Seed sequence polymorphism rs2168518 and allele-specific target gene regulation of hsa-miR-4513.

MicroRNAs (miRNAs) are small post-transcriptional regulators that offer promising targets for treating complex diseases. To this end, hsa-miR-4513 is an excellent candidate as this gene harbors within its conserved heptametrical seed sequence a frequent polymorphism (rs2168518), which has previously been associated with several complex phenotypes. So far, little is known about the biological mechanism(s) underlying these associations. In an initial step, we now aimed to identify allele-specific target genes of hsa-miR-4513. We performed RNA sequencing in a miRNA overexpression model in human umbilical vein endothelial cells transfected with separated hsa-miR-4513 alleles at rs2168518, namely hsa-miR-4513-G and hsa-miR-4513-A. Genes specifically regulated by the rs2168518 alleles were independently verified by quantitative reverse transcription PCR (qRT-PCR), western blot analysis and allele-specific miRNA binding via a luciferase reporter assay. By a text-based search publicly available databases such as Online Mendelian Inheritance in Man and Mouse Genome Informatics were utilized to link target genes of hsa-miR-4513 to previously described phenotypes. Overall, we identified 23 allele-specific hsa-miR-4513 target genes and replicated 19 of those independently via qRT-PCR. Western blot analysis and luciferase reporter assays conducted for an exemplary subsample further confirmed the allele-specific regulation of these genes by hsa-miR-4513. Remarkably, multiple allele-specific target genes identified are linked via text retrieval to several phenotypes previously reported to be associated with hsa-miR-4513. These genes offer promising candidates for ongoing research on the functional pathobiological impact of hsa-miR-4513 and its seed polymorphism rs2168518. This could give rise to therapeutic applications targeting this miRNA.

The glucocorticoid receptor as a master regulator of the Müller cell response to diabetic conditions in mice.

Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell response to diabetic conditions. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.

Mitochondrial and Cellular Function in Fibroblasts, Induced Neurons, and Astrocytes Derived from Case Study Patients: Insights into Major Depression as a Mitochondria-Associated Disease.

The link between mitochondria and major depressive disorder (MDD) is increasingly evident, underscored both by mitochondria's involvement in many mechanisms identified in depression and the high prevalence of MDD in individuals with mitochondrial disorders. Mitochondrial functions and energy metabolism are increasingly considered to be involved in MDD's pathogenesis. This study focused on cellular and mitochondrial (dys)function in two atypical cases: an antidepressant non-responding MDD patient ("Non-R") and another with an unexplained mitochondrial disorder ("Mito"). Skin biopsies from these patients and controls were used to generate various cell types, including astrocytes and neurons, and cellular and mitochondrial functions were analyzed. Similarities were observed between the Mito patient and a broader MDD cohort, including decreased respiration and mitochondrial function. Conversely, the Non-R patient exhibited increased respiratory rates, mitochondrial calcium, and resting membrane potential. In conclusion, the Non-R patient's data offered a new perspective on MDD, suggesting a detrimental imbalance in mitochondrial and cellular processes, rather than simply reduced functions. Meanwhile, the Mito patient's data revealed the extensive effects of mitochondrial dysfunctions on cellular functions, potentially highlighting new MDD-associated impairments. Together, these case studies enhance our comprehension of MDD.

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors.

The RS1 gene on Xp 22.13 encodes retinoschisin which is known to directly interact with the retinal Na/K-ATPase at the photoreceptor inner segments. Pathologic mutations in RS1 cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy in young males. To further delineate the retinoschisin-Na/K-ATPase complex, co-immunoprecipitation was performed with porcine and murine retinal lysates targeting the ATP1A3 subunit. This identified the voltage-gated potassium (Kv) channel subunits Kv2.1 and Kv8.2 as direct interaction partners of the retinal Na/K-ATPase. Colocalization of the individual components of the complex was demonstrated at the membrane of photoreceptor inner segments. We further show that retinoschisin-deficiency, a frequent consequence of molecular pathology in XLRS, causes mislocalization of the macromolecular complex during postnatal retinal development with a simultaneous reduction of Kv2.1 and Kv8.2 protein expression, while the level of retinal Na/K-ATPase expression remains unaffected. Patch-clamp analysis revealed no effect of retinoschisin-deficiency on Kv channel mediated potassium ion currents in vitro. Together, our data suggest that Kv2.1 and Kv8.2 together with retinoschisin and the retinal Na/K-ATPase are integral parts of a macromolecular complex at the photoreceptor inner segments. Defective compartmentalization of this complex due to retinoschisin-deficiency may be a crucial step in initial XLRS pathogenesis.

A mega-analysis of expression quantitative trait loci in retinal tissue.

Significant association signals from genome-wide association studies (GWAS) point to genomic regions of interest. However, for most loci the causative genetic variant remains undefined. Determining expression quantitative trait loci (eQTL) in a disease relevant tissue is an excellent approach to zoom in on disease- or trait-associated association signals and hitherto on relevant disease mechanisms. To this end, we explored regulation of gene expression in healthy retina (n = 311) and generated the largest cis-eQTL data set available to date. Genotype- and RNA-Seq data underwent rigorous quality control protocols before FastQTL was applied to assess the influence of genetic markers on local (cis) gene expression. Our analysis identified 403,151 significant eQTL variants (eVariants) that regulate 3,007 genes (eGenes) (Q-Value < 0.05). A conditional analysis revealed 744 independent secondary eQTL signals for 598 of the 3,007 eGenes. Interestingly, 99,165 (24.71%) of all unique eVariants regulate the expression of more than one eGene. Filtering the dataset for eVariants regulating three or more eGenes revealed 96 potential regulatory clusters. Of these, 31 harbour 130 genes which are partially regulated by the same genetic signal. To correlate eQTL and association signals, GWAS data from twelve complex eye diseases or traits were included and resulted in identification of 80 eGenes with potential association. Remarkably, expression of 10 genes is regulated by eVariants associated with multiple eye diseases or traits. In conclusion, we generated a unique catalogue of gene expression regulation in healthy retinal tissue and applied this resource to identify potentially pleiotropic effects in highly prevalent human eye diseases. Our study provides an excellent basis to further explore mechanisms of various retinal disease etiologies.

