High-throughput analysis of ANRIL circRNA isoforms in human pancreatic islets

The antisense non-coding RNA in the INK locus (ANRIL) is a hotspot for genetic variants associated with cardiometabolic disease. We recently found increased ANRIL abundance in human pancreatic islets from donors with certain Type II Diabetes (T2D) risk-SNPs, including a T2D risk-SNP located within ANRIL exon 2 associated with beta cell proliferation. Recent studies have found that expression of circular species of ANRIL is linked to the regulation of cardiovascular phenotypes. Less is known about how the abundance of circular ANRIL may influence T2D phenotypes. Herein, we sequence circular RNA in pancreatic islets to characterize circular isoforms of ANRIL. We identify highly expressed circular ANRIL isoforms whose expression is correlated across dozens of individuals and characterize ANRIL splice sites that are commonly involved in back-splicing. We find that samples with the T2D risk allele in ANRIL exon 2 had higher ratios of circular to linear ANRIL compared to protective-allele carriers, and that higher circular:linear ANRIL was associated with decreased beta cell proliferation. Our study points to a combined involvement of both linear and circular ANRIL species in T2D phenotypes and opens the door for future studies of the molecular mechanisms by which ANRIL impacts cellular function in pancreatic islets.

An HIV Vaccine Protective Allele in FCGR2C Associates With Increased Odds of Perinatal HIV Acquisition

In the Thai RV144 HIV-1 vaccine trial, a three-variant haplotype within the Fc gamma receptor 2C gene (FCGR2C) reduced the risk of HIV-1 acquisition. A follow-on trial, HVTN702, of a similar vaccine candidate found no efficacy in South Africa, where the predominant population is polymorphic for only a single variant in the haplotype, c.134-96C>T (rs114945036). To investigate a role for this variant in HIV-1 acquisition in South Africans, we used the model of maternal-infant HIV-1 transmission. A nested case-control study was conducted of infants born to mothers living with HIV-1, comparing children with perinatally-acquired HIV-1 (cases, n = 176) to HIV-1-exposed uninfected children (controls, n = 349). All had received nevirapine for prevention of mother-to-child transmission. The FCGR2C copy number and expression variants (c.−386G>C, c.−120A>T c.169T>C, and c.798+1A>G) were determined using a multiplex ligation-dependent probe amplification assay and the c.134-96C>T genotype with Sanger sequencing. The copy number, genotype and allele carriage were compared between groups using univariate and multivariate logistic regression. The FCGR2C c.134-96C>T genotype distribution and copy number differed significantly between HIV-1 cases and exposed-uninfected controls (P = 0.002, PBonf = 0.032 and P = 0.010, PBonf = > 0.05, respectively). The FCGR2C c.134-96T allele was overrepresented in the cases compared to the controls (58% vs 42%; P = 0.001, PBonf = 0.016). Adjusting for birthweight and FCGR2C copy number, perinatal HIV-1 acquisition was associated with the c.134-96C>T (AOR = 1.89; 95% CI 1.25-2.87; P = 0.003, PBonf = 0.048) and c.169C>T (AOR = 2.39; 95% CI 1.45-3.95; P = 0.001, PBonf = 0.016) minor alleles but not the promoter variant at position c.−386G>C. The c.134-96C>T variant was in strong linkage disequilibrium with the c.169C>T variant, but remained significantly associated with perinatal acquisition when adjusted for c.169C>T in multivariate analysis. In contrast to the protective effect observed in the Thai RV144 trial, we found the FCGR2C variant c.134-96T-allele associated with increased odds of perinatal HIV-1 acquisition in South African children. These findings, taken together with a similar deleterious association found with HIV-1 disease progression in South African adults, highlight the importance of elucidating the functional relevance of this variant in different populations and vaccination/disease contexts.

POS0354 DETECTION OF ABCG2 VARIANTS IN ENCODING OF URATE TRANSPORTERS ASSOCIATED WITH THE HYPERURICEMIA IN HAEMODIALYSIS PATIENTS

