Functional assays of non-canonical splice-site variants in inherited retinal dystrophies genes

Inherited retinal dystrophies are a group of disorders characterized by the progressive degeneration of photoreceptors leading to loss of the visual function and eventually to legal blindness. Although next generation sequencing (NGS) has revolutionized the molecular diagnosis of these diseases, the pathogenicity of some mutations casts doubts. After the screening of 208 patients with a panel of 117 genes, we obtained 383 variants that were analysed in silico with bioinformatic prediction programs. Based on the results of these tools, we selected 15 variants for their functional assessment. Therefore, we carried out minigene assays to unveil whether they could affect the splicing of the corresponding gene. As a whole, seven variants were found to induce aberrant splicing in the following genes: BEST1, CACNA2D4, PRCD, RIMS1, FSCN2, MERTK and MAK. This study shows the efficacy of a workflow, based on the association of the Minimum Allele Frequency, family co-segregation, in silico predictions and in vitro assays to determine the effect of potential splice site variants identified by DNA-based NGS. These findings improve the molecular diagnosis of inherited retinal dystrophies and will allow some patients to benefit from the upcoming gene-based therapeutic strategies.

An inherited night blindness in Wiltshire sheep

Twelve cases of adult-onset blindness were identified in a flock of 130 polled Wiltshire sheep in New Zealand over a 3-year period. Affected sheep developed night blindness between 2 and 3 years of age, which progressed to complete blindness by 4 to 5 years of age. Fundic examination findings included progressive tapetal hyperreflectivity and attenuation of retinal blood vessels. Histologically, the retinas had a selective loss of rod photoreceptors with initial preservation of cone photoreceptors. Retinal degeneration was not accompanied by any other ocular or central nervous system abnormalities, and pedigree analysis suggested an inherited basis for the disease. Mating an affected Wiltshire ram to 2 affected Wiltshire ewes resulted in 6 progeny that all developed retinal degeneration by 2 years of age, while mating of the same affected ram to 6 unaffected ewes resulted in 8 unaffected progeny, consistent with autosomal recessive inheritance. Homozygosity mapping of 5 affected Wiltshire sheep and 1 unaffected Wiltshire sheep using an OvineSNP50 Genotyping BeadChip revealed an identical-by-descent region on chromosome 5, but none of the genes within this region were considered plausible candidate genes. Whole-genome sequencing of 2 affected sheep did not reveal any significant mutations in any of the genes associated with retinitis pigmentosa in humans or progressive retinal atrophy in dogs. Inherited progressive retinal degeneration affecting rod photoreceptors has not been previously reported in sheep, but this disease has several similarities to inherited retinal dystrophies in other species.

Identification of a PROM1 mutation in a Spanish family with inherited retinal dystrophies

Background: We report a Spanish family, comprising an affected mother and daughter, respectively diagnosed with retinitis pigmentosa and Stargardt-like macular dystrophy, in whom we identified a PROM1 mutation. Methods: A custom gene panel consisting of 119 inherited retinal dystrophies (IRD)-genes was applied in the two affected individuals of this family and sequenced using the Illumina´s NextSeq500 platform. Results: The analysis of the resulting data allowed us to identify the pathogenic PROM1 mutation c.1117C>T (p.Arg373Cys) as the primary cause of the disease in both patients. No additional variants contributing to the extent of retinal dysfunction were detected. Conclusion: The variable expressivity of the detected PROM1 mutation is the most likely responsible for the intrafamilial phenotypic variability observed in this family. Screening of this mutation should be considered in patients with compatible clinical manifestations, especially when accompanied by an autosomal dominant family history.

VarGenius-HZD Allows Accurate Detection of Rare Homozygous or Hemizygous Deletions in Targeted Sequencing Leveraging Breadth of Coverage

Homozygous deletions (HDs) may be the cause of rare diseases and cancer, and their discovery in targeted sequencing is a challenging task. Different tools have been developed to disentangle HD discovery but a sensitive caller is still lacking. We present VarGenius-HZD, a sensitive and scalable algorithm that leverages breadth-of-coverage for the detection of rare homozygous and hemizygous single-exon deletions (HDs). To assess its effectiveness, we detected both real and synthetic rare HDs in fifty exomes from the 1000 Genomes Project obtaining higher sensitivity in comparison with state-of-the-art algorithms that each missed at least one event. We then applied our tool on targeted sequencing data from patients with Inherited Retinal Dystrophies and solved five cases that still lacked a genetic diagnosis. We provide VarGenius-HZD either stand-alone or integrated within our recently developed software, enabling the automated selection of samples using the internal database. Hence, it could be extremely useful for both diagnostic and research purposes.

Clinical Characteristics of Comorbid Retinal Dystrophies and Primary Angle Closure Disease

Purpose: To assess the clinical characteristics of comorbid retinal dystrophies and primary angle closure disease.Design: Retrospective study from January 1992 to June 2020.Methods: This descriptive study included 92 eyes of 46 patients with comorbid retinal dystrophies and primary angle closure disease (PACD) that included eyes with primary angle closure suspect, primary angle closure and primary angle closure glaucoma. Demographic profile, clinical characteristics of PACD and its association with retinal dystrophies are described. Results: The study included 46 patients (92 eyes). Males were majority, 63%. Mean (± standard deviation) age when retinal dystrophy was diagnosed was 29.6 ± 9.4 years and PACD was diagnosed at 32.23±7.92 years. Mean BCVA at presentation was 1.07±0.87 log MAR (95% confidence interval (CI):0.87,1.26). Mean Intraocular pressure at diagnosis of glaucoma was 27±16 mmHg (95% CI:23.5,31.5 mmHg). The most common retinal dystrophy associated with PACD was retinitis pigmentosa (RP) followed by RP with retinoschisis. The hospital-based prevalence of PACD among all patients with RP and retinoschisis was 0.19% and 0.15% respectively. Laser peripheral iridotomy (LPI) was performed in 74 eyes (80.5%). Glaucoma was managed medically in majority of the eyes (58 eyes, 63.04%) and minority required surgical management with trabeculectomy (11, 11.9%). Conclusion: Retinitis pigmentosa is the most common retinal dystrophy associated with PACD. Comorbid PACD in eyes with retinal dystrophies was observed in 2nd to 3rd decade of life. This calls for screening for angle closure in eyes with retinal dystrophies from second decade onwards to identify the comorbid PACD and treat or refer them appropriately.

