Human Genetics

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO), account for [~]25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (1) show advanced retinal degeneration in a male patient (60-70 year old) harboring four transcriptionally active intact copies of rhodopsin, (2) recapitulated the clinical phenotypes using retinal organoids, and (3) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (qRT-PCR and bulk RNA-sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300-days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that adRP due to RHO-CNV develops due protein overexpression overloading the photoreceptor post-translational modification machinery.

Meniere disease (MD) is a polygenic condition defined by episodes of vertigo associated with sensorineural hearing loss and tinnitus. Genetic studies in familial MD in East Asian population are limited and the potential MD genes remain to be established in non-Finnish European populations. By exome sequencing and rare variant analysis, we search for existing and novel genes associated with MD in a South Korean cohort of 16 MD individuals with bilateral sensorineural hearing loss. We have found one individual with two rare missense variants in the OTOP2 gene, a new candidate gene for MD and three heterozygous variants in the MYO7A gene, supporting the hypothesis of biallelic inheritance. Protein modelling was conducted on three rare missense variants in OTOP2 to further elucidate functional consequences. The structural and functional implications inferred from these models suggest a likely pathogenic role, providing additional insights into the molecular mechanisms underlying MD.

BACKGROUNDCongenital heart defects (CHD) appear in almost one percent of live births. Asian countries have the highest birth prevalence of CHD in the world. Recessive genotypes may represent a significant CHD risk factor in Asian populations, because Asian populations have a high degree of consanguineous marriages, which increases the risk of CHD. Genetic analysis of consanguineous families may represent a relatively unexplored source for investigating CHD etiology. METHODSTo obtain insight into the contribution of recessive genotypes in CHD we analysed a cohort of forty-nine Pakistani CHD probands, originating from consanguineous unions. The majority (82%) of patients malformations were septal defects. We identified protein altering, rare homozygous variants (RHVs) in the patients coding genome by whole exome sequencing. RESULTSThe patients had a median of seven damaging RHVs each, and our analysis revealed a total of 758 RHVs in 693 different genes. By prioritizing these genes based on variant severity, loss-of-function intolerance and specific expression in the developing heart, we identified a set of 23 candidate disease genes. These candidate genes were significantly enriched for genes known to cause heart defects in recessive mouse models (P<2.4e-06). In addition, we found a significant enrichment of cilia genes in both the initial set of 693 genes (P<5.4e-04) and the 23 candidate disease genes (P<5.2e-04). Functional investigation of ADCY6 in cell- and zebrafish-models verified its role in heart development. CONCLUSIONSOur results confirm a significant role for cilia genes in recessive forms of CHD and suggest important functions of cilia genes in cardiac septation.

Meniere disease (MD) is a chronic inner ear disorder with significant heritability. This study aims to compare the burden of rare high-and moderate-impact protein-coding variants in a MD cohort to determine whether the genetic burden in sporadic MD (SMD) overlaps familial MD (FMD), potentially revealing hidden inheritance in SMD. In this study exome sequencing identified rare variants in unrelated FMD (N=93) and SMD (N=287) patients. Gene Burden Analysis (GBA) was performed, and candidate genes were prioritized using the number of individuals with variants, inner-ear gene expression, and hearing and balance-related phenotypic annotations. In FMD patients, a higher accumulation of missense and loss-of-function variants was observed compared to SMD, particularly in genes associated with auditory and vestibular functions. GBA identified 269 enriched genes in SMD, with 31 annotated for inner ear phenotypes, while FMD had 432 genes with 51 pinpointed. Sporadic and FMD overlapped in 28.1% of the enriched genes, with ADGRV1, MEGF8 and MYO7A the most commonly found. In conclusion, SMD and FMD have a divergent genetic architecture. Both SMD and FMD have an overload of missense variants in stria vascularis and hair cell stereocilia genes that suggests different mechanisms in MD pathogenesis and a multiallelic-recessive inheritance pattern.