Background:Hyperuricemia is associated with gout, hypertension, cardiovascular diseases and renal disease. The presence of chronic kidney disease (CKD) is associated with low excretion of the uric acid as the homeostasis in maintaining of serum levels of uric acid is impaired. Progression of CKD is connected to hyperuricemia and lowering levels of the uric acid is one of the most important goals in clinical treatment. Dysfunctional variants of ATP-binding cassette transporter subfamily G member 2 (ABCG2), a major urate transporter in the kidney and intestine, are the major causes of hyperuricemia and gout.A recent studies found that ABCG2 is also a major transporter of uremic toxins and ABCG2 dysfunctional variants are risk genetics factors for mortality in hemodialysis patients.Objectives:The aim of the present study was to investigate the frequencies of ABCG2 variants in cohort of hemodialysis patients.Methods:In this retrospective one centre study 167 patients (age=79,8±11,03;female=74) with end-stage CKD (pre-dialysis n=86; dialysis n=79) were collected. Peak urate levels were 456,3±113,6 μmol/l in pre-dialysis and 572,93±114,56 μmol/l in dialysis. ABCG2 coding regions were analyzed from genomic DNA, as we described previously (1). The reference sequence was defined as version ENST00000237612.7, the reference protein sequence was defined as Q9UNQ0. The chi-square goodness-of-fit test was used to compare minor allele frequencies (MAF), and the log-rank test was used to compare empirical distribution functions.Results:In the CKD cohort, 15 intronic and seven non-synonymous allelic exonic variants were detected: two common (rs2231137/p.V12M; rs2231142/p.Q141K), five ultra rare and/or rare (rs142634180/p.R45Q, rs759726272/p.M131I, rs140207606/p.R236X, rs138606116/p.G354R, rs138892154/p.A607V), and one novel (p.E344D). Common variant p.V12M, previously reported as protective allele, was detected in 12 heterozygous patients with. The p.Q141K variant, detected in the homozygous stage in three patients and in 31 patients as heterozygous variant. Heterozygous rare variants p.R45Q, p. G354R, p.A607V, and novel variant p.E344D were detected in one heterozygous patient each; heterozygous p.M131I and p.R236X in two patients.Conclusion:In this study, significantly higher frequency of dysfunctional ABCG2 variants, common and rare, in comparison with common European population, were identified. On the other hand, the frequency of probably protective allele variant p.V12M was significantly lower in CKD cohort (MAF in our cohort = 0,036/MAF in the European population = 0,061). Further analysis of ABCG2 association with CKD events via ABCG2 inflammation role is necessary. In conclusions, our finding of one novel, five rare and two common non-synonymous ABCG2 allelic variants in a sample of 167 CKD patients suggests that the ABCG2 variants should be considered a risk factor for CKD.References:[1]Toyoda Y, et al. Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort. Cells. 2019;18;8(4).Acknowledgements:This study was supported by the grant from the Czech Republic Ministry of Health RVO 00023728.Disclosure of Interests:None declared

The Weight of HLA-DPA1 rs3077 Single Nucleotide Polymorphism in Prostate Cancer, a Multicenter Study

Prostate cancer (PCa) has almost the highest genetic transmission that mimics an autosomal dominance hereditary pattern of cancers in some families. Its incidence in Arab countries was reported to be steadily increasing. Aim. To determine the relevance of HLA-DPA1 rs3077 (A/G) SNP with prostate cancer’s risk and/or severity. Subjects and Methods. Forty PCa patients and forty age matched patients with benign prostatic hyperplasia (BPH), as a control group, were enrolled in the study. Serum levels of urea, creatinine, total prostate-specific antigen (PSA), and free PSA were measured. PSA ratio was determined as well. Genotyping of HLA-DPA1 rs3077 (A/G) SNP was done using real-time PCR. Results. The measured lab parameters, except free PSA, were significantly higher among PCa patients in comparison to controls ( P < 0.001 ∗ ). Moreover, PSA ratio was significantly high among PCa patients ( P < 0.001 ∗ ). HLA-DPA1 rs3077 GG genotype was more frequent in PCa patients and the associated OR was 2.546 ( P = 0.059 ), while AA genotype was more frequent in the control group and the associated OR was 0.145 ( P = 0.081 ). Frequency of G allele was higher among PCa patients than the control group while A allele frequency was significantly decreased ( P = 0.034 ∗ ) (protective allele). On multivariate analysis, there is no significant correlation found between HLA-DPA1 rs3077 SNP and PSA ratio (OR = 4.5, 95% CI = 1.2–17.4, P = 0.856 ). Conclusion. HLA-DPA1 rs3077 G allele could be a risk factor for prostate cancer. However, HLA-DPA1 rs3077 SNP has no relation to PCa severity.

