-sectional study. In individuals with biallelic disease-causing variants in USH2A, clinical diagnosis of Usher syndrome type 2 (USH2) or autosomal recessive nonsyndromic retinitis pigmentosa (ARRP) was based on history of hearing loss and audiology examinations. The VALVVFQ-48 was administered verbally to participants ≥18 years old. VA was measured in both eyes; VTOT was determined from static perimetry in the study eye (better VA). FV scores were calculated using Rasch analysis. Median age of 121 participants (76 with USH2, 45 with ARRP) was 41 years (range: 19-80); 54% were female. FV scores varied from -2.0 to 7.6 logits (median [interquartile range (IQR)]: 2.8 [1.5-3.8]). ARRP and USH2 participants had similar FV scores, both before [mean (95% CI): 2.8 (2.3-3.4) and 2.7 (2.3-3.2), respectively], and after [mean (95% CI): 2.5 (2.1-3.0) and 2.9 (2.6-3.3), respectively; P = .24] adjusting for age, VA, disease duration, and VTOT. VA and VTOT accounted for 29% and 26% of the variance in FV scores, respectively (P < .001 for each). Together, they accounted for 36% of variance observed. Biallelic USH2A variants were associated with a large range of FV, yet similar in ARRP and USH2, despite hearing loss in USH2. The modified VALVVFQ-48 we evaluated is not ideal for detecting the impact of USH2A-associated retinal degenerations on activities of daily living.

Does Background Matter? A Comparative Characterization of Mouse Models of Autosomal Retinitis Pigmentosa rd1 and Pde6b-KO.

Many retinal degenerative diseases result in vision impairment or permanent blindness due to photoreceptor loss or dysfunction. It has been observed that Pde6brd1 mice (rd1), which carry a spontaneous nonsense mutation in the pde6b gene, have a strong phenotypic similarity to patients suffering from autosomal recessive retinitis pigmentosa. In this study, we present a novel mouse model of retinitis pigmentosa generated through pde6b gene knockout using CRISPR/Cas9 technology. We compare this Pde6b-KO mouse model to the rd1 mouse model to gain insights into the progression of retinal degeneration. The functional assessment of the mouse retina and the tracking of degeneration dynamics were performed using electrophysiological methods, while retinal morphology was analyzed through histology techniques. Interestingly, the Pde6b-KO mouse model demonstrated a higher amplitude of photoresponse than the rd1 model of the same age. At postnatal day 12, the thickness of the photoreceptor layer in both mouse models did not significantly differ from that of control animals; however, by day 15, a substantial reduction was observed. Notably, the decline in the number of photoreceptors in the rd1 model occurred at a significantly faster rate. These findings suggest that the C3H background may play a significant role in the early stages of retinal degeneration.

RETINITIS PIGMENTOSA ASSOCIATED WITH THE EYS C2139Y VARIANT : An Important Cause of Blindness in East Asian Populations.

The study aimed to describe the phenotypic features of retinitis pigmentosa (RP) associated with the previously described EYS C2139Y variant in Singaporeans and establish the importance of this variant as a prevalent cause of RP among East Asians. A clinical phenotyping and exome-sequencing study was conducted on consecutive patients with nonsyndromic RP. Epidemiological analysis was performed using Singaporean and global population-based genetic data. A study of 150 consecutive unrelated individuals with nonsyndromic RP found that 87 (58%) of cases had plausible genotypes. A previously described missense variant in the EYS gene, 6416G>A (C2139Y), occurred heterozygously or homozygously in 17 of 150 families (11.3%), all with autosomal recessive RP. Symptom onset in EYS C2139Y-related RP ranged from 6 to 45 years, with visual acuity ranging from 20/20 at 21 years to no light perception by 48 years. C2139Y-related RP had typical findings, including sectoral RP in cases with EYS E2703X in trans . The median age at presentation was 45 years and visual fields declined to less than 20° (Goldmann V4e isopter) by age 65 years. Intereye correlation for visual acuity, fields, and ellipsoid band width was high (r 2 = 0.77-0.95). Carrier prevalence was 0.66% (allele frequency of 0.33%) in Singaporean Chinese and 0.34% in East Asians, suggesting a global disease burden exceeding 10,000 individuals. The EYS C2139Y variant is common in Singaporean RP patients and other ethnic Chinese populations. Targeted molecular therapy for this single variant could potentially treat a significant proportion of RP cases worldwide.