Cataract is the leading cause of blindness among the elderly worldwide and cataract surgery is one of the most common operations performed in the United States1-3. The etiology remains largely unclear, and to contribute to its elucidation we conducted a multiethnic genome-wide association meta-analysis of cataract, combining results from the GERA and UK Biobank cohorts, and tested for replication in the research cohort from 23andMe, Inc.. We report 54 genome-wide significant loci, 37 of which were previously unknown. Sex-stratified analyses identified two additional novel loci (CASP7 and GSTM2) specific to women and sex differences in effect sizes and significance of association at five other loci. We show that genes within or near 80% of the cataract-associated loci are significantly expressed and/or enriched-expressed in the mouse lens across various spatiotemporal stages. Further, 32 candidate genes in the associated loci have altered gene expression in 9 different gene perturbation mouse models of lens defects/cataract, suggesting their relevance to lens biology.

Meniere disease (MD) is an inner ear disorder characterised by episodic vertigo, tinnitus, and sensorineural hearing loss. Previous sequencing studies have identified rare mutations in 16 genes expressed in hair cells, many of which are involved in maintaining the structure and stability of stereocilia. Experimental evidence supports that noise-induced hearing loss is associated with structural damage in the hair cell stereocilia. We hypothesise that rare variants in genes expressed in hair cell stereocilia may predispose individuals to MD with occupational noise exposure. To explore this, we analysed whole exome sequencing data from MD patients with documented histories of occupational noise exposure. A rare missense variant chr22:37769343C>T in the TRIOBP gene was identified in three individuals (OR=1,846 (203-8192), p=3.39x10-5). In silico protein modelling suggests this variant (p.R2273C) interacts with the Actin C chain, potentially affecting cytoskeletal integrity in hair cells. Immunolabelling confirmed TRIOBP expression in the stereocilia rootlets of cochlear and vestibular hair cells, supporting its structural function in both sensory epithelia. Furthermore, retrieved MD single-cell ATAC-seq data revealed reduced chromatin accessibility at the RFX3 transcription factor locus, suggesting a transcriptional TRIOBP downregulation. Our results support that occupational noise exposure may trigger MD in the carriers of TRIOBP mutations.

