Associations of ANGPTL6, DOCK6, FABP1, and PCSK9 single-nucleotide variants with hypercholesterolemia in the Polish population: a cross-sectional study.

INTRODUCTION: Hypercholesterolemia is a chronic noncommunicable disease predisposing to cardiovascular diseases. Genome­wide association studies have shown that more than 500 common nucleotide variants are associated with dyslipidemia. OBJECTIVES: We evaluated associations between selected nucleotide variants in ANGPTL6, DOCK6, FABP1, and PCSK9 genes and hypercholesterolemia in the Polish adult population sample. PATIENTS AND METHODS: The study included 109 patients with hypercholesterolemia and 251 individuals with no diagnosed lipid disorder. Genotyping of ANGPTL6 rs8112063, DOCK6 rs737337 and rs17699089, FABP1 rs2241883 and rs2919872, and PCSK9 rs562556 and rs11206510 was carried out using highresolution melting curve analysis. Serum concentrations of FABP1, PCSK9, ANGPTL6, and ANGPTL8 were determined in 51 individuals by enzyme­linked immunosorbent assay. RESULTS: Carriers of the FABP1 rs2919872 CC genotype were over 2.5­fold less likely to be diagnosed with hypercholesterolemia than carriers of the T allele (odds ratio [OR], 0.386; 95% CI, 0.203-0.735; P = 0.003; Pcorr = 0.006). There were no associations between rs2919872 and serum lipid concentrations. Carriers of the ANGPTL6 rs8112063 C allele had an almost 2­fold higher risk of developing hypercholesterolemia than carriers of the T allele (OR, 1.820; 95% CI, 1.053-3.144; P = 0.03; Pcorr = 0.046). Moreover, the carriers of the ANGPTL6 rs8112063 C allele had higher serum concentrations of high-density lipoprotein cholesterol than those with TT genotype (P = 0.009). There were no significant associations between the other tested variants and hypercholesterolemia. CONCLUSIONS: FABP1 rs2919872 and ANGPTL6 rs8112063 are associated with a risk of hypercholesterolemia in the Polish population.

Paraoxonase 1 gene variants concerning cardiovascular mortality in conventional cigarette smokers and non-smokers treated with hemodialysis.

Cigarette smoking effects might correspond with paraoxonase 1 gene (PON1) single nucleotide variants (SNVs). We investigated the association of PON1 rs705379, rs854560, and rs662 with cardiovascular mortality in hemodialysis (HD) patients concerning conventional cigarette smoking. Cardiovascular, cardiac, coronary heart disease (CHD)- and non-CHD-related deaths were analyzed in 206 HD cigarette smokers and 659 HD non-smokers. P-values were adjusted for sex, age, and high-density lipoprotein cholesterol. Among all smokers, the rs705379 TT genotype was associated with cardiovascular (P = 0.028), cardiac (P = 0.046), and cardiac non-CHD-related (P = 0.001) mortality. Non-diabetic smokers showed similar qualitative significance to all smokers concerning mentioned death rates (P-values 0.011, 0.044, and 0.009, respectively). In diabetic non-smokers, the rs705379 T allele correlated with CHD-related deaths (P = 0.020). The rs854560 T allele was associated with lower cardiovascular mortality in non-diabetic smokers (P = 0.008). The rs854560 TT genotype showed a negative non-significant correlation with non-CHD-related cardiac death in all non-smokers (P = 0.079). In diabetic smokers, the rs662 G allele was associated with higher cardiac mortality (P = 0.005). In all non-smokers and non-diabetic non-smokers, the rs662 G correlated with cardiovascular deaths (P = 0.020 and P = 0.018, respectively). Genotyping PON1 SNVs may help argue HD smokers harboring the rs705379 TT genotype or T allele and non-smokers possessing the rs662 G allele for prevention against cardiovascular diseases. These groups are more burdened genetically for cardiovascular mortality.

Polymorphism rs368234815 of interferon lambda 4 gene and spontaneous clearance of hepatitis C virus in haemodialysis patients: a case-control study.

