Identification of known and novel long non-coding RNAs potentially responsible for the effects of BMD GWAS loci

Osteoporosis, characterized by low bone mineral density (BMD), is the most common complex disease affecting bone and constitutes a major societal health problem. Genome-wide association studies (GWASs) have identified over 1100 associations influencing BMD. It has been shown that perturbations to long non-coding RNAs (lncRNAs) influence BMD and the activities of bone cells; however, the extent to which lncRNAs are involved in the genetic regulation of BMD is unknown. Here, we combined the analysis of allelic imbalance (AI) in human acetabular bone fragments with a transcriptome-wide association study (TWAS) and expression quantitative trait loci (eQTL) colocalization analysis using data from the Genotype-Tissue Expression (GTEx) project to identify lncRNAs potentially responsible for GWAS associations. We identified 27 lncRNAs in bone that are located in proximity to a BMD GWAS association and harbor SNPs demonstrating AI. Using GTEx data we identified an additional 31 lncRNAs whose expression was associated (FDR correction<0.05) with BMD through TWAS and had a colocalizing eQTL (regional colocalization probability (RCP)>0.1). The 58 lncRNAs are located in 43 BMD associations. To further support a causal role for the identified lncRNAs, we show that 23 of the 58 lncRNAs are differentially expressed as a function of osteoblast differentiation. Our approach identifies lncRNAs that are potentially responsible for BMD GWAS associations and suggest that lncRNAs play a role in the genetics of osteoporosis.

Functional non-coding SNPs in human endothelial cells fine-map vascular trait associations

Functional consequences of genetic variation in the non-coding human genome are difficult to ascertain despite demonstrated associations to common, complex disease traits. To elucidate properties of functional non-coding SNPs with effects in human endothelial cells (EC), we utilized molecular Quantitative Trait Locus (molQTL) analysis for transcription factor binding, chromatin accessibility, and H3K27 acetylation to nominate a set of likely functional non-coding SNPs. Together with information from genome-wide association studies for vascular disease traits, we tested the ability of 34,344 variants to perturb enhancer function in ECs using the highly multiplexed STARR-seq assay. Of these, 5,592 variants validated, whose enriched attributes included: 1) mutations to TF binding motifs for ETS or AP1 that are regulators of EC state, 2) location in accessible and H3K27ac-marked EC chromatin, and 3) molQTLs associations whereby alleles associate with differences in chromatin accessibility and TF binding across genetically diverse ECs. Next, using pro-inflammatory IL1B as an activator of cell state, we observed robust evidence (>50%) of context-specific SNP effects, underscoring the prevalence of non-coding gene-by-environment (GxE) effects. Lastly, using these cumulative data, we fine-mapped vascular disease loci and highlight evidence suggesting mechanisms by which non-coding SNPs at two loci affect risk for Pulse Pressure/Large Artery Stroke, and Abdominal Aortic Aneurysm through respective effects on transcriptional regulation of POU4F1 and LDAH. Together, we highlight the attributes and context dependence of functional non-coding SNPs, and provide new mechanisms underlying vascular disease risk.

Genetic Determinants of Peripheral Artery Disease

Peripheral artery disease—atherosclerosis of the abdominal aorta and lower extremity vascular bed—is a complex disease with both environmental and genetic determinants. Unmitigated disease is associated with major functional decline and can lead to chronic limb-threatening ischemia, amputation, and increased mortality. Over the last 10 years, major advances have been made in identifying the genetic basis of this common, complex disease. In this review, we provide an overview of the primary types of genetic analyses performed for peripheral artery disease, including heritability and linkage studies, and more recently biobank-based genome-wide association studies. Looking forward, we highlight areas of future study including efforts to identify causal peripheral artery disease genes, rare variant and structural variant analyses using whole-exome and whole-genome sequencing data, and the need to include individuals of diverse genetic ancestries.

Colocalization analysis of polycystic ovary syndrome to identify potential disease-mediating genes and proteins

Polycystic ovary syndrome (PCOS) is a common complex disease in women with a strong genetic component and downstream consequences for reproductive, metabolic and psychological health. There are currently 19 known PCOS risk loci, primarily identified in women of Han Chinese or European ancestry, and 14 of these risk loci were identified or replicated in a genome-wide association study of PCOS performed in up to 10,074 cases and 103,164 controls of European descent. However, for most of these loci the gene responsible for the association is unknown. We therefore use a Bayesian colocalization approach (Coloc) to highlight genes in PCOS-associated regions that may have a role in mediating the disease risk. We evaluated the posterior probabilities of evidence consistent with shared causal variants between 14 PCOS genetic risk loci and intermediate cellular phenotypes in one protein (N = 3301) and two expression quantitative trait locus datasets (N = 31,684 and N = 80–491). Through these analyses, we identified seven proteins or genes with evidence of a possibly shared causal variant for almost 30% of known PCOS signals, including follicle stimulating hormone and ERBB3, IKZF4, RPS26, SUOX, ZFP36L2, and C8orf49. Several of these potential effector proteins and genes have been implicated in the hypothalamic–pituitary–gonadal signalling pathway and provide an avenue for functional follow-up in order to demonstrate a causal role in PCOS pathophysiology.