The splicing factor DHX38 enables retinal development through safeguarding genome integrity

DEAH-Box Helicase 38 (DHX38) is a pre-mRNA splicing factor and also a disease-causing gene of autosomal recessive retinitis pigmentosa (arRP). The role of DHX38 in the development and maintenance of the retina remains largely unknown. In this study, by using the dhx38 knockout zebrafish model, we demonstrated that Dhx38 deficiency causes severe differentiation defects and apoptosis of retinal progenitor cells (RPCs) through disrupted mitosis and increased DNA damage. Furthermore, we found a significant accumulation of R-loops in the dhx38-deficient RPCs and human cell lines. Finally, we found that DNA replication stress is the prerequisite for R-loop-induced DNA damage in the DHX38 knockdown cells. Taken together, our study demonstrates a necessary role of DHX38 in the development of retina and reveals a DHX38/R-loop/replication stress/DNA damage regulatory axis that is relatively independent of the known functions of DHX38 in mitosis control.

USH2A variants causing retinitis pigmentosa or Usher syndrome provoke differential retinal phenotypes in disease-specific organoids

There is an emblematic clinical and genetic heterogeneity associated with inherited retinal diseases (IRDs). The most common form is retinitis pigmentosa (RP), a rod-cone dystrophy caused by pathogenic variants in over 80 different genes. Further complexifying diagnosis, different variants in individual RP genes can also alter the clinical phenotype. USH2A is the most prevalent gene for autosomal-recessive RP and one of the most challenging because of its large size and, hence, large number of variants. Moreover, USH2A variants give rise to non-syndromic and syndromic RP, known as Usher syndrome (USH) type 2, which is associated with vision and hearing loss. The lack of a clear genotype-phenotype correlation or prognostic models renders diagnosis highly challenging. We report here a long-awaited differential non-syndromic RP and USH phenotype in three human disease-specific models: fibroblasts, induced pluripotent stem cells (iPSCs), and mature iPSC-derived retinal organoids. Moreover, we identified distinct retinal phenotypes in organoids from multiple RP and USH individuals, which were validated by isogenic-corrected controls. Non-syndromic RP organoids showed compromised photoreceptor differentiation, whereas USH organoids showed a striking and unexpected cone phenotype. Furthermore, complementary clinical investigations identified macular atrophy in a high proportion of USH compared with RP individuals, further validating our observations that USH2A variants differentially affect cones. Overall, identification of distinct non-syndromic RP and USH phenotypes in multiple models provides valuable and robust readouts for testing the pathogenicity of USH2A variants as well as the efficacy of therapeutic approaches in complementary cell types.

Case analysis of epilepsy, neurodevelopmental disorder, and motor disorders associated with mutations in the dehydrodolichyl diphosphate synthase gene.

Using "DHDDS; epilepsy; neurodevelopmental disorder" as the keywords, the literature relevant to DHDDS gene mutations up until February 2022 was reviewed. A total of 25 cases were retrieved, among which 21 cases with complete data were included in the chi-squared test. The clinical characteristics of DHDDS gene-related cases were summarized and analyzed. The onset of epilepsy caused by mutations of the DHDDS gene typically occurs during infancy. Predominantly, the mutation occurs in the locus of c.632G>A p.R211Q. Myoclonus is frequently the initial manifestation of epilepsy; it frequently coexists with neurodevelopmental disorder and intellectual disability, and patients have no specific type of motor disorder. Cranial magnetic resonance imaging (MRI) reveals no abnormalities, whereas electroencephalogram (EEG) frequently exhibits abnormalities. Valproic acid (VPA) yields good curative effects. Mutations in the DHDDS gene are associated with congenital glycosylation disorder, autosomal recessive retinitis pigmentosa, and epilepsy. According to statistical analysis using the chi-squared test, for pediatric patients with mutations in this gene locus, most of the epilepsy types are myoclonic epilepsies with intellectual disability and neurodevelopmental disorders. They have normal brain MRIs and abnormal EEGs. VPA produces beneficial therapeutic results and the differences are all statistically significant. The current diagnosis still relies on next-generation sequencing or whole-exome sequencing.

Clinical and Molecular Aspects of C2orf71/PCARE in Retinal Diseases.

