Background Familial exudative vitreoretinopathy (FEVR) is an inherited eye disease characterised by the incomplete development of the retinal vasculature. Over 10 genes have been associated with FEVR, but there are still a substantial number of genetically unsolved cases. The aim of this study was to analyse whole genome sequencing (WGS) data from the FEVR cases in the Genomics England (GEL) 100 000 genomes project to identify the causative variants. Methods WGS was performed by GEL and accessed within the GEL Research Environment. FEVR cases were identified using LabKey and candidate variants were extracted using the ‘gene-variant workflow’ and ‘CNV/SV workflow’ and by using BCFtools in unfiltered VCF files. Results Fifty-nine FEVR probands were submitted to GEL. We found six novel and eight previously reported pathogenic variants in six genes known to underlie FEVR ( TSPAN12, LRP5, FZD4, CTNNB1, KIF11 and NDP ), as well as structural variants in TSPAN12 and KIF11 . These accounted for 15/59 (25.4%) of FEVR cases. We also found candidate heterozygous variants in CTNND1 in three unsolved FEVR cases. Expanding the list of genes examined to include all genes reported to be mutated in ocular disorders likely solved a further four cases, indicating that these individuals may be misclassified as FEVR in GEL. Conclusion By performing bespoke reanalysis of the FEVR GEL cohort, this study has highlighted additional heterozygous variants in CTNND1 in FEVR cases and increased the diagnostic yield from 20% solved by the GEL analysis pipeline to 37% (22/59), but the majority of FEVR cases remain without a molecular diagnosis.

Purpose:Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EOSRD) are inherited retinal diseases that are characterized by severe visual loss very early in life. Whereas LCA is characterized by loss of vision in the first year of life, nystagmus, and absent or abnormal electrical signals on electroretinogram, persons with EOSRD show onset of disease between 1 and 5 years of age, with better preserved visual acuity and some signals on an electroretinogram. We investigated the genetic cause of disease and clinical characteristics in three probands with EOSRD. Methods:All patients were examined by at least two ophthalmologists to reach a clinical diagnosis. APEX microarray screening, smMIP-based sequencing, and whole exome and whole genome sequencing were used to obtain a genetic diagnosis and investigate potential modifiers. Results:The EOSRD phenotype of these three probands was established through ophthalmological investigation. Biallelic severe ABCA4 variants were identified after the phenotypic diagnosis of EOSRD in these probands. We then asked whether additional gene defects may be involved and worsen the phenotype. Through whole genome sequencing we identified two NBAS variants in patient 1 and a well-known homozygous, hypomorphic missense variant in CNGB3 in patient 3. Conclusions:We propose that biallelic severe ABCA4 variants can be implicated in EOSRD. We hypothesize that the ABCA4 and CNGB3 variants could have an additive effect given the colocalization of the encoded proteins in cone photoreceptors cell membranes. Whether the CNGB3 and NBAS variants play a modifying role remains to be investigated.

Purpose:To determine the profile of inflammation-related proteins and complement system factors in the plasma of CRB1-associated inherited retinal dystrophies (CRB1-IRDs). Methods:We used the Olink Explore 384 Inflammation II panel for targeted proteomics in 30 cases and 29 controls (cohort I) to identify immune pathways involved in CRB1-IRDs. Genotyping was performed in cohort I and a second cohort of 123 patients from 14 countries and 1292 controls (cohort II). Results:A significant shift in complement cascade factors was observed in plasma proteomes of CRB1-IRD patients (enrichment for complement cascade, Padj = 3.03 × 10-15). We detected higher plasma levels of complement factor I and complement factor H [CFH] (q = 0.008 and q = 0.046, respectively, adjusted for age and sex), inhibitors of complement component 3 (C3), which correlated significantly (Pearson's coefficient >0.6) with elevated levels of C3 (q = 0.064). The CRB1 missense variants frequently found in patients showed a strong linkage disequilibrium with the common CFH variant rs7535263 (D' = 0.97 for p.(Cys948Tyr); D' = 1.0 for p.(Arg764Cys)), known to be linked with altered plasma CFH-related protein levels. Correction for the CFH genotype revealed significantly elevated plasma levels of CFH-related 2 (CFHR2) in CRB1-IRD patients (q = 0.041). Conclusions:CRB1-IRDs are characterized by changes in plasma levels of complement factors and proteins of the innate immune system, and linkage between CRB1 and CFH genes implicates functional variants of the CFH-CFHR locus with specific pathogenic variants of CRB1.