Systems genomics in age-related macular degeneration.

Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are 'omics' technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD.

Genetic Association Analysis of Anti-VEGF Treatment Response in Neovascular Age-Related Macular Degeneration.

Anti-VEGF treatment for neovascular age-related macular degeneration (nAMD) has been FDA-approved in 2004, and since then has helped tens of thousands of patients worldwide to preserve vision. Still, treatment responses vary widely, emphasizing the need for genetic biomarkers to robustly separate responders from non-responders. Here, we report the findings of an observational study compromising 179 treatment-naïve nAMD patients and their reaction to treatment after three monthly doses of anti-VEGF antibodies. We show that established criteria of treatment response such as visual acuity and central retinal thickness successfully divides our cohort into 128 responders and 51 non-responders. Nevertheless, retinal thickness around the fovea revealed significant reaction to treatment even in the formally categorized non-responders. To elucidate genetic effects underlying our criteria, we conducted an undirected genome-wide association study followed by a directed replication study of 30 previously reported genetic variants. Remarkably, both approaches failed to result in significant findings, suggesting study-specific effects were confounding the present and previous discovery studies. Of note, all studies so far are greatly underpowered, hampering interpretation of genetic findings. In consequence, we highlight the need for an extensive phenotyping study with sample sizes exceeding at least 15,000 to reliably assess anti-VEGF treatment responses in nAMD.

Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family.

Structural variation and single-nucleotide variation of the complement factor H (CFH) gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ∼360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four CFH-related (CFHR) gene paralogs (CFHR2 and CFHR4 ∼25-35 Mya and CFHR1 and CFHR3 ∼7-13 Mya). Remarkably, all evolutionary breakpoints share a common ∼4.8-kbp segment corresponding to an ancestral CFHR gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral CFH, creating four CFHR fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of CFH [P = 5.81 × 10-8, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD (P < 10-3) and AHUS (P = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new CFHR genes but also in the predisposition to complex human genetic disease phenotypes.

On the differences between mega- and meta-imputation and analysis exemplified on the genetics of age-related macular degeneration.

While current genome-wide association analyses often rely on meta-analysis of study-specific summary statistics, individual participant data (IPD) from multiple studies increase options for modeling. When multistudy IPD is available, however, it is unclear whether this data is to be imputed and modeled across all participants (mega-imputation and mega-analysis) or study-specifically (meta-imputation and meta-analysis). Here, we investigated different approaches toward imputation and analysis using 52,189 subjects from 25 studies of the International Age-related Macular Degeneration (AMD) Genomics Consortium including, 16,144 AMD cases and 17,832 controls for association analysis. From 27,448,454 genetic variants after 1,000-Genomes-based imputation, mega-imputation yielded ~400,000 more variants with high imputation quality (mostly rare variants) compared to meta-imputation. For AMD signal detection (P < 5 × 10-8 ) in mega-imputed data, most loci were detected with mega-analysis without adjusting for study membership (40 loci, including 34 known); we considered these loci genuine, since genetic effects and P-values were comparable across analyses. In meta-imputed data, we found 31 additional signals, mostly near chromosome tails or reference panel gaps, which disappeared after accounting for interaction of whole-genome amplification (WGA) with study membership or after excluding studies with WGA-participants. For signal detection with multistudy IPD, we recommend mega-imputation and mega-analysis, with meta-imputation followed by meta-analysis being a computationally appealing alternative.

BEST1 protein stability and degradation pathways differ between autosomal dominant Best disease and autosomal recessive bestrophinopathy accounting for the distinct retinal phenotypes.

Mutations in bestrophin-1 (BEST1) are associated with distinct retinopathies, notably three forms with autosomal dominant inheritance and one condition with an autosomal recessive mode of transmission. The molecular mechanisms underlying their distinct retinal phenotypes are mostly unknown. Although heterozygous missense mutations in BEST1 reveal dominant-negative effects in patients with autosomal dominant Best disease (BD), heterozygous mutations associated with autosomal recessive bestrophinopathy (ARB) display no disease phenotype. Here we show that the recessive mutations trigger a strong and fast protein degradation process in the endoplasmic reticulum (ER), thereby favoring a decreased stoichiometry of mutant versus normal BEST1 subunits in the assembly of the homo-pentameric BEST1 chloride channel. In contrast, dominant mutations escape ER-associated degradation and are subjected to a slightly delayed post-ER degradation via the endo-lysosomal degradation pathway. As a result, increased formation of a non-functional BEST1 channel occurs due to a roughly equimolar incorporation of normal and mutant BEST1 subunits into the channel complex. Taken together, our data provide insight into the molecular pathways of dominantly and recessively acting BEST1 missense mutations suggesting that the site of subcellular protein quality control as well as the rate and degree of mutant protein degradation are ultimately responsible for the distinct retinal disease phenotypes in BD and ARB.