Mutations in the photoreceptor-specific C2orf71 gene (also known as photoreceptor cilium actin regulator protein PCARE) cause autosomal recessive retinitis pigmentosa type 54 and cone-rod dystrophy. No treatments are available for patients with C2orf71 retinal ciliopathies exhibiting a severe clinical phenotype. Our understanding of the disease process and the role of PCARE in the healthy retina significantly limits our capacity to transfer recent technical developments into viable therapy choices. This study summarizes the current understanding of C2orf71-related retinal diseases, including their clinical manifestations and an unclear genotype-phenotype correlation. It discusses molecular and functional studies on the photoreceptor-specific ciliary PCARE, focusing on the photoreceptor cell and its ciliary axoneme. It is proposed that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane during the development of a new outer segment disk in photoreceptor cells. This review also introduces various cellular and animal models used to model these diseases and provides an overview of potential treatments.

Successful large gene augmentation of USH2A with non-viral episomal vectors

USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.

Association of ABCA4 Gene Variants in Patients with Autosomal Recessive Cone-Rod Dystrophy and Retinitis Pigmentosa Cohorts from South India

Association of ABCA4 Gene Variants in Patients with Autosomal Recessive Cone-Rod Dystrophy and Retinitis Pigmentosa Cohorts from South India

A nonsense mutation in C8orf37 linked with retinitis pigmentosa, early macular degeneration, cataract, and myopia in an arRP family from North India

ObjectiveThis study aimed at identifying the underlying genetic defect in a consanguineous autosomal recessive retinitis pigmentosa (arRP) (RP-1175) family having RP with early macular degeneration, cataract, and myopia.MethodsWhole-exome sequencing (WES) was performed on the DNA of the proband, and variants observed were validated in the rest of the affected and unaffected family members by Sanger sequencing. Different bioinformatics tools were applied to access the pathogenicity of the observed variant.ResultsA nonsense mutation i.e., c.555G > A (p.Trp185Ter) in C8orf37 in homozygous form, has been identified that segregated with the disease in the affected members. c.555G > A was absent in unaffected family members and in 107 ethnically matched controls, therefore ruling out its possibility of being a polymorphism.ConclusionsPresent study identifies a nonsense mutation (c.555G > A) at codon 185 in C8orf37 linked with arRP, early macular degeneration, posterior subcapsular cataract, and myopia. The identical mutation has previously been reported in a Pakistani family with isolated RP and in a Chinese family with RP and macular degeneration. This variable expressivity of the identified mutation c.555G > A in C8orf37 in the analyzed Indian family may be attributed to the presence of the modifier alleles. Also, Trp185 might be a mutation hotspot in Asian arRP patients and in the future, p.Trp185Ter in C8orf37 may be tested during initial screening in arRP cases especially belonging to a similar population.

Molecular Genetic Analysis of the Autosomal Recessive Non-Syndromic Inherited Retinitis Pigmentosa.

90% of visually impaired people live in developing countries. There are various types of vision impairment, but the focus of the current study is retinitis pigmentosa (RP). Up to now, 150 mutations have been reported that are linked with RP. Healthy and affected members from two Pakistani families (RP01 and RP02) segregating autosomal recessive RP were selected for DNA extraction. PCR was conducted, and the amplified PCR products were analyzed using Polyacrylamide Gel Electrophoresis (PAGE) andvisualized in the Gel Doc systemfor linkage analysis.The Gene Hunter 2.1r5 tool in the Simple Linkage v5.052 beta software suite was used to conduct multipoint parametric linkage analysis on the two consanguineous families examined on the 6K Illumina array. Exons and intron-exon borders of all known arRP genes found in homozygous areas were sequenced in the matching probands using a 3130 automated sequencer and the Big Dye Terminator Cycle Sequencing Kit v3.1. The mutation study was carried out using the AlaMut 1.5 program. In both families, linkage analysis was performed using microsatellite marker DIS422 for genecrumbs homolog 1 (CRB1)and microsatellite marker D8S2332 for geneRetinitis Pigmentosa 1 (RP1). Multipoint linkage analysis identifies genomic regions that could potentially contain the genetic defect. In family RP01, only a single peak with a maximal multipoint LOD score of 3.00 was identified on chromosome 1, whereas in family RP02, multiple peaks with multipoint LOD scores of 1.80 were identified on chromosome 8. Analysis of theCRB1gene revealed a homozygous substitution of glycine for valine (c.1152T>G; p.V243G), whereas theRP1gene demonstrated that leucine was substituted for proline as a result of cytosine to thymine transfer (c.3419C>T; p. P1035L). Conclusion: Homozygosity mapping is a powerful method for finding genetic abnormalities that are both precise and comprehensive for identifying harmful variations in consanguineous families. This method is invaluable for providing accurate clinical diagnostic and genetic advice in remote regions of Pakistanwhile also increasing knowledge about autosomal recessive diseases and the dangers of mixing.