BACKGROUND: In non-uremic subjects, IFNL4 rs368234815 predicts HCV clearance. We investigated whether rs368234815 is associated with spontaneous HCV clearance in haemodialysis patients and whether it is a stronger predictor of HCV resolution than the IFNL polymorphisms already associated with HCV clearance in dialysis subjects. We also evaluated an association of rs368234815 with patients` survival and alterations in transcription factor binding sites (TFBS) caused by IFNL polymorphisms. METHODS: Among 161 haemodialysis patients with positive anti-HCV antibodies, 68 (42.2%) spontaneously resolved HCV infection, whereas 93 remained HCV RNA positive. Patients were tested for near IFNL3 rs12980275, IFNL3 rs4803217, IFNL4 rs12979860, IFNL4 rs368234815, and near IFNL4 rs8099917. IFNL4 rs368234815 polymorphism (TT/TT, ΔG/TT, ΔG/ΔG) was genotyped by restriction fragment length polymorphism analysis; other IFNL polymorphisms - by high resolution melting curve analysis. We used the Kaplan-Meier method with the log-rank test for survival analysis. In silico analysis included the use of ENCODE TFBS ChIP-seq data, HOCOMOCO, JASPAR CORE, and CIS-BP databases, and FIMO software. RESULTS: The probability (OR, 95%CI, P) of spontaneous HCV clearance for rs368234815 TT/TT patients was higher than for the ΔG allele carriers (2.63, 1.38-5.04, 0.003). This probability for other major homozygotes varied between 2.80, 1.45-5.43, 0.002 for rs12980275 and 2.44, 1.27-4.69, 0.007 for rs12979860. In the additive model, rs368234815 TT/TT was the strongest predictor of HCV clearance (6.38, 1.69-24.2, 0.003). Survival analysis suggested an association of the ΔG allele with mortality due to neoplasms (log-rank P = 0.005). The rs368234815 ∆G allele caused TFBS removal for PLAGL1. CONCLUSIONS: In haemodialysis patients, the association of rs368234815 with the spontaneous HCV clearance is better than that documented for other IFNL3/IFNL4 polymorphisms only in the additive mode of inheritance. However, identifying the homozygosity in the variant ∆G allele of rs368234815 means a more potent prediction of persistent HCV infection in haemodialysis subjects that we observe in the case of the variant homozygosity of other tested IFNL3/IFNL4 polymorphisms. Removal of PLAGL1 TFBS in subjects harbouring the rs368234815 ∆G allele may contribute to cancer susceptibility. The association of rs368234815 with cancer-related mortality needs further studies in HCV-exposed subjects.

Polymorphism rs368234815 of interferon-λ4 gene and generation of antibodies to hepatitis B virus surface antigen in extracorporeal dialysis patients.

Background: The rs368234815 polymorphism of interferon-λ4 (IFN-λ4) gene (IFNL4) is involved in HBV surface antigen (HBsAg) clearance in non-uremic subjects. The rs368234815 ΔG/ΔG genotype can express IFN-λ4 while the TT/TT genotype cannot. We investigated whether rs368234815 is associated with the development of HBsAg antibodies (anti-HBs) in response to vaccination or infection, and HBsAg loss after infection in uremic patients on extracorporeal dialysis.Research design and methods: Dialyzed patients (n = 467) were genotyped for rs368234815 by the polymerase chain reaction-restriction fragment length polymorphism method. Non-responders to HBV vaccination we compared with responders. HBsAg positive patients not able to develop anti-HBs we compared with individuals who eliminated HBsAg and generated anti-HBs. HBsAg positive patients we compared with subjects who eliminated HBsAg.Results: The ∆G allele was associated with the 1.6-fold higher risk not to develop anti-HBs titers ≥10 IU/L in response to HBV vaccination and infection (P = 0.016 adjusted for gender, age at dialysis onset, HCV RNA). The ∆G/∆G genotype indicated a higher probability of non-responsiveness to HBV vaccination than the TT/TT genotype (OR 2.64, 95%CI 1.01-6.87, adjusted P = 0.048).Conclusions: In extracorporeal dialysis patients, IFNL4 rs368234815 is associated with the capacity to produce protective anti-HBs titers in response to HBV vaccination.

Circulating interferon-λ3 and post-vaccination antibodies against the surface antigen of hepatitis B virus in hemodialysis patients exposed to hepatitis E virus.