PurposeGenetic variants in complement genes are associated with age-related macular degeneration (AMD). However, many rare variants have been identified in these genes, but have an unknown significance, and their impact on protein function and structure is still unknown. We set out to address this issue by evaluating the spatial placement and impact on protein structureof these variants by developing an analytical pipeline and applying it to the International AMD Genomics Consortium (IAMDGC) dataset (16,144 AMD cases, 17,832 controls). MethodsThe IAMDGC dataset was imputed using the Haplotype Reference Consortium (HRC), leading to an improvement of over 30% more imputed variants, over the original 1000 Genomes imputation. Variants were extracted for the CFH, CFI, CFB, C9, and C3 genes, and filtered for missense variants in solved protein structures. We evaluated these variants as to their placement in the three-dimensional structure of the protein (i.e. spatial proximity in the protein), as well as AMD association. We applied several pipelines to a) calculate spatial proximity to known AMD variants versus gnomAD variants, b) assess a variants likelihood of causing protein destabilization via calculation of predicted free energy change (ddG) using Rosetta, and c) whole gene-based testing to test for statistical associations. Gene-based testing using seqMeta was performed using a) all variants b) variants near known AMD variants or c) with a ddG >|2|. Further, we applied a structural kernel adaptation of SKAT testing (POKEMON) to confirm the association of spatial distributions of missense variants to AMD. Finally, we used logistic regression on known AMD variants in CFI to identify variants leading to >50% reduction in protein expression from known AMD patient carriers of CFI variants compared to wild type (as determined by in vitro experiments) to determine the pipelines robustness in identifying AMD-relevant variants. These results were compared to functional impact scores, ie CADD values > 10, which indicate if a variant may have a large functional impact genomewide, to determine if our metrics have better discriminative power than existing variant assessment methods. Once our pipeline had been validated, we then performed a priori selection of variants using this pipeline methodology, and tested AMD patient cell lines that carried those selected variants from the EUGENDA cohort (n=34). We investigated complement pathway protein expression in vitro, looking at multiple components of the complement factor pathway in patient carriers of bioinformatically identified variants. ResultsMultiple variants were found with a ddG>|2| in each complement gene investigated. Gene-based tests using known and novel missense variants identified significant associations of the C3, C9, CFB, and CFH genes with AMD risk after controlling for age and sex (P=3.22x10-5;7.58x10-6;2.1x10-3;1.2x10-31). ddG filtering and SKAT-O tests indicate that missense variants that are predicted to destabilize the protein, in both CFI and CFH, are associated with AMD (P=CFH:0.05, CFI:0.01, threshold of 0.05 significance). Our structural kernel approach identified spatial associations for AMD risk within the protein structures for C3, C9, CFB, CFH, and CFI at a nominal p-value of 0.05. Both ddG and CADD scores were predictive of reduced CFI protein expression, with ROC curve analyses indicating ddG is a better predictor (AUCs of 0.76 and 0.69, respectively). A priori in vitro analysis of variants in all complement factor genes indicated that several variants identified via bioinformatics programs PathProx/POKEMON in our pipeline via in vitro experiments caused significant change in complement protein expression (P=0.04) in actual patient carriers of those variants, via ELISA testing of proteins in the complement factor pathway, and were previously unknown to contribute to AMD pathogenesis. ConclusionWe demonstrate for the first time that missense variants in complement genes cluster together spatially and are associated with AMD case/control status. Using this method, we can identify CFI and CFH variants of previously unknown significance that are predicted to destabilize the proteins. These variants, both in and outside spatial clusters, can predict in-vitro tested CFI protein expression changes, and we hypothesize the same is true for CFH. A priori identification of variants that impact gene expression allow for classification for previously classified as VUS. Further investigation is needed to validate the models for additional variants and to be applied to all AMD-associated genes.

Familial Meniere Disease (FMD) is a rare polygenic disorder of the inner ear. Mutations in the connexin gene family, which encodes gap junction proteins, can also cause hearing loss, but their role in FMD is largely unknown. Here, we found an enrichment of rare missense variants in the GJD3 gene when comparing allelic frequencies in FMD (N=94) with the Spanish reference population (OR=3.9[1.92-7.91], FDR=2.36E-03). In the GJD3 sequence, we identified a rare haplotype (TGAGT) composed of two missense, two synonymous, and one downstream variants. This haplotype was found in five individuals with FMD, segregating in three unrelated families with a total of ten individuals; and in another eight Meniere Disease individuals. GJD3 encodes the gap junction protein delta 3, also known as human connexin 31.9 (CX31.9). The protein model predicted that the NP_689343.3:p.(His175Tyr) missense variant could modify the interaction between connexins and the connexon assembly, affecting the homotypic GJD3 gap junction between cells. Our studies in mice revealed that the mouse ortholog Gjd3 - encoding Gjd3 or mouse connexin 30.2 (Cx30.2) - was expressed in the organ of Corti and vestibular organs, particularly in the tectorial membrane, the base of inner and outer hair cells and the nerve fibers. The present results describe a novel association between GJD3 and familial FMD, providing evidence that FMD is related to changes in the inner ear channels; in addition, it supports a new role of tectorial membrane proteins in FMD.