Clinical Heterogeneity in Two Siblings Harbouring a Heterozygous PRPH2 Pathogenic Variant.

The aim of the study was to describe the clinical and genetic correlation of a c.469 G>A p.(Asp157Asn) heterozygous pathogenic variant in PRPH2 in two siblings of Italian origin. Both patients underwent ophthalmic examination, electrophysiological testing, autofluorescence imaging, and optical coherence tomography (OCT). Screening for pathogenic variants of the obtained DNA from the family members was carried out. The 52-year-old (♀, index patient) and 50-year-old (♂) siblings had BCVA (OD and OS) of 20/20 and 20/16 (♀) and 20/25 and 20/40 (♂), respectively, and suffered increased sensitivity to glare. Yellow irregular macular deposits, numerous small irregular hypo- and hyperreflective spots at the posterior pole, a patchy loss of photoreceptors, and retinal pigment epithelium (RPE) in the perifoveal region were seen. Electrophysiology showed dysfunction of rods and cones, with more affected cone dysfunction in the index patient, contrary to the generalised rod dysfunction in the brother of the index patient. The clinical, electrophysiological, and multimodal imaging findings of both siblings pointed towards Stargardt retinopathy with heterogenic presentation. The DNA analysis identified an autosomal dominant c.469 G>A p.(Asp157Asn) heterozygous pathogenic variant in PRPH2 associated with autosomal dominant cone-rod dystrophy and rod-cone dystrophy. PRPH2 codes for peripherin-2, a membrane protein that consists of 346 amino acids. Our findings confirm a heterogeneity in clinical presentation associated with pathogenic variants in PRPH2. It may follow either an autosomal dominant or an autosomal recessive mode of inheritance and show a very heterogeneous clinical manifestation of retinal degeneration, e.g., autosomal dominant retinitis pigmentosa (♂ sibling; II-3) and autosomal dominant cone-rod dystrophy (index ♀ sibling; II-2), autosomal dominant macular dystrophy, and also autosomal recessive retinitis pigmentosa.

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction.

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and invitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Mutational Spectrum, Ocular and Olfactory Phenotypes of CNGB1-Related RP-Olfactory Dysfunction Syndrome in a Multiethnic Cohort.

CNGB1 gene mutations are a well-known cause of autosomal recessive retinitis pigmentosa (RP), which was recently associated with olfactory dysfunction. The purpose of this study was to report the molecular spectrum and the ocular and olfactory phenotypes of a multiethnic cohort with CNGB1-associated RP. A cross-sectional case series was conducted at two ophthalmic genetics referral centers. Consecutive patients with molecularly confirmed CNGB1-related RP were included. All patients underwent a complete ophthalmological examination complemented by psychophysical olfactory evaluation. Fifteen patients (10 families: 8 Portuguese, 1 French, and 1 Turkish), mean aged 57.13 ± 15.37 years old (yo), were enrolled. Seven disease-causing variants were identified, two of which are reported for the first time: c.2565_2566del and c.2285G > T. Although 11/15 patients reported onset of nyctalopia before age 10, diagnosis was only established after 30 yo in 9/15. Despite widespread retinal degeneration being present in 14/15 probands, a relatively preserved visual acuity was observed throughout follow-up. Olfactory function was preserved in only 4/15 patients, all of whom carried at least one missense variant. Our study supports previous reports of an autosomal recessive RP-olfactory dysfunction syndrome in association with certain disease-causing variants in the CNGB1 gene and expands the mutational spectrum of CNGB1-related disease by reporting two novel variants.