Variants in six pre-mRNA processing factors cause autosomal dominant Retinitis Pigmentosa (adRP). The RP9 gene encodes a seventh splicing factor, and in 2002, we published RP9 variants c.410A>T; p.(H137L) and c.509A>G; p.(D170G) as likely causes of adRP in a large multigenerational RP9-linked family and a single case, respectively. It has since been suggested these variants might be artefacts due to simultaneous amplification of the RP9P pseudogene, and no further pathogenic variants have been reported. We therefore rescreened two members of the RP9-linked family by genome sequencing. Examination of the 2 Mb locus defined by crossovers in the original family revealed no other plausible causative variants. Alignment of both short and long-read sequences confirmed that p.(H137L) is in the RP9 gene, not the pseudogene. Screening for p.(H137L) in 1961 RP/Rod-cone dystrophy (RCD) cases from the Leeds patient cohort and UK 100,000 Genomes Project (100kGP) database revealed four further carriers. Including the original family, this variant was therefore present in 5/1962 RP/RCD probands, and is absent from gnomAD, constituting statistically significant enrichment in RP cases. Long-read sequencing of p.(H137L) in available carriers showed this is a UK founder allele. The RP9 p.(D170G) allele was also confirmed as gene, not pseudogene, derived, but is present in 22 individuals in the 100kGP cohort, none with RP, as well as >200 individuals in gnomAD and Biobank, suggesting it is non-pathogenic. In conclusion, RP9 p.(H137L) is strongly associated with RP and remains the only plausible variant accounting for the condition in a large multi-generation adRP family.

Inherited retinal diseases (IRDs) are rare disorders, typically presenting as Mendelian traits, that result in stationary or progressive visual impairment. They are characterized by extensive genetic heterogeneity, possibly the highest among all human genetic diseases, as well as diverse inheritance patterns. Despite advances in gene discovery, limited understanding of gene function and challenges in accurately interpreting variants continue to hinder both molecular diagnosis and genetic research in IRDs. One key problem is the absence of a comprehensive and widely accepted catalog of disease-associated genes, which would ensure consistent genetic testing and reliable molecular diagnoses. With the rapid pace of IRD gene discovery, gene catalogs require frequent validation and updates to remain clinically and scientifically useful. To address these gaps, we developed RetiGene, an expert-curated gene atlas that integrates variant data, bulk and single-cell RNA sequencing, and functional annotations. Through the integration of diverse data sources, RetiGene supports candidate gene prioritization, functional studies, and therapeutic development in IRDs.

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.

Trinucleotide repeat expansion in CTG18.1, in intron 2 of TCF4 (MIM *602272, #613267), is the main cause of Fuchs endothelial corneal dystrophy (FECD), accounting for around 75

Amelogenesis is the process of tooth enamel formation, and genetic variants disrupting it cause the Mendelian inherited disorder amelogenesis imperfecta (AI). AI patients have weak, discoloured or brittle enamel, caused by reduced enamel quantity or mineralisation. AI can occur in isolation or, less commonly, as part of a syndrome. Pathogenic variants in at least 38 genes have been shown to cause AI. Current genetic screening studies typically use exome sequencing, but this is expensive and involves complex data analysis workflows. Target enrichment using smMIPs (single molecule molecular inversion probes) provides a flexible alternative, allowing the creation of a disease-specific reagent for low cost, robust, high-throughput screening. Here, we describe the development of an smMIP reagent targeting 19 genes implicated in isolated AI and assess its use in screening a cohort of 181 UK probands with nonsyndromic AI. While this was intended only as a prescreen to prioritise exome sequencing more efficiently, it nevertheless led to molecular diagnoses for 63 probands (35%). Cost per sample screened was approximately £40. Variants in three genes, COL17A1, FAM83H (both dominant) and MMP20 (recessive), accounted for approximately half of solved cases. There is scope to further improve the smMIP reagent by adding additional probes targeting regions of low coverage or additional genes, including those involved in syndromic AI, as well as accommodating new information about the genetic basis of AI. The smMIP reagent provides a robust, flexible, high-throughput, low-cost approach to AI screening, and it is available as a resource to the international AI research community.