Primary open-angle glaucoma (POAG) disproportionately affects individuals of African ancestry, yet rare coding variation in this population remains understudied. To address this gap, we performed a multi-cohort exome-wide meta-analysis across POAAGG, PMBB, All of Us, and UK Biobank, including 4,815 POAG cases and 22,922 controls of genetically inferred African ancestry. Although no gene reached exome-wide significance, we identified several suggestive gene-level associations driven by rare variants (minor allele frequency [&le;]0.1% or singletons),including signals in SRF, BLTP3A, METTL2A, and KRT10. Among these, SRF demonstrated the strongest association and was driven by rare missense variants with moderate effect sizes. Given its role in cytoskeletal organization and actin dynamics; processes central to trabecular meshwork function and intraocular pressure regulation SRF represents a biologically plausible candidate gene. Notably, these genes have not been previously highlighted in predominantly European ancestry POAG association studies, suggesting potential ancestry-specific rare variant contributions. Overall, our findings highlight the critical importance of investigating rare coding variation in POAG, in disproportionately affected populations to deepen understanding of POAG etiology and genetic risk.

PurposeRetinitis pigmentosa (RP) is an inherited retinal disorder that results in the degeneration of photoreceptor cells, ultimately leading to severe visual impairment. We characterized a consanguineous family from Southern India wherein an individual in his 20s presented with night blindness since childhood. The purpose of this study was to identify the causative mutation for RP in this individual as well as characterize how the mutation may ultimately affect protein function. MethodsWe performed a complete ophthalmologic examination of the proband followed by exome sequencing. The identified mutation was then modeled in cultured cells, evaluating its expression, solubility (both by western blot), subcellular distribution (confocal microscopy), and testing whether this variant induced endoplasmic reticulum (ER) stress (qPCR and western blotting). ResultsThe proband presented with generalized and parafoveal retinal pigment epithelial atrophy with bone spicule pigmentation in the mid periphery and arteriolar attenuation. Optical coherence tomography scans through the macula of both eyes showed atrophy of outer retinal layers with loss of the ellipsoid zone, whereas systemic examination of this individual was normal. The probands parents and sibling were asymptomatic and had normal funduscopic examinations. We discovered a novel homozygous p.Pro388Ser mutation in the tubby-like protein 1 (TULP1) gene in the individual with RP. In cultured cells, the P388S mutation does not alter the subcellular distribution of TULP1 or induce ER stress when compared to wild-type TULP1, but instead significantly lowers protein stability as indicated by steady-state and cycloheximide-chase experiments. ConclusionsThese results add to the list of known TULP1 mutations associated with RP and suggest a unique pathogenic mechanism in TULP1-induced RP, which may be shared amongst select mutations in TULP1.

ImportanceGenome-wide association studies have identified hundreds of common single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) associated with primary open-angle glaucoma (POAG) risk, though these variants have modest effect sizes and individually may have minor contributions to disease development. As whole-genome sequencing data is becoming more readily available, structural variants and other complex genomic features can be interrogated for contribution to disease risk. ObjectiveTest the association of structural variants in known glaucoma loci with disease risk. DesignCross-sectional study. SettingA multicenter cohort of individuals from the United States who contributed genomic and electronic health record data to the All of Us Research Program. ParticipantsPOAG case/control cohorts were generated in the All of Us Researcher Workbench using age (>40 for cases, >65 for controls) and ICD 9/10 diagnosis codes. Main Outcomes and MeasuresLogistic regression analyses adjusted for age, sex, and the top 10 principal components of ancestry were used to test association of structural variants within 500 kilobases of 309 known open-angle glaucoma risk loci. The significance threshold after Bonferroni correction was set at p<1.6x10-4. Results516 POAG cases and 18,716 controls of European ancestry from the All of Us v8 data release were included in the analysis. Mean age was 77.0 years among cases and 74.7 years among controls. Females comprised 45.7% of cases and 56.5% of controls. An 8,732 base pair deletion upstream of PITX2 (chr4:110680827-110689558) was associated with 7.3-fold higher odds of POAG (95% confidence interval: 2.9-18.5, p= 2.4x10-5, variant carrier frequency= 1.6% in cases and 0.25% in controls). Functional annotation identified multiple enhancers overlapping the deletion, suggesting that this structural variant likely impacts gene regulation and expression. Conclusion and RelevanceWhole genome sequencing data captures rare structural variants with large effect sizes that are missed by conventional SNP and indel genotyping approaches, enabling improved POAG risk stratification. These data also expand the phenotypic spectrum of structural variation in the PITX2 locus from childhood glaucoma to adult-onset disease, where age at diagnosis and clinical severity may be influenced by the extent of disrupted regulatory elements.

