Mutations in SAMD7 cause autosomal-recessive macular dystrophy with or without cone dysfunction.

Sterile alpha motif domain containing 7 (SAMD7) is a component of the Polycomb repressive complex 1, which inhibits transcription of many genes, including those activated by the transcription factor Cone-Rod Homeobox (CRX). Here we report bi-allelic mutations in SAMD7 as a cause of autosomal-recessive macular dystrophy with or without cone dysfunction. Four of these mutations affect splicing, while another mutation is a missense variant that alters the repressive effect of SAMD7 on CRX-dependent promoter activity, as shown by in vitro assays. Immunostaining of human retinal sections revealed that SAMD7 is localized in the nuclei of both rods and cones, as well as in those of cells belonging to the inner nuclear layer. These results place SAMD7 as a gene crucial for human retinal function and demonstrate a significant difference in the role of SAMD7 between the human and the mouse retina.

Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4.

The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters the pre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1.

Autozygome-guided exome-first study in a consanguineous cohort with early-onset retinal disease uncovers an isolated RIMS2 phenotype and a retina-enriched RIMS2 isoform.

Leber congenital amaurosis (LCA) and early-onset retinal degeneration (EORD) are inherited retinal diseases (IRD) characterized by early-onset vision impairment. Herein, we studied 15 Saudi families by whole exome sequencing (WES) and run-of-homozygosity (ROH) detection via AutoMap in 12/15 consanguineous families. This revealed (likely) pathogenic variants in 11/15 families (73%). A potential founder variant was found in RPGRIP1. Homozygous pathogenic variants were identified in known IRD genes (ATF6, CRB1, CABP4, RDH12, RIMS2, RPGRIP1, SPATA7). We established genotype-driven clinical reclassifications for ATF6, CABP4, and RIMS2. Specifically, we observed isolated IRD in the individual with the novel RIMS2 variant, and we found a retina-enriched RIMS2 isoform conserved but not annotated in mouse. The latter illustrates potential different phenotypic consequences of pathogenic variants depending on the particular tissue/cell-type specific isoforms they affect. Lastly, a compound heterozygous genotype in GUCY2D in one non-consanguineous family was demonstrated, and homozygous variants in novel candidate genes ATG2B and RUFY3 were found in the two remaining consanguineous families. Reporting these genes will allow to validate them in other IRD cohorts. Finally, the missing heritability of the two unsolved IRD cases may be attributed to variants in non-coding regions or structural variants that remained undetected, warranting future WGS studies.

Intermediate uveitis in common variable immunodeficiency (CVID) associated with a heterozygous variant in the TNFRSF13B gene.

PURPOSE: To report on a rare case of intermediate uveitis occurring in a patient with common variable immunodeficiency (CVID) and a heterozygous TNFRSF13B variant. METHODS: Observational case report. RESULTS: A 23-year-old male presented with a 3-month history of increasing floaters and blurred vision to both eyes. He had been treated with topical and intravitreal corticosteroids by his local ophthalmologist nine months before. Ocular examination demonstrated bilateral intermediate uveitis with retinal vasculitis. He had been treated with intravenous immunoglobulins during childhood, due to primary humoral immunodeficiency. Systemic work-up for other causes of intermediate uveitis was unremarkable, notably no features of systemic sarcoid-like disease were detected. Initial treatment with mycophenolate mofetil showed insufficient response, and upon switching to adalimumab, clinical remission was achieved. Immunocytometry and genetic work-up revealed a smB+CD21norm subtype of CVID and a heterozygous TNFRSF13B variant. CONCLUSION: This report of CVID-associated intermediate uveitis in a patient with a heterozygous TNFRSF13B variant highlights the potential involvement of the eye within CVID-associated autoimmunity and the role for anti-TNF blockade in this challenging group of patients.

Multi-omics analysis in human retina uncovers ultraconserved cis-regulatory elements at rare eye disease loci.

Cross-species genome comparisons have revealed a substantial number of ultraconserved non-coding elements (UCNEs). Several of these elements have proved to be essential tissue- and cell type-specific cis-regulators of developmental gene expression. Here, we characterize a set of UCNEs as candidate CREs (cCREs) during retinal development and evaluate the contribution of their genomic variation to rare eye diseases, for which pathogenic non-coding variants are emerging. Integration of bulk and single-cell retinal multi-omics data reveals 594 genes under potential cis-regulatory control of UCNEs, of which 45 are implicated in rare eye disease. Mining of candidate cis-regulatory UCNEs in WGS data derived from the rare eye disease cohort of Genomics England reveals 178 ultrarare variants within 84 UCNEs associated with 29 disease genes. Overall, we provide a comprehensive annotation of ultraconserved non-coding regions acting as cCREs during retinal development which can be targets of non-coding variation underlying rare eye diseases.

Evolutionary origin of Hoxc13-dependent skin appendages in amphibians.

Cornified skin appendages, such as hair and nails, are major evolutionary innovations of terrestrial vertebrates. Human hair and nails consist largely of special intermediate filament proteins, known as hair keratins, which are expressed under the control of the transcription factor Hoxc13. Here, we show that the cornified claws of Xenopus frogs contain homologs of hair keratins and the genes encoding these keratins are flanked by promoters in which binding sites of Hoxc13 are conserved. Furthermore, these keratins and Hoxc13 are co-expressed in the claw-forming epithelium of frog toe tips. Upon deletion of hoxc13, the expression of hair keratin homologs is abolished and the development of cornified claws is abrogated in X. tropicalis. These results indicate that Hoxc13-dependent expression of hair keratin homologs evolved already in stem tetrapods, presumably as a mechanism for protecting toe tips, and that this ancestral genetic program was coopted to the growth of hair in mammals.

Insight into the role of TXNRD2 in steroidogenesis through a novel homozygous TXNRD2 splice variant.