Responsiveness to the hepatitis B virus (HBV) vaccination in hemodialysis (HD) patients who had been exposed to the hepatitis E virus (HEV) and persistently generate antibodies against HEV remains unknown. Interferon (IFN)-λ3 positively correlates with the surface HBV antibodies (anti-HBs) in both healthy and HD subjects. We aimed to show whether HD patients differ in circulating IFN-λ3 and vaccine-induced anti-HBs titers concerning natural HEV immunization. HBV/HCV negative HD patients (31 HEV IgG positive, 45 HEV negative), HBV vaccinated and receiving booster doses as needed, had been tested for anti-HBs titers (CMIA) and IFN-λ3 concentrations (ELISA) in the blood collected before a dialysis session. There were no differences in circulating IFN-λ3 and anti-HBs titers between both groups. In responders to the HBV vaccine, there was a positive correlation between plasma IFN-λ3 levels and anti-HBs titers (r = 0.505, adjusted P = 0.01 in HEV exposed subjects; r = 0.523, adjusted P = 0.001 in controls). HEV past infection does not attenuate post-vaccination anti-HBs generation and does not influence a correlation between circulating IFN-λ3 levels and anti-HBs titers.

ENHO, RXRA, and LXRA polymorphisms and dyslipidaemia, related comorbidities and survival in haemodialysis patients.

BACKGROUND: The energy homeostasis-associated gene (ENHO), retinoid X receptor alpha gene (RXRA), and liver X receptor alpha gene (LXRA) are involved in adipogenic/lipogenic regulation. We investigated whether single-nucleotide polymorphisms in these genes (ENHO rs2281997, rs72735260; RXRA rs749759, rs10776909, rs10881578; LXRA rs2279238, rs7120118, rs11039155) are associated with dyslipidaemia, related comorbidities and survival of haemodialysis (HD) patients also tested for T-helper (Th) cell interleukin genes (IL). METHODS: The study was carried out in 873 HD patients. Dyslipidaemia was diagnosed by the recommendations of the Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines (2003); atherogenic dyslipidaemia was referred to if the TG/HDL cholesterol ratio was equal to or higher than 3.8. Genotyping of ENHO SNPs, LXRA SNPs, and IL12A rs568408 was carried out using HRM analysis. RXRA SNPs, IL12B rs3212227, and IL18 rs360719 were genotyped using PCR-RFLP analysis. The circulating adropin concentration was determined in 126 patients by enzyme-linked immunosorbent assay. Survival probability was analysed using the Kaplan-Meier method in 440 patients followed through 7.5 years. RESULTS: Dyslipidaemia by K/DOQI was diagnosed in 459 patients (91% revealed hyper-LDL- cholesterolaemia), atherogenic dyslipidaemia was diagnosed in 454 patients, and 231 patients were free of dyslipidaemia by both criteria. The variant allele (T) of ENHO rs2281997 was associated with the hyper-LDL cholesterolaemic pattern of dyslipidaemia by K/DOQI. The frequency of atherogenic dyslipidaemia was lower in T-allele bearers than in CC-genotype patients. The rs2281997 T allele was associated with lower cardiovascular mortality in HD patients showing atherogenic dyslipidaemia. ENHO, RXRA, and LXRA showed epistatic interactions in dyslipidaemia. Circulating adropin was lower in atherogenic dyslipidaemia than in non-atherogenic conditions. RXRA rs10776909 was associated with myocardial infarction. Bearers of LXRA rs2279238, rs7120118 or rs11039155 minor alleles showed higher mortality. ENHO SNP positions fell within the same DNase 1 hypersensitivity site expressed in the Th1 cell line. Epistatic interactions occurred between rs2281997 and Th1 IL SNPs (rs360719, rs568408). CONCLUSIONS: Atherogenic dyslipidaemia occurs in HD patients in whom ENHO encodes less adropin. ENHO, RXRA, and LXRA SNPs, separately or jointly, are associated with dyslipidaemia, myocardial infarction, and survival in HD patients. Differences in the availability of transcription binding sites may contribute to these associations.

Interferon­λ4 gene polymorphisms, circulating interferon λ3, and clinical variables in hemodialysis patients exposed to hepatitis E virus.