PurposeThe transcription factor TBX2 plays a critical role in inner hair cells development in mice. Yet, the link between TBX2 malfunction and human hearing-related disorders remains unexplored. MethodsLinkage analysis combined with whole genome sequencing was applied to identify the causative gene in two autosomal dominant Chinese families characterized by late-onset progressive sensorineural hearing loss and incomplete penetrance of horizontal oscillatory nystagmus. Functional evaluation of TBX2 variants was performed through protein expression, localization, and transcriptional activity analysis in vitro, phenotypic analysis and mechanism study in knockout mice model in vivo. ResultsMultipoint parametric linkage analysis of Family 1 revealed a maximum LOD score of 3.01 on chromosome 17q23.2. Whole genome sequencing identified distinct TBX2 variants, c.977delA (p.Asp326Alafs*42) and c.987delC (p.Ala330Argfs*38) in each family, co-segregating with hearing loss. These variants resulted in premature termination and the generation of a new peptide segment, reducing transcriptional activity. Further, heterozygous Tbx2 knockout mice exhibited late-onset progressive hearing loss, along with ectopic expression of Prestin in IHCs and a gradual decrease in expression from P7 to P42. ConclusionOur findings indicate that heterozygous TBX2 frameshift variants are the genetic cause of late-onset progressive hearing loss and incomplete penetrance of nystagmus. The heterozygous Tbx2 knockout mouse model mirrored the human hearing loss phenotype, further validating TBX2s role in auditory function. These insights enhance our understanding of TBX2 in the auditory system, providing valuable information for molecular diagnostics and genetic counseling in related hearing disorders.

PurposeThe human 10q26 locus is a major genetic risk factor for age-related macular degeneration (AMD). Fine mapping by linkage and large-scale genome-wide association studies (GWAS) has narrowed this region to a 30-kb interval encompassing the ARMS2 and HTRA1 genes. However, the causative gene(s), risk variants, and underlying pathogenic mechanisms remain unresolved. MethodsLong non-coding RNA (lncRNA) candidates within the ARMS2-HTRA1 region were identified using human postmortem retinal RNA-seq data and public databases (NCBI, Ensembl). Candidate transcripts were validated by RT-PCR and Sanger sequencing. Published single-cell RNA-seq datasets were analysed to define cell type-specific expression, and RNA levels were compared between AMD and non-AMD donor retinas. Additionally, expression changes were assessed in human iPSC-derived retinal pigment epithelium (RPE) cells exposed to cigarette smoke extract (CSE) and paraquat (PQT). ResultsWe identified and validated a lncRNA, HTRA1-AS1, and its transcript variants (ENST00000647969.1) within the ARMS2 locus. HTRA1-AS1 overlaps ARMS2 and is transcribed in the antisense orientation. It is predominantly expressed in rod photoreceptors, Muller glia and Choroid/RPE, and its retinal expression was significantly reduced in AMD compared with controls (43 AMD donors vs. 44 controls, p = 0.007). By contrast, HTRA1 mRNA showed no significant difference (p = 0.121). Furthermore, ENST00000647969.1, HTRA1-AS1 and ARMS2 expression increased dramatically, up to 101-fold, 8-fold and 75-fold, respectively, in induced pluripotent stem cells (iPSC)-derived RPE cells following cigarette smoke extract (CSE)-induced oxidative stress but showed no significant change after paraquat treatment. ConclusionThese findings suggest that HTRA1-AS1, a dysregulated lncRNA within the ARMS2 locus, may act as a non-coding element contributing to transcriptional mis-regulation underlying AMD pathogenesis.