OBJECTIVE: Adrenal cortisol production occurs through a biosynthetic pathway which depend on NADH and NADPH for energy supply. The mitochondrial respiratory chain and the reactive oxygen species (ROS) detoxification system are therefore important for steroidogenesis. Mitochondrial dysfunction leading to oxidative stress has been implicated in the pathogenesis of several adrenal conditions. Nonetheless, only very few patients with variants in one gene of the ROS detoxification system, Thioredoxin Reductase 2 (TXNRD2), have been described with variable phenotypes. DESIGN: Clinical, genetic, structural, and functional characterization of a novel, biallelic TXNRD2 splice variant. METHODS: On human biomaterial, we performed whole exome sequencing to identify and RNA analysis to characterize the specific TXNRD2 splice variant. Amino acid conservation analysis and protein structure modeling were performed in silico. Using patient's fibroblast-derived human induced pluripotent stem cells, we generated adrenal-like cells (iALC) to study the impact of wild-type (WT) and mutant TXNRD2 on adrenal steroidogenesis and ROS production. RESULTS: The patient had a complex phenotype of primary adrenal insufficiency (PAI), combined with genital, ophthalmological, and neurological features. He carried a homozygous splice variant c.1348-1G > T in TXNRD2 which leads to a shorter protein lacking the C-terminus and thereby affecting homodimerization and flavin adenine dinucleotide binding. Patient-derived iALC showed a loss of cortisol production with overall diminished adrenal steroidogenesis, while ROS production was significantly increased. CONCLUSION: Lack of TXNRD2 activity for mitochondrial ROS detoxification affects adrenal steroidogenesis and predominantly cortisol production.

Comparative 3D genome analysis between neural retina and retinal pigment epithelium reveals differential cis-regulatory interactions at retinal disease loci.

BACKGROUND: Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. RESULTS: To understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4. CONCLUSIONS: Through comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.

Spleen function is reduced in individuals with NR5A1 variants with or without a difference of sex development: a cross-sectional study.

OBJECTIVE: NR5A1 is a key regulator of sex differentiation and has been implicated in spleen development through transcription activation of TLX1. Concerns exist about hypo- or asplenism in individuals who have a difference of sex development (DSD) due to an NR5A1 disease-causing variant. We aimed to assess spleen anatomy and function in a clinical cohort of such individuals and in their asymptomatic family member carriers. DESIGN: Cross-sectional assessment in 22 patients with a DSD or primary ovarian insufficiency and 5 asymptomatic carriers from 18 families, harboring 14 different NR5A1 variants. METHODS: Spleen anatomy was assessed by ultrasound, spleen function by peripheral blood cell count, white blood cell differentiation, percentage of nonswitched memory B cells, specific pneumococcal antibody response, % pitted red blood cells, and Howell-Jolly bodies. RESULTS: Patients and asymptomatic heterozygous individuals had significantly decreased nonswitched memory B cells compared to healthy controls, but higher than asplenic patients. Thrombocytosis and spleen hypoplasia were present in 50% of heterozygous individuals. Four out of 5 individuals homozygous for the previously described p.(Arg103Gln) variant had asplenia. CONCLUSIONS: Individuals harboring a heterozygous NR5A1 variant that may cause DSD have a considerable risk for functional hyposplenism, irrespective of their gonadal phenotype. Splenic function should be assessed in these individuals, and if affected or unknown, prophylaxis is recommended to prevent invasive encapsulated bacterial infections. The splenic phenotype associated with NR5A1 variants is more severe in homozygous individuals and is, at least for the p.(Arg103Gln) variant, associated with asplenism.

Combining a prioritization strategy and functional studies nominates 5'UTR variants underlying inherited retinal disease.

BACKGROUND: 5' untranslated regions (5'UTRs) are essential modulators of protein translation. Predicting the impact of 5'UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5'UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). METHODS: We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5'UTRs by different whole exome sequencing (WES) kits. The selected 5'UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5'UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches. RESULTS: Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5'UTR compared to that of their canonical isoform. Depending on the probe design, 3-20% of IRD genes have 5'UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5'UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. CONCLUSIONS: This study demonstrates the importance of 5'UTR variants implicated in IRDs and provides a systematic approach for 5'UTR annotation and validation that is applicable to other inherited diseases.

Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

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.

Characterising the refractive error in paediatric patients with congenital stationary night blindness: a multicentre study.

BACKGROUND/AAIMS: Congenital stationary night blindness (CSNB) is an inherited retinal disease that is often associated with high myopia and can be caused by pathological variants in multiple genes, most commonly CACNA1F, NYX and TRPM1. High myopia is associated with retinal degeneration and increased risk for retinal detachment. Slowing the progression of myopia in patients with CSNB would likely be beneficial in reducing risk, but before interventions can be considered, it is important to understand the natural history of myopic progression. METHODS: This multicentre, retrospective study explored CSNB caused by variants in CACNA1F, NYX or TRPM1 in patients who had at least 6 measurements of their spherical equivalent of refraction (SER) before the age of 18. A mixed-effect model was used to predict progression of SER overtime and differences between genotypes were evaluated. RESULTS: 78 individuals were included in this study. All genotypes showed a significant myopic predicted SER at birth (-3.076D, -5.511D and -5.386D) for CACNA1F, NYX and TRPM1 respectively. Additionally, significant progression of myopia per year (-0.254D, -0.257D and -0.326D) was observed for all three genotypes CACNA1F, NYX and TRPM1, respectively. CONCLUSIONS: Patients with CSNB tend to be myopic from an early age and progress to become more myopic with age. Patients may benefit from long-term myopia slowing treatment in the future and further studies are indicated. Additionally, CSNB should be considered in the differential diagnosis for early-onset myopia.