A novel homozygous missense substitution p.Thr313Ile in the PDE6B gene underlies autosomal recessive retinitis pigmentosa in a consanguineous Pakistani family

BackgroundRetinitis pigmentosa (RP) is one of the most frequent hereditary retinal diseases that often starts with night blindness and eventually leads to legal blindness. Our study aimed to identify the underlying genetic cause of autosomal recessive retinitis pigmentosa (arRP) in a consanguineous Pakistani family.MethodsFollowing a detailed ophthalmological examination of the patients by an ophthalmologist, whole-exome sequencing was performed on the proband’s DNA to delineate the genetic cause of RP in the family. In-depth computational methods, in-silico analysis, and familial co-segregation study were performed for variant detection and validation.ResultsWe studied an inbred Pakistani family with two siblings affected by retinitis pigmentosa. The proband, a 32 years old female, was clinically diagnosed with RP at the age of 6 years. A classical night blindness symptom was reported in the proband since her early childhood. OCT report showed a major reduction in the outer nuclear layer and the ellipsoid zone width, leading to the progression of the disease. Exome sequencing revealed a novel homozygous missense mutation (c.938C > T;p.Thr313Ile) in exon 12 of the PDE6B gene. The mutation p.Thr313Ile co-segregated with RP phenotype in the family. The altered residue (p.Thr313) was super conserved evolutionarily across different vertebrate species, and all available in silico tools classified the mutation as highly pathogenic.ConclusionWe present a novel homozygous pathogenic mutation in the PDE6B gene as the underlying cause of arRP in a consanguineous Pakistani family. Our findings highlight the importance of missense mutations in the PDE6B gene and expand the known mutational repertoire of PDE6B-related RP.

Retinitis Pigmentosa Associated with EYS Gene Mutations: Disease Severity Staging and Central Retina Atrophy

Eyes shut homolog (EYS) gene mutations are estimated to affect at least 5% of patients with autosomal recessive retinitis pigmentosa. Since there is no mammalian model of human EYS disease, it is important to investigate its age-related changes and the degree of central retinal impairment. A cohort of EYS patients was studied. They underwent full ophthalmic examination as well as assessment of retinal function and structure, by full-field and focal electroretinograms (ERGs) and spectral domain optical coherence tomography (OCT), respectively. The disease severity stage was determined by the RP stage scoring system (RP-SSS). Central retina atrophy (CRA) was estimated from the automatically calculated area of the sub-retinal pigment epithelium (RPE) illumination (SRI). The RP-SSS was positively correlated with age, showing an advanced severity score (≥8) at an age of 45 and a disease duration of 15 years. The RP-SSS was positively correlated with the CRA area. LogMAR visual acuity and ellipsoid zone width, but not ERG, were correlated with CRA. In EYS-related disease, the RP-SSS showed advanced severity at a relative early age and was correlated with the central area of the RPE/photoreceptor atrophy. These correlations may be relevant in view of therapeutic interventions aimed at rescuing rods and cones in EYS-retinopathy.

Novel compound heterozygous variants in the USH2A gene associated with autosomal recessive retinitis pigmentosa without hearing loss.

Background: Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies characterized by the primary degeneration of rod photoreceptors and the subsequent loss of cone photoreceptors because of cell death. It is caused by different mechanisms, including inflammation, apoptosis, necroptosis, pyroptosis, and autophagy. Variants in the usherin gene (USH2A) have been reported in autosomal recessive RP with or without hearing loss. In the present study, we aimed to identify causative variants in a Han-Chinese pedigree with autosomal recessive RP. Methods: A six-member, three-generation Han-Chinese family with autosomal recessive RP was recruited. A full clinical examination, whole exome sequencing, and Sanger sequencing, as well as co-segregation analysis were performed. Results: Three heterozygous variants in the USH2A gene, c.3304C>T (p.Q1102*), c.4745T>C (p.L1582P), and c.14740G>A (p.E4914K), were identified in the proband, which were inherited from parents and transmitted to the daughters. Bioinformatics analysis supported the pathogenicity of the c.3304C>T (p.Q1102*) and c.4745T>C (p.L1582P) variants. Conclusions: Novel compound heterozygous variants in the USH2A gene, c.3304C>T (p.Q1102*) and c.4745T>C (p.L1582P), were identified as the genetic causes of autosomal recessive RP. The findings may enhance the current knowledge of the pathogenesis of USH2A-associated phenotypes, expand the spectrum of the USH2A gene variants, and contribute to improved genetic counseling, prenatal diagnosis, and disease management.