With continued advances in gene sequencing technologies comes the need to develop better tools to understand which mutations cause disease. Here we validate structure-based network analysis (SBNA)1, 2 in well-studied human proteins and report results of using SBNA to identify critical amino acids that may cause retinal disease if subject to missense mutation. We computed SBNA scores for genes with high-quality structural data, starting with validating the method using 4 well-studied human disease-associated proteins. We then analyzed 47 inherited retinal disease (IRD) genes. We compared SBNA scores to phenotype data from the ClinVar database and found a significant difference between benign and pathogenic mutations with respect to network score. Finally, we applied this approach to 65 patients at Massachusetts Eye and Ear (MEE) who were diagnosed with IRD but for whom no genetic cause was found. Multivariable logistic regression models built using SBNA scores for IRD-associated genes successfully predicted pathogenicity of novel mutations, allowing us to identify likely causative disease variants in 37 patients with IRD from our clinic. In conclusion, SBNA can be meaningfully applied to human proteins and may help predict mutations causative of IRD.

Mutations in PRPF31 are the second most common cause of the degenerative retinal condition autosomal dominant retinitis pigmentosa. Difficulty in characterising missense variants in this gene presents a significant challenge in providing accurate diagnosis for patients to enable targeted testing of other family members, aid family planning, allow pre-implantation diagnosis and inform eligibility for gene therapy trials. With PRPF31 gene therapy in development, there is an urgent need for tools for accurate molecular diagnosis. Here we present a high-throughput high content imaging assay providing quantitative measure of effect of missense variants in PRPF31 which meets the recently published criteria for a baseline standard in vitro test for clinical variant interpretation. This assay utilizes a new and well-characterized PRPF31+/- human retinal cell line generated using CRISPR gene editing, which allows testing of PRPF31 variants which may be causing disease through either haploinsufficiency or dominant negative effects, or a combination of both. The mutant cells have significantly fewer cilia than wild-type cells, allowing rescue of ciliogenesis with benign or mild variants, but do not totally lack cilia, so dominant negative effects can be observed. The results of the assay provide BS3_supporting evidence to the benign classification of two novel uncharacterized PRPF31 variants and suggest that one novel uncharacterized PRPF31 variant may be pathogenic. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of unknown significance, and can be extended to variant classification in other ciliopathies.

Age-related hearing loss (ARHL) is a progressive, bilateral decline in hearing ability that affects one in four individuals over 60 years of age worldwide. While previous genome-wide association studies (GWAS) have identified distinct single-nucleotide variants (SNVs) associated with metabolic and sensory ARHL phenotypes, the contribution of short tandem repeats (STRs) - a neglected yet important class of genetic variants - remains poorly understood. To address this gap, TRTools was used to impute STRs from a high quality, sequencing-derived SNV-STR reference panel to investigate the association between STRs and metabolic and sensory estimates. Heritability analyses revealed that while STRs contribute to estimates of both ARHL components, this class of variation plays a more important role in metabolic hearing loss (6%), which typically increases with age, compared to sensory hearing loss (4%). Further, the inclusion of this class of variant into GWAS analyses uncovered an association between a haplotype consisting of two missense variants (rs7714670 and rs6453022) and an intronic STR (chr5:73778077:A16) in ARHGEF28 (P=3.30x10-9), proving further insight into the variants driving this previously identified signal. Notably, burden analyses revealed that rare and longer repeats were associated with an increased risk of the metabolic phenotype and a reduced risk of the sensory phenotype. Functional annotation of significant and nominally significant STRs revealed potential effects on gene expression and splicing of nearby genes. Our findings provide the first evidence that STRs explain some of the missing heritability of ARHL phenotypes and create an STR resource for researchers to use in future analyses.