CRB1-Related Retinal Dystrophies in a Cohort of 50 Patients: A Reappraisal in the Light of Specific Müller Cell and Photoreceptor CRB1 Isoforms

Pathogenic variants in CRB1 lead to diverse recessive retinal disorders from severe Leber congenital amaurosis to isolated macular dystrophy. Until recently, no clear phenotype-genotype correlation and no appropriate mouse models existed. Herein, we reappraise the phenotype-genotype correlation of 50 patients with regards to the recently identified CRB1 isoforms: a canonical long isoform A localized in Müller cells (12 exons) and a short isoform B predominant in photoreceptors (7 exons). Twenty-eight patients with early onset retinal dystrophy (EORD) consistently had a severe Müller impairment, with variable impact on the photoreceptors, regardless of isoform B expression. Among them, two patients expressing wild type isoform B carried one variant in exon 12, which specifically damaged intracellular protein interactions in Müller cells. Thirteen retinitis pigmentosa patients had mainly missense variants in laminin G-like domains and expressed at least 50% of isoform A. Eight patients with the c.498_506del variant had macular dystrophy. In one family homozygous for the c.1562C>T variant, the brother had EORD and the sister macular dystrophy. In contrast with the mouse model, these data highlight the key role of Müller cells in the severity of CRB1-related dystrophies in humans, which should be taken into consideration for future clinical trials.

Identification of a novel RPGR mutation associated with X-linked cone-rod dystrophy in a Chinese family

Background Cone-rod dystrophy (CORD) is a group of inherited retinal dystrophies, characterized by decreased visual acuity, color vision defects, photophobia, and decreased sensitivity in the central visual field. Our study has identified a novel pathogenic variant associated with X-linked cone-rod dystrophy (XLCORD) in a Chinese family. Methods All six family members, including the proband, affected siblings, cousins and female carriers, have underwent thorough ophthalmic examinations. The whole exome sequencing was performed for the proband, followed by Sanger sequencing for spilt-sample validation. A mammalian expression vector (AAV-MCS) with mutated retinitis pigmentosa GTPase regulator (RPGR) sequence was expressed in HEK293 T cells. The mutated protein was verified by Western blotting and immunohistochemistry. Results A novel mutation in the RPGR gene (c.2383G > T, p.E795X) is identified to be responsible for CORD pathogenesis. Conclusions Our findings have expanded the spectrum of CORD-associated mutations in RPGR gene and serve as a basis for genetic diagnosis for X-linked CORD.

The Genetic Landscape of Inherited Retinal Diseases in a Mexican Cohort: Genes, Mutations and Phenotypes

In this work, we aimed to provide the genetic diagnosis of a large cohort of patients affected with inherited retinal dystrophies (IRDs) from Mexico. Our data add valuable information to the genetic portrait in rare ocular diseases of Mesoamerican populations, which are mostly under-represented in genetic studies. A cohort of 144 unrelated probands with a clinical diagnosis of IRD were analyzed by next-generation sequencing using target gene panels (overall including 346 genes and 65 intronic sequences). Four unsolved cases were analyzed by whole-exome sequencing (WES). The pathogenicity of new variants was assessed by in silico prediction algorithms and classified following the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic or likely pathogenic variants were identified in 105 probands, with a final diagnostic yield of 72.9%; 17 cases (11.8%) were partially solved. Eighteen patients were clinically reclassified after a genetic diagnostic test (17.1%). In our Mexican cohort, mutations in 48 genes were found, with ABCA4, CRB1, RPGR and USH2A as the major contributors. Notably, over 50 new putatively pathogenic variants were identified. Our data highlight cases with relevant clinical and genetic features due to mutations in the RAB28 and CWC27 genes, enrich the novel mutation repertoire and expand the IRD landscape of the Mexican population.

A Rare Case of Juvenile X-Linked Retinoschisis

Aim: To present a rare clinical case of X-linked retinoschisis, confirmed clinically, electrophysiologically and genetically. Material and methods: A 12-year-old boy underwent detailed ophthalmic examination including fundus photography, full-field, multifocal and pattern electroretinography, visual field testing, optical coherence tomography, which established the clinical diagnosis, confirmed also genetically. Results: The clinical findings included a slight loss of vision, central and paracentral scotomas, a characteristic spoke-wheel pattern appearance of the macula in fundoscopy and the pathognomic appearance of splitting of the retinal layers in the macula with foveal schisis with cystic spaces on OCT. Reduced cone and rod ERG responds were detected with the characteristic decreasing of b-ware near the isoelectric line. The genetic analysis found that the patient was hemizygous for the missense mutation c.598G>A (p.Arg200Cys) of RS1 gene, coming from his asymptomatic mother. Conclusion: The comprehensive clinical, electrophysiological and genetic testing of patients with rare hereditary retinal dystrophies is essential for the correct diagnosis and the choice of therapeutic approach.