Erica G. M. Boonen

Purpose: A significant proportion of cases with rare inherited retinal disease (IRD) remain genetically unresolved following short-read whole genome sequencing (WGS). This study aimed to increase the diagnostic yield of two cohorts in a research setting. One cohort was previously screened by short-read WGS (n=120 cases), while the second cohort (n=28 cases) was not included in any previous short-read WGS studies. In contrast to the larger cohort, these cases only underwent either exome sequencing (ES) or single-molecule molecular inversion probe (smMIPs) sequencing as pre-screening in earlier studies. For all cases, we performed a stepwise, case-by-case reanalysis in which short-read WGS was generated (n=28 cases) and (re)analyzed short-read WGS data using an optimized approach. For a subset of cases (n=20) that remained unresolved, this was followed by long-read WGS. Together, these steps aimed to improve the diagnostic yield in these cohorts. Methods: Short-read WGS data of 148 IRD-cases were (re)analyzed using updated allele frequency databases, new variant caller and variant predictor tools, as well as updated and extended gene-panels to identify causal single nucleotide variants (SNVs) and structural variants (SVs). For 20 genetically unresolved cases with sufficient high-quality DNA available, long-read WGS was performed. Results: The (re)analysis of short-read WGS data in the 120 previously WGS-screened unresolved cases resulted in a diagnostic yield of 15% (18/120). For the 28 individuals without prior WGS, performing WGS analysis within this study provided an additional yield of 32% (9/28). Furthermore, long-read WGS contributed two genetic diagnoses among the 20 long-read WGS-sequenced probands. Amongst other reasons, new genetic diagnoses were attributed to pathogenic variants in genes newly associated with IRDs and in genes that were not included in the gene panels applied in previous WGS studies. The Mobile Element Locator Tool facilitated the identification of two pathogenic Alu insertions in short-read WGS. Long-read WGS identified two additional pathogenic variants. Conclusion: These results support periodic reanalysis of existing short-read WGS data as a key component of IRD genetic diagnostics, with selective long-read WGS representing a valuable additional approach for unresolved cases. Together, these complementary strategies offer a practical framework for improving molecular diagnosis and narrowing the remaining diagnostic gap in IRDs.

Background/objectives: Obtaining a genetic diagnosis for patients with inherited retinal diseases has become even more important since gene-specific therapies have become available. When genetic screening reveals variants of uncertain significance (VUS), additional evidence is required to determine genetic eligibility for therapy. Confirming the effect on splicing that is predicted by SpliceAI could change their classification to either likely pathogenic or pathogenic and would therefore be of great importance when interpreting these variants when geneticists worldwide are trying to reach a diagnosis. Methods: Using minigene assays, we established a pipeline to assess the effect on splicing for all variants. We selected 73 RPE65 variants that were classified as either VUS or likely benign in the RPE65 Leiden Open Variant Database (LOVD) or ClinVar and were predicted to affect splicing by SpliceAI with a delta score of >0.1 and by using an analysis window of 5000 bp up- and downstream of the variant. Results: Using four wild-type vectors, we generated 59 constructs containing the variants of interest. Through these minigene assays, we assessed the effect on splicing of these VUS to enable reclassification. Upon quantification, we identified seven variants with a full, aberrant splicing effect without residual wild-type transcript. Eleven variants had between 5% and 20% remaining wild-type transcript. Forty-one variants had ≥20% residual wild-type transcript, among which fifteen variants showed no effect on splicing. Conclusions: Following the 2023 established ClinGen specific ACMG guidelines for RPE65 (Criteria Specification Registry), evidence from splice assays enabled reclassification of seven RPE65 variants from VUS to pathogenic through an assigned PVS1-very-strong criterium, as less than 5% of wild-type transcript was present. These findings contribute to the interpretation of variants observed in patients, which will in turn dictate their eligibility for gene therapy.