Generalised Lymphatic Dysplasia (GLD) is characterised by widespread lymphoedema, with at least one of the following: fetal hydrops, intestinal or pulmonary lymphangiectasia, pleural effusions, pericardial effusions and ascites. Satisfactory medical therapies are lacking. A genetic association has been identified that prevents expression or surface trafficking of PIEZO1, a subunit of mechanically activated calcium-permeable channels. However, PIEZO1 is a large and highly polymorphic gene and interpretation of variants identified in this gene can be challenging. PIEZO1-related GLD with non-immune fetal hydrops is autosomal recessive, however, heterozygous variants in PIEZO1 (often gain-of-function) causing Dehydrated Hereditary Stomatocytosis (DHS) (a relative mild anaemia), may also present with perinatal non-immune hydrops (not caused by anaemia). Here we sought to develop methods to confirm pathogenicity of missense variants of uncertain significance in PIEZO1, to gain deeper understanding and pharmacological solutions. Four novel GLD-associated missense variants in PIEZO1 are identified that express and surface localise as full-length protein but with reduced or abolished mechanically activated channel function. Yoda1, a small-molecule agonist, functionally rescues the channels and their physiological regulation by mechanical force and hypo-osmolality. The GLD-associated variants mediate intracellular calcium release as well as calcium entry, suggesting two pools of channels and opportunity for increased rescue through access to the intracellular pool. New Yoda1 analogues are also identified that improve rescue. The functional assays have assisted the interpretation of the variants of uncertain significance as the data suggest loss of PIEZO1 force sensing as a cause of the GLD observed in the patients. The potential to pharmacologically overcome the loss of force sensing was demonstrated and supports the concept of stimulation of PIEZO1 with an agonist to address wide-ranging problems of lymphatic insufficiency. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/23292554v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@2f514eorg.highwire.dtl.DTLVardef@831480org.highwire.dtl.DTLVardef@1b6ee1forg.highwire.dtl.DTLVardef@cb6757_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIPreviously unrecognised variants in PIEZO1 that associate with GLD are identified and characterised and pathogenicity confirmed C_LIO_LIThe variants encode single amino acid changes that inhibit PIEZO1 channel activation by physiological mechanical forces C_LIO_LIA small-molecule agonist rescues the channels and their physiological regulation C_LIO_LIVariants are partly intracellular, suggesting an opportunity for improved rescue through the use of intracellular-acting agonists C_LIO_LINew agonists are identified that improve rescue, suggesting routes to medical therapies for GLD and potentially other disorders of lymphatic insufficiency C_LI

Genetic disorders impacting vision affect millions of individuals worldwide, including age-related macular degeneration (common) and inherited retinal disorders (rare). There is incomplete understanding of the impact of genetic variation on gene expression in the human retina, and its role in genetic disorders. Through the generation of whole genome sequencing and bulk RNA-sequencing of neurosensory retina (NSR) and retinal pigment epithelium (RPE) from 201 post-mortem eyes, we uncovered common and rare genetic variants shaping retinal expression profiles. This includes 1,483,595 significant cis-expression quantitative trait loci (eQTLs) impacting 9,959 and 3,699 genes in NSR and RPE, respectively, with associated genetic variants enriched to cis-candidate regulatory elements and notable shared eGenes between NSR and RPE. We also detected 1051 expression outliers and prioritised 299 rare non-coding single-nucleotide, structural variants or copy number variants as plausible drivers for 28% of outlier events. This study increases understanding of gene expression regulation in the human retina. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/25326445v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@153c017org.highwire.dtl.DTLVardef@16827b5org.highwire.dtl.DTLVardef@1071537org.highwire.dtl.DTLVardef@1ddcb_HPS_FORMAT_FIGEXP M_FIG C_FIG