Introduction Factors associated with hepatitis E virus (HEV) infection are rarely recognized in patients on renal replacement therapy (RRT), and the results of studies are inconsistent. Objectives We aimed to search for determinants of HEV seroprevalence among polymorphisms of the interferon­λ4 gene (IFNL4) associated with seroclearance of hepatotropic viruses (IFNL4 rs12979860, rs8099917 near IFNL4), circulating interferon λ3 (IFN­λ3), and clinical variables of patients treated with hemodialysis (HD) in a HEV­endemic region. Patients and methods The study was carried out in 90 HD patients. HEV open reading frame 2 antigen (HEV Ag), immunoglobulin M and G antibodies to HEV (anti­HEV IgM and anti­HEV IgG, respectively) and IFN­λ3 were tested, and IFNL4 polymorphic variants (rs8099917, rs12979860) were genotyped. Survival analysis was conducted concerning anti­HEV IgG positivity. Results In the study group, there were 37.8% anti­HEV IgG­positive subjects. None was HEV Ag or anti­HEV IgM positive. HD modalities utilizing high­flux dialyzers (adjusted odds ratio [OR], 3.586; 95% confidence interval [CI], 1.142-11.263; P = 0.03) as well as major homozygosity in rs8099917 (adjusted OR, 4.933; 95% CI, 1.516-16.054; P = 0.008) and rs12979860 (adjusted OR, 3.537; 95% CI, 1.136-11.014, P = 0.03), but not circulating IFN­λ3 levels, were positive determinants of anti­HEV IgG positivity. Liver enzyme activities and C­reactive protein levels tested as response variables to HEV exposure, as well as survival probability, were not different between patients stratified by anti­HEV IgG positivity. Conclusions Among HD patients, IFNL4 polymorphisms and treatment with high­flux HD are explanatory variables for isolated anti­HEV IgG positivity indicating spontaneous HEV resolution.

Circulating Interferon-λ3, Responsiveness to HBV Vaccination, and HBV/HCV Infections in Haemodialysis Patients.

The IFN-λ3 gene (IFNL3) plays a role in HCV clearance. We investigated circulating IFN-λ3 and IFNL3 SNPs in haemodialysis patients who differed in their response to HBV vaccination and their HBV/HCV infection status. In 201 patients, plasma IFN-λ3 was determined using ELISA. IFNL3 SNPs (rs12979860, rs8099917) were genotyped using HRM analysis. Differences in IFN-λ3 levels were shown between responders and nonresponders to HBV vaccination and between HBsAg-positive patients and those who developed anti-HBs after infection and became HBsAg negative. HBV vaccine responders without HCV resolution revealed lower IFN-λ3 than noninfected responders. HBsAg/HCV RNA-positive subjects showed lower IFN-λ3 than patients positive only for HCV RNA or subjects who resolved both infections. Circulating IFN-λ3 correlated positively with anti-HBs and negatively with positive HCV RNA testing in the adjusted regression analyses. HBV vaccine nonresponders, HBsAg-positive patients, and subjects with replicating HCV composed a group with unfavourable outcomes. Responders to HBV vaccination, subjects who became HBsAg negative, and those who cleared HCV were analysed as having favourable outcomes. The latter showed higher IFN-λ3 but did not differ in distribution of IFNL3 SNPs compared with subjects with unfavourable outcomes. Higher IFN-λ3 concentrations are associated with response to HBV vaccination, self-limited HBV infection, and HCV resolution.

Polymorphisms of T helper cell cytokine-associated genes and survival of hemodialysis patients - a prospective study.