GSR (glutathione reductase) gene as a possible candidate gene for predisposition to uterine fibroids

Миома матки (ММ), или лейомиома, - это наиболее распространенная опухоль репродуктивной системы у женщин. Окислительный стресс является одним из основных патогенетических механизмов развития ММ. Глутатион (GSH) - неферментный антиоксидант, играющий ведущую роль в защите организма от окислительного стресса. Восстановление окисленного глутатиона осуществляется ферментом глутатионредуктазой, кодируемой геном GSR. Целью исследования было изучение ассоциации однонуклеотидного полиморфизма rs2551715 гена GSR с риском развития ММ. В исследование было включено 978 неродственных жителей Центральной России: 590 пациенток с ММ и 388 здоровых женщин соответствующего возраста. Генотипирование однонуклеотидного полиморфизма rs2551715 гена GSR проводили методом ПЦР в режиме реального времени путем дискриминации аллелей с помощью TaqMan-зондов. Для анализа ассоциаций генотипов с развитием заболевания пользовались лог-аддитивной регрессионной моделью. Все расчеты выполнены относительно минорного аллеля; введены поправки на возраст. Аллель Т rs2551715 GSR (OR=0,81, 95%CI=0,67-0,97; р=0,02) ассоциировался со сниженным риском развития ММ. Анализ частот генотипов показал, что SNP rs2551715 GSR был связан со сниженным риском ММ (OR=0,82, 95% CI=0,69-0,98; р=0,028). Биоинформатический анализ выявил, что транскрипционные факторы, связывающиеся с аллелем Т, участвуют в регуляции сигнального пути гладкомышечных клеток, ответ на фактор роста, регуляции дифференцировки клеток и пролиферации клеточной популяции. Таким образом, впервые установлена ассоциация rs2551715 GSR с предрасположенностью к развитию ММ. Uterine fibroids (UF), or leiomyoma, is the most common tumor of the reproductive system in female. Oxidative stress is one of the basic pathogenetic mechanisms for uterine fibroids development. Glutathione (GSH) is a non-enzymatic antioxidant that plays a key role in the protection against oxidative stress. The reduction of oxidized glutathione is carried out by the enzyme glutathione reductase, encoded by the GSR gene. The aim of the study was to investigate the association of common single nucleotide polymorphism rs2551715 in gene encoding glutathione reductase with the risk of UF. A total of 978 unrelated individuals from Central Russia were included for this study: 590 patients with uterine fibroids (MM) and 388 age-matched healthy females. Genotyping of the single nucleotide polymorphism rs2551715 GSR was performed using TaqMan-based PCR. To analyze the associations of genotypes with the risk of diseases, a log-additive regression model was used. All calculations were performed relative to the minor allele; corrections for gender and age have been introduced. Allele T rs2551715 GSR (OR = 0.81, 95% CI = 0.67-0.97; P = 0.02) was associated with a decreased risk of UF. Analysis of genotype frequencies showed that rs2551715 GSR was associated with a decreased risk of UF (OR = 0.82, 95% CI = 0.69-0.98 ; P = 0.028). Bioinformatic analysis revealed that the spectrum of transcription factors binding with the protective allele T is involved in the regulation of the of smooth muscle cells signaling pathway, response to growth factor, regulation of cell differentiation, regulation of cell population proliferation. Thus, for the first time, the association of rs2551715 GSR with a predisposition to development of uterine fibroids has been established.

The Interaction of Human and Epstein–Barr Virus miRNAs with Multiple Sclerosis Risk Loci

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein–Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.

Five-Year Outcome of Aflibercept Monotherapy for Exudative Age-Related Macular Degeneration with Good Baseline Visual Acuity

We investigated the long-term visual and anatomical outcomes of aflibercept monotherapy for exudative age-related macular degeneration (AMD) with good baseline best-corrected visual acuity (BCVA). A medical chart review was performed for 40 consecutive patients with baseline decimal BCVA ≥ 0.6 secondary to exudative AMD. Three monthly injections were administrated, and thereafter additional injection was performed if needed over 5 years. In total, 13 eyes with neovascular AMD (nAMD) and 27 eyes with polypoidal choroidal vasculopathy (PCV) were enrolled. In both groups, the mean BCVA significantly improved at the 12-month visit (p < 0.05). However, the significant improvement in BCVA disappeared at the 24-month visit, and the final mean BCVA was equivalent to that at baseline (p = 0.17 in the nAMD group and p = 0.15 in the PCV group). The median number of injections required after the loading dose was 15.0 during the 5-year follow-up (nAMD:15.0 vs. PCV:15). During the study period, 37 (92.5%) eyes required retreatment(s). Cox regression analysis demonstrated that the protective allele of ARMS2 A69S was associated with a retreatment-free period from the initial injection (p = 0.041, repeated forward selection method). As-needed aflibercept monotherapy is a preferable treatment option for exudative AMD with good initial visual acuity regardless of nAMD or PCV during the 5-year study period.