Dysregulated Bone Metabolism Is Related to High Expression of miR-151a-3p in Severe Adolescent Idiopathic Scoliosis

Adolescent idiopathic scoliosis (AIS) is a common complex disease, and bone homeostasis plays an important role in its pathogenesis. Recent advances in epigenetic research show that dysregulated miRNAs may participate in the development of orthopedic diseases and AIS. The aim of this study was to detect differentially expressed miRNAs in severe AIS and elucidate the mechanism of miRNA deregulation in the pathogenesis of AIS. In the present study, miRNA expression profiles were detected in severe and mild AIS patients as well as healthy controls by miRNA sequencing. Candidate miRNAs were validated in a larger cohort. Primary osteoblasts from severe AIS patients were extracted and isolated to determine the effect of the candidate miRNAs on bone metabolism. Finally, we determined the methylation level in primary osteoblasts from severe AIS patients. The result showed that miR-151a-3p was overexpressed in severe AIS patients. Reduced GREM1 expression was observed in primary osteoblasts from severe AIS patients. miR-151a-3p directly inhibited GREM1 in primary osteoblasts. Relatively lower methylation levels were detected in primary osteoblasts from severe AIS patients. In conclusion, our study revealed that plasma miR-151a-3p levels may serve as a biomarker for severe AIS. Overexpression of miR-151a-3p may interrupt bone homeostasis via inhibiting GREM1 expression. Our result may provide a new biomarker for the early detection of AIS and increase our understanding of the pathogenesis of AIS.

Water Stress Enhances the Progression of Branch Dieback and Almond Decline under Field Conditions

Branch dieback and tree decline have been described as a common complex disease worldwide in woody crops, with Botryosphaeriaceae and Diaporthaceae being considered the most frequent fungi associated with the disease symptoms. Their behaviour is still uncertain, since they are considered endophytes becoming pathogenic in weakened hosts when stress conditions, such as water deficiency occur. Therefore, the main goal of this study was to determine if water stress enhances general decline on weakened almond trees subjected to different irrigation treatments under natural field conditions. In parallel, the occurrence of fungal species associated with almond decline was also determined in relation to disease progression by fungal isolation, and morphological and molecular based-methods. The symptoms of branch dieback and general decline were observed over time, mainly in the experimental plots subjected to high water deficiency. Botryosphaeriaceae were the most consistently isolated fungi, and Botryosphaeria dothidea was the most frequent. Collophorina hispanica was the second most frequent species and Diaporthe and Cytospora species were isolated in a low frequency. Most of them were recovered from both asymptomatic and symptomatic trees, with their consistency of isolation increasing with the disease severity. This work reveals the need to elucidate the role of biotic and abiotic factors which increase the rate of infection of fungal trunk pathogens, in order to generate important knowledge on their life cycle.

Attenuation of Dupuytren’s fibrosis via targeting of the STAT1 modulated IL-13Rα1 response

Fibrotic disorders represent common complex disease pathologies that are therapeutically challenging. Inflammation is associated with numerous fibrotic pathogeneses; however, its role in the multifaceted mechanisms of fibrosis remains unclear. IL-13 is implicated in aberrant responses involved in fibrotic disease, and we aimed to understand its role in the inflammatory processes of a common fibrotic disorder, Dupuytren’s disease. We demonstrated T-cells produced IFN-g, which induced IL-13 secretion from mast cells and up-regulated IL-13Ra1 on fibroblasts, rendering them more reactive to IL-13. Consequently, diseased myofibroblasts demonstrated enhanced fibroproliferative effects upon IL-13 stimulation. We established IFN-g and IL-13 responses involved STAT dependent pathways, and STAT targeting (tofacitinib) could inhibit IL-13 production from mast cells, IL-13Ra1 up-regulation in fibroblasts and fibroproliferative effects of IL-13 on diseased myofibroblasts. Accordingly, utilizing Dupuytren’s as an accessible human model of fibrosis, we propose targeting STAT pathways may offer previously unidentified therapeutic approaches in the management of fibrotic disease.