POPDC2 encodes for the Popeye domain-containing protein 2 which has an important role in cardiac pacemaking and conduction, due in part to its cAMP-dependent binding and regulation of TREK-1 potassium channels. Loss of Popdc2 in mice results in sinus pauses and bradycardia and morpholino knockdown of popdc2 in zebrafish results in atrioventricular (AV) block. We identified bi-allelic variants in POPDC2 in 4 families that presented with a phenotypic spectrum consisting of sinus node dysfunction, AV conduction defects and hypertrophic cardiomyopathy. Using homology modelling we show that the identified POPDC2 variants are predicted to diminish the ability of POPDC2 to bind cAMP. In in vitro electrophysiological studies we demonstrated that, while co-expression of wild-type POPDC2 with TREK-1 increased TREK-1 current density, POPDC2 variants found in the patients failed to increase TREK-1 current density. While patient muscle biopsy did not show clear myopathic disease, it showed significant reduction of the expression of both POPDC1 and POPDC2, suggesting that stability and/or membrane trafficking of the POPDC1-POPDC2 complex is impaired by pathogenic variants in any of the two proteins. Single-cell RNA sequencing from human hearts demonstrated that co-expression of POPDC1 and 2 was most prevalent in AV node, AV node pacemaker and AV bundle cells. Sinoatrial node cells expressed POPDC2 abundantly, but expression of POPDC1 was sparse. Together, these results concur with predisposition to AV node disease in humans with loss-of-function variants in POPDC1 and POPDC2 and presence of sinus node disease in POPDC2, but not in POPDC1 related disease in human. Using population-level genetic data of more than 1 million individuals we showed that none of the familial variants were associated with clinical outcomes in heterozygous state, suggesting that heterozygous family members are unlikely to develop clinical manifestations and therefore might not necessitate clinical follow-up. Our findings provide evidence for POPDC2 as the cause of a novel Mendelian autosomal recessive cardiac syndrome, consistent with previous work showing that mice and zebrafish deficient in functional POPDC2 display sinus and AV node dysfunction. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/24309755v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@803f01org.highwire.dtl.DTLVardef@1a89147org.highwire.dtl.DTLVardef@1d9d145org.highwire.dtl.DTLVardef@13a1645_HPS_FORMAT_FIGEXP M_FIG C_FIG

Primary ciliopathies are a group of inherited developmental disorders resulting from defects in the primary cilium. Mutations in CEP290 (Centrosomal protein of 290kDa) are the most frequent cause of recessive ciliopathies (incidence up to 1:15,000). Pathogenic variants span the full length of this large (93.2kb) 54 exon gene, causing phenotypes ranging from isolated inherited retinal dystrophies (IRDs; Leber Congenital Amaurosis, LCA) to a pleiotropic range of severe syndromic multi-organ ciliopathies affecting retina, kidney and brain. Most pathogenic CEP290 variants are predicted null (37% nonsense, 42% frameshift), but there is no clear genotype-phenotype association. Almost half (26/53) of the coding exons in CEP290 are in-phase "skiptic" (or skippable) exons. Variants located in skiptic exons could be removed from CEP290 transcripts by skipping the exon, and nonsense-associated altered splicing (NAS) has been proposed as a mechanism that attenuates the pathogenicity of nonsense or frameshift CEP290 variants. Here, we have used in silico bioinformatic techniques to study the propensity of CEP290 skiptic exons for NAS. We then used CRISPR-Cas9 technology to model CEP290 frameshift mutations in induced pluripotent stem cells (iPSCs) and analysed their effects on splicing and ciliogenesis. We identified exon 36, a hotspot for LCA mutations, as a strong candidate for NAS that we confirmed in mutant iPSCs that exhibited sequence-specific exon skipping. Exon 36 skipping did not affect ciliogenesis, in contrast to a larger frameshift mutant that significantly decreased cilia size and incidence in iPSCs. We suggest that sequence-specific NAS provides the molecular basis of genetic pleiotropy for CEP290-related disorders.