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 in vitro. 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.

Beyond nephronophthisis: Retinal dystrophy in the absence of kidney dysfunction in childhood expands the clinical spectrum of CEP83 deficiency.

The CEP83 protein is an essential part in the first steps of ciliogenesis, causing a ciliopathy if deficient. As a core component of the distal appendages of the centriole, CEP83 is located in almost all cell types and is involved in the primary cilium assembly. Previously reported CEP83 deficient patients all presented with nephronophthisis and kidney dysfunction. Despite retinal degeneration being a common feature in ciliopathies, only one patient also had retinitis. Here, we present two unrelated patients, who both presented with retinitis pigmentosa, without nephronophthisis or any form of kidney dysfunction. Both patients harbor bi-allelic variants in CEP83. This report expands the current clinical spectrum of CEP83 deficiency. For timely diagnosis of CEP83 deficiency, we advocate that CEP83 should be included in gene panels for inherited retinal diseases.

Long-term outcomes of vitrectomy for proliferative diabetic retinopathy.

PURPOSE: To investigate the long-term outcomes of patients who underwent vitrectomy for proliferative diabetic retinopathy. METHODS: Cumulative incidences were calculated for low vision (<0.3), re-vitrectomy in the study eye and fellow eye vitrectomy. To identify potential prognostic factors that associate with these outcomes, we used multivariable Cox regression models. RESULTS: In a total of 217 patients, we found 1-, 5- and 10-year cumulative incidences of low vision in the study eye of 24%, 31% and 39%, respectively. For both eyes, these rates were, respectively, 10%, 14% and 14%. Low vision in both eyes was associated with higher age and worse contralateral visual acuity. The 1-, 5- and 10-year cumulative incidences for re-vitrectomy in the study eye were 16%, 27% and 27%, respectively, and for a vitrectomy in the fellow eye 24%, 40% and 54%, respectively. Re-vitrectomy of the study eye was associated with worse contralateral visual acuity, while vitrectomy of the fellow eye was associated with shorter diabetes duration, worse contralateral visual acuity, higher HbA1c level and worse diabetic retinopathy severity stage of the fellow eye. CONCLUSION: Functional visual acuity in at least one eye was achieved or preserved in most patients. After 10 years, about a quarter of all patients underwent a re-vitrectomy, while more than half of the patients needed a vitrectomy of the fellow eye. Knowledge of these long-term outcomes is essential when counselling patients for a vitrectomy.

Heterozygous deep-intronic variants and deletions in ABCA4 in persons with retinal dystrophies and one exonic ABCA4 variant.

Variants in ABCA4 are responsible for autosomal-recessive Stargardt disease and cone-rod dystrophy. Sequence analysis of ABCA4 exons previously revealed one causative variant in each of 45 probands. To identify the "missing" variants in these cases, we performed multiplex ligation-dependent probe amplification-based deletion scanning of ABCA4. In addition, we sequenced the promoter region, fragments containing five deep-intronic splice variants, and 15 deep-intronic regions containing weak splice sites. Heterozygous deletions spanning ABCA4 exon 5 or exons 20-22 were found in two probands, heterozygous deep-intronic variants were identified in six probands, and a deep-intronic variant was found together with an exon 20-22 deletion in one proband. Based on ophthalmologic findings and characteristics of the identified exonic variants present in trans, the deep-intronic variants V1 and V4 were predicted to be relatively mild and severe, respectively. These findings are important for proper genetic counseling and for the development of variant-specific therapies.