When genetic burden reaches threshold

Rare cardiac genetic diseases have generally been considered to be broadly Mendelian in nature, with clinical genetic testing for these conditions predicated on the detection of a primary causative rare pathogenic variant that will enable cascade genetic screening in families. However, substantial variability in penetrance and disease severity among carriers of pathogenic variants, as well as the inability to detect rare Mendelian variants in considerable proportions of patients, indicates that more complex aetiologies are likely to underlie these diseases. Recent findings have suggested genetic variants across a range of population frequencies and effect sizes may combine, along with non-genetic factors, to determine whether the threshold for expression of disease is reached and the severity of the phenotype. The availability of increasingly large genetically characterized cohorts of patients with rare cardiac diseases is enabling the discovery of common genetic variation that may underlie both variable penetrance in Mendelian diseases and the genetic aetiology of apparently non-Mendelian rare cardiac conditions. It is likely that the genetic architecture of rare cardiac diseases will vary considerably between different conditions as well as between patients with similar phenotypes, ranging from near-Mendelian disease to models more akin to common, complex disease. Uncovering the broad range of genetic factors that predispose patients to rare cardiac diseases offers the promise of improved risk prediction and more focused clinical management in patients and their families.

P18 Investigating the role of rare genetic variants and susceptibility to juvenile idiopathic arthritis highlights the importance of monogenic disease genes

Background Juvenile idiopathic arthritis (JIA) is a rheumatic disease, onset before the age of sixteen. JIA is a common complex disease, with contributions from both genetic and environmental risk factors. However, given the young age of onset for JIA, it is possible that genetic risk factors influence susceptibility to a greater extent than environmental risk factors. While several susceptibility loci have been identified for JIA, they are mainly genetic variants that are common in the population. The role of rare genetic variants has not yet been fully explored in JIA. Therefore, the aims of this research were to use rare (&lt;5%MAF) variant based analysis to further define the genetic architecture of JIA. Methods A total of 2,162 UK JIA cases were genotyped using the Illumina HumanCoreExome array and the Understanding Society’s UK household longitudinal study provided the genotype data for 9196 controls. Single variant analysis was used to calculate the association of variants to JIA susceptibility. Gene-based aggregation analysis, specifically the SKAT-O method, were used to calculate the association of whole gene regions to JIA cases. This gene-based aggregation method increases the statistical power of associations, which is valuable when attempting to identify rare variants. Study-wide significance for this research was calculated using a Bonferroni corrected threshold (P = 3.34x10-6). Results Several strongly associated candidate gene regions were identified using SKAT-O analysis. Firstly, the region mevalonate kinase (MVK) (P = 3.95x10-10) was found to be strongly associated. MVK has been reported in mevalonate kinase deficiency, a rare juvenile disease with a phenotype akin to systemic JIA. PYCR1 (P = 1.97x10-9) was an additional region of interest. Variants in this region have been reported in geroderma osteodysplasticum; a disease that results in dysplasia of the hips, hyperextensible joints and skeletal changes. The region WISP3 (P = 8.52x10-9) was also strongly associated. Variants from this region have previously been reported in progressive pseudorheumatoid dysplasia; a disease that can be clinically misdiagnosed with JIA due to similarities in phenotypes. Furthermore, LILRA2 (P = 2.89x10-7) presented as a strong candidate region, with the gene being upregulated in synovial tissue of rheumatoid arthritis patients. Conclusion This analysis has identified promising candidate genes that influence susceptibility to JIA and highlights two key points. Firstly, gene-burden tests of rare variants have the potential to identify novel susceptibility genes for juvenile-onset disease. While MVK and WISP3 have previously been reported to be associated with JIA, these associations were modest, and the current study provides compelling evidence to support their importance. Secondly, the results highlight the importance of investigating the contribution of monogenic genes in juvenile-onset common complex disease. Disclosures M.K. Tordoff None. T.A. Briggs None. S.L. Smith None. A. Yarwood None. W. Thomson None. J. Bowes None.

Identification and functional analysis of genetic variants in TBX5 gene promoter in patients with acute myocardial infarction

Background Coronary artery disease (CAD), including acute myocardial infarction (AMI), is a common complex disease. Although a great number of genetic loci and variants for CAD have been identified, genetic causes and underlying mechanisms remain largely unclear. Epidemiological studies have revealed that CAD incidence is strikingly higher in patients with congenital heart disease than that in normal population. T-box transcription factors play critical roles in embryonic development. In particular, TBX5 as a dosage-sensitive regulator is required for cardiac development and function. Thus, dysregulated TBX5 gene expression may be involved in CAD development. Methods TBX5 gene promoter was genetically and functionally analysed in large groups of AMI patients (n = 432) and ethnic-matched healthy controls (n = 448). Results Six novel heterozygous DNA sequence variants (DSVs) in the TBX5 gene promoter (g.4100A > G, g.4194G > A, g.4260 T > C, g.4367C > A, g.4581A > G and g.5004G > T) were found in AMI patients, but in none of controls. These DSVs significantly changed the activity of TBX5 gene promoter in cultured cells (P < 0.05). Furthermore, three of the DSVs (g.4100A > G, g.4260 T > C and g.4581A > G) evidently modified the binding sites of unknown transcription factors. Conclusions The DSVs identified in AMI patients may alter TBX5 gene promoter activity and change TBX5 level, contributing to AMI development as a rare risk factor.