Background Collagen XVII is most typically associated with human disease when biallelic COL17A1 variants (>230) cause junctional epidermolysis bullosa (JEB), a rare, genetically heterogeneous, mucocutaneous blistering disease with amelogenesis imperfecta (AI), a developmental enamel defect. Despite recognition that heterozygous carriers in JEB families can have AI, and that heterozygous COL17A1 variants also cause dominant corneal epithelial recurrent erosion dystrophy (ERED), the importance of heterozygous COL17A1 variants causing dominant non-syndromic AI is not widely recognised. Methods Probands from an AI cohort were screened by single molecule molecular inversion probes or targeted hybridisation capture (both a custom panel and whole exome sequencing) for COL17A1 variants. Patient phenotypes were assessed by clinical examination and analyses of affected teeth. Results Nineteen unrelated probands with isolated AI (no co-segregating features) had 17 heterozygous, potentially pathogenic COL17A1 variants, including missense, premature termination codons, frameshift and splice site variants in both the endo-domains and the ecto-domains of the protein. The AI phenotype was consistent with enamel of near normal thickness and variable focal hypoplasia with surface irregularities including pitting. Conclusion These results indicate that COL17A1 variants are a frequent cause of dominantly inherited non-syndromic AI. Comparison of variants implicated in AI and JEB identifies similarities in type and distribution, with five identified in both conditions, one of which may also cause ERED. Increased availability of genetic testing means that more individuals will receive reports of heterozygous COL17A1 variants. We propose that patients with isolated AI or ERED, due to COL17A1 variants, should be considered as potential carriers for JEB and counselled accordingly, reflecting the importance of multidisciplinary care.

Background Plexins are large transmembrane receptors for the semaphorin family of signalling proteins. Semaphorin-plexin signalling controls cellular interactions that are critical during development as well as in adult life stages. Nine plexin genes have been identified in humans, but despite the apparent importance of plexins in development, only biallelic PLXND1 and PLXNA1 variants have so far been associated with Mendelian genetic disease. Methods Eight individuals from six families presented with a recessively inherited variable clinical condition, with core features of amelogenesis imperfecta (AI) and sensorineural hearing loss (SNHL), with variable intellectual disability. Probands were investigated by exome or genome sequencing. Common variants and those unlikely to affect function were excluded. Variants consistent with autosomal recessive inheritance were prioritised. Variant segregation analysis was performed by Sanger sequencing. RNA expression analysis was conducted in C57Bl6 mice. Results Rare biallelic pathogenic variants in plexin B2 ( PLXNB2 ), a large transmembrane semaphorin receptor protein, were found to segregate with disease in all six families. The variants identified include missense, nonsense, splicing changes and a multiexon deletion. Plxnb2 expression was detected in differentiating ameloblasts. Conclusion We identify rare biallelic pathogenic variants in PLXNB2 as a cause of a new autosomal recessive, phenotypically diverse syndrome with AI and SNHL as core features. Intellectual disability, ocular disease, ear developmental abnormalities and lymphoedema were also present in multiple cases. The variable syndromic human phenotype overlaps with that seen in Plxnb2 knockout mice, and, together with the rarity of human PLXNB2 variants, may explain why pathogenic variants in PLXNB2 have not been reported previously.

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.

Loss of glycogen myophosphorylase (PYGM) expression results in an inability to break down muscle glycogen, leading to McArdle disease-an autosomal recessive metabolic disorder characterized by exercise intolerance and muscle cramps. While previously considered relatively benign, this condition has recently been associated with pattern dystrophy in the retina, accompanied by variable sight impairment, secondary to retinal pigment epithelial (RPE) cell involvement. However, the pathomechanism of this condition remains unclear. In this study, we generated a PYGM-null induced pluripotent stem cell line and differentiated it into mature RPE to examine structural and functional defects, along with metabolite release into apical and basal media. Mutant RPE exhibited normal photoreceptor outer segment phagocytosis but displayed elevated glycogen levels, reduced transepithelial resistance, and increased cytokine secretion across the epithelial layer compared to isogenic WT controls. Additionally, decreased expression of the visual cycle component, RDH11, encoding 11-cis-retinol dehydrogenase, was observed in PYGM-null RPE. While glycolytic flux and oxidative phosphorylation levels in PYGM-null RPE were near normal, the basal oxygen consumption rate was increased. Oxygen consumption rate in response to physiological levels of lactate was significantly greater in WT than PYGM-null RPE. Inefficient lactate utilization by mutant RPE resulted in higher glucose dependence and increased glucose uptake from the apical medium in the presence of lactate, suggesting a reduced capacity to spare glucose for photoreceptor use. Metabolic tracing confirmed slower 13C-lactate utilization by PYGM-null RPE. These findings have key implications for retinal health since they likely underlie the vision impairment in individuals with McArdle disease.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

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)