The U4 small nuclear RNA (snRNA) forms a duplex with the U6 snRNA and, together with U5 and ∼30 proteins, is part of the U4/U6.U5 tri-snRNP complex, located at the core of the major spliceosome. Recently, recurrent de novo variants in the U4 RNA, transcribed from the RNU4-2 gene, and in at least two other RNU genes were discovered to cause neurodevelopmental disorder. We detected inherited and de novo heterozygous variants in RNU4-2 (n.18_19insA and n.56T>C) and in four out of the five RNU6 paralogues (n.55_56insG and n.56_57insG) in 135 individuals from 62 families with non-syndromic retinitis pigmentosa (RP), a rare form of hereditary blindness. We show that these variants are recurrent among RP families and invariably cluster in close proximity within the three-way junction (between stem-I, the 5’ stem-loop and stem-II) of the U4/U6 duplex, affecting its natural conformation. Interestingly, this region binds to numerous splicing factors of the tri-snRNP complex including PRPF3, PRPF8 and PRPF31, previously associated with RP as well. The U4 and U6 variants identified seem to affect snRNP biogenesis, namely the U4/U6 di-snRNP, which is an assembly intermediate of the tri-snRNP. Based on the number of positive cases observed, deleterious variants in RNU4-2 and in RNU6 paralogues could be a significant cause of isolated or dominant RP, accounting for up to 1.2% of all undiagnosed RP cases. This study highlights the role of non-coding genes in rare Mendelian disorders and uncovers pleiotropy in RNU4-2 , where different variants underlie neurodevelopmental disorder and RP.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing—a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Macular dystrophies are a group of individually rare but collectively common inherited retinal dystrophies characterised by central vision loss and loss of visual acuity. Single molecule Molecular Inversion Probes (smMIPs) have proved effective in identifying genetic variants causing macular dystrophy. Here, a previously established smMIPs panel tailored for genes associated with macular diseases has been used to examine 57 UK macular dystrophy cases, achieving a high solve rate of 63.2% (36/57). Among 27 bi-allelic STGD1 cases, only three novel ABCA4 variants were identified, illustrating that the majority of ABCA4 variants in Caucasian STGD1 cases are currently known. We examined cases with ABCA4-associated disease in detail, comparing our results with a previously reported variant grading system, and found this model to be accurate and clinically useful. In this study, we showed that ABCA4-associated disease could be distinguished from other forms of macular dystrophy based on clinical evaluation in the majority of cases (34/36)

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.

Background: Macular diseases (MDs) are a subgroup of retinal disorders characterized by central vision loss that represent a major cause of vision impairment. Despite the identification of numerous genes associated with inherited MD (iMD) and risk factors associated with age-related MD (AMD), the extent of genetic and non-genetic factors that influence the expression of MD is still not fully explained. Single molecule Molecular Inversion Probes (smMIPs) have proven effective in sequencing the ABCA4 gene in patients with Stargardt disease in a cost-effective manner to identify associated coding and non-coding variation, however a large portion of patients with MDs still remain genetically unexplained. Methods: For MD patients, we hypothesized that the missing heritability may be revealed by smMIPs-based sequencing of all MD-associated genes and risk factors. We used 17,394 smMIPs to sequence the coding regions of 105 genes and non-coding or regulatory loci associated with iMD and AMD, known pseudo-exons, and the mitochondrial genome in two test cohorts that had previously been screened for variants in the entire ABCA4 gene. Results: Sequencing of 379 probands achieved an average nucleotide coverage of 431× across all targets. Following detailed sequencing analysis of 110 probands, a diagnostic yield of 38% was observed. This established an ‘‘MD-smMIPs panel’’ that allows a genotype-first approach in a high-throughput and cost-effective manner, whilst achieving uniform and high coverage across targets. Conclusions: Further analysis will identify known and novel variants in MD-associated genes to offer an accurate clinical diagnosis to patients and their families. Furthermore, this will reveal new genetic associations for MD and potential genetic overlaps between iMD and AMD.