Clinical Relevance of HLA-DRB1 and -DQB1 Alleles in Iranian Systemic Lupus Erythematosus Patients

Given the potential link between genetic risk factors and clinical features of systemic lupus erythematosus (SLE), this study aimed to explore the relationship between human leukocyte antigen (HLA)-DRB1/DQB1 alleles and haplotypes and clinical sub-phenotypes of the disease in a group of Iranian SLE patients. HLA-DRB1 and HLA-DQB1 alleles were determined by PCR-SSP in 127 SLE patients and 153 ethnically-matched healthy controls. The relationships between various clinical manifestations and HLA alleles/haplotypes were analyzed in the patients. We observed the positive associations of DRB1*07 and DRB1*07-DQB1*02 haplotypes with articular and pulmonary involvement (p=0.006 and p<0.001 respectively), DRB1*03 and DQB1*02 alleles, and DRB1*03-DQB1*02 haplotypes with cutaneous (p=0.03, p=0.004 and p=0.02 respectively) and renal involvement, and DRB1*13 as well as DRB1*13-DQB1*06 haplotypes with renal involvement. Conversely, negative associations of DRB1*13 with cutaneous and gastrointestinal disorders (p=0.004 and p=0.02 respectively) and DRB1*01 with renal involvement (p=0.03) were found in our patients. Patients carrying susceptible HLA-DRB1 alleles had a higher risk for expression of cutaneous involvement (p=0.03), anti-coagulant antibody development (p=0.01), and a lower risk for pulmonary disorders compared to patients' negatives for susceptible alleles (p=0.04). Our findings on associations between HLA risk allele (DRB1*03) as well as non-risk alleles with particular clinical manifestations and between the potentially protective allele (DRB1*01) and protection against renal involvement indicate the important role of HLA class II genes in predisposing of specific serological and clinical features of SLE disease which could be implicative for therapeutic applications and better management of SLE patients.

SLE non-coding genetic risk variant determines the epigenetic dysfunction of an immune cell specific enhancer that controls disease-critical microRNA expression

Since most variants that impact polygenic disease phenotypes localize to non-coding genomic regions, understanding the consequences of regulatory element variants will advance understanding of human disease mechanisms. Here, we report that the systemic lupus erythematosus (SLE) risk variant rs2431697 as likely causal for SLE through disruption of a regulatory element, modulating miR-146a expression. Using epigenomic analysis, genome-editing and 3D chromatin structure analysis, we show that rs2431697 tags a cell-type dependent distal enhancer specific for miR-146a that physically interacts with the miR-146a promoter. NF-kB binds the disease protective allele in a sequence-specific manner, increasing expression of this immunoregulatory microRNA. Finally, CRISPR activation-based modulation of this enhancer in the PBMCs of SLE patients attenuates type I interferon pathway activation by increasing miR-146a expression. Our work provides a strategy to define non-coding RNA functional regulatory elements using disease-associated variants and provides mechanistic links between autoimmune disease risk genetic variation and disease etiology.

Characterizing the function of EPB41L4A in the predisposition to papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is the most common histotype of thyroid carcinoma. The heritability of PTC is high compared to other cancers, but its underlying causes are unknown. A recent genome-wide association study revealed the association of a variant at the 5q22 locus, rs73227498, with PTC predisposition. We report that rs17134155, a variant in high linkage disequilibrium with rs73227498, is located in an enhancer region downstream of coding transcripts of EPB41L4A. Rs17134155 showed significant enhancer activity in luciferase assays, and haplotypes containing the protective allele of this variant conferred a significantly lower risk of PTC. While the index SNP, rs73227498, acted as a significant cis-eQTL for expression of EPB41L4A, rs17134155 was a significant cis-sQTL for the alternative splicing of a non-coding transcript of EPB41L4A, called EPB41L4A-203. We also performed knockdown of EPB41L4A followed by microarray analysis. Some of the top differentially-expressed genes were represented among regulators of the WNT/β-catenin signaling pathway. Our results indicate that an enhancer region at 5q22 regulates the expression and splicing of EPB41L4A transcripts. We also provide evidence that EPB41L4A expression is involved in regulating growth and differentiation pathways, suggesting that decreased expression of EPB41L4A is a mechanism in the predisposition to PTC.