BACKGROUND: Circulating pro-inflammatory cytokines were associated with increased relative mortality risk, while immune parameters reflecting improved T-cell function were predictors of survival in hemodialysis (HD) patients. We evaluated in the prospective study whether variants in T helper cell cytokine-associated genes are determinants of mortality in HD patients. METHODS: The study was carried out in 532 prevalent HD subjects who were followed-up for 7 years. HRM analysis was used for IFNL3, IL12A, IL13, and IL4R genotyping. CCL2, IL12B, and IL18 were genotyped using PCR-RFLP analysis. Survival analyses were conducted using the Kaplan-Meier method and the Cox proportional hazard model. RESULTS: In univariate analyses, IFNL3 rs8099917 was associated with all-cause mortality in recessive model of inheritance (log-rank test P = 0.044), IL12A rs568408 - in dominant model (log-rank test P = 0.029). Minor homozygotes (the genotype GG) in IFNL3 rs8099917 showed shorter survival during the study (3.6, 1.0-7.0 years vs 4.7, 0.1-7.0 years, P = 0.009) than the major allele (T) bearers. The rs8099917 GG patients demonstrated higher risk of death than the remaining patients (GT + TT) (OR 1.94, 95%CI 1.11-3.40, P = 0.020). Major homozygosity (the genotype GG) in IL12A rs568408 was associated with higher mortality than that shown in bearers of the minor allele (AA + AG) (HR 1.31, 95%CI 1.02-1.69, P = 0.035). In multivariate analyses, however, the mentioned polymorphisms were not independent predictors of survival. CONCLUSIONS: Polymorphisms of IFNL3 rs8099917 and IL12A rs568408 contribute to survival of HD patients, but not as independent factors.

Antibodies to HBV surface antigen in relation to interferon-λ3 in hemodialysis patients.

AIM: To investigate circulating IFN-λ3 and IFNL3 polymorphisms in hemodialysis (HD) patients differing in HBV surface antigen antibody (anti-HBs) production. METHODS: The study included 106 HBV-vaccinated HD patients (88 developed anti-HBs) and 36 HBV-infected HD subjects (27 developed anti-HBs). Plasma IFN-λ3 (enzyme-linked immunosorbent assay) and rs12979860 (C>T) and rs8099917 (T>G) in IFNL3 (high-resolution melting curve analysis) were analyzed with regard to the association with anti-HBs production in response to HBV vaccination or infection. The results were adjusted for gender, age, cause of renal disease, dialysis vintage, dialysis modality, IFN-λ3, and 25(OH)D as appropriate. RESULTS: HBV vaccine responders had higher circulating IFN-λ3 (ng/L) than non-responders (120, 36-233 vs. 53, 33-109, P<0.000001). Patients who generated anti-HBs after HBV infection also had higher circulating IFN-λ3 levels than those who did not (133, 35-215 vs. 71, 9-229, P=0.043). The IFN-λ3 concentration correlated with the anti-HBs titer in vaccinated (r=0.614, P<0.000001) and infected patients (r=0.589, P=0.0002). Plasma IFN-λ3 was the only significant indicator of responsiveness to HBV vaccination (adjusted P=0.018) and remained the only significant associate for the development of post-infection anti-HBs (adjusted P=0.049). A plasmaIFN-λ3 level of 85.5ng/L was thecut-off value for theprognosis of an anti-HBs titer below vs. equal to or over 10IU/L in the entire group of HD patients (ROC sensitivity 68.7%, specificity 85.2%, and AUC 0.827). Significant associations were not found between IFN-λ3 and IFNL3 rs12979860. Subjects treated with low flux HD that harbored the TT genotype in rs8099917 showed higher IFN-λ3 levels than patients bearing the G allele in rs8099917 (139, 68-233 vs. 103, 9-208, P=0.049). CONCLUSION: In HD patients, circulating IFN-λ3 strongly correlates with anti-HBs production after HBV vaccination and infection. IFNL3 rs8099917 polymorphisms seem to be associated with IFN-λ3 plasma levels in HD subjects.

Polymorphisms of Vitamin D Signaling Pathway Genes and Calcium-Sensing Receptor Gene in respect to Survival of Hemodialysis Patients: A Prospective Observational Study.

We evaluated in the 7-year prospective study whether variants in vitamin D pathway genes and calcium-sensing receptor gene (CASR) are determinants of mortality in hemodialysis (HD) patients (n = 532). HRM analysis was used for GC rs2298849, GC rs1155563, RXRA rs10776909, RXRA rs10881578, and CASR rs7652589 genotyping. GC rs7041, RXRA rs749759, VDR rs2228570, and VDR rs1544410 were genotyped using PCR-RFLP analysis. The minor allele in GC rs2298849 was associated with all-cause mortality in univariate analysis (HR 1.330, 95% CI 1.046-1.692, P = 0.020). Bearers of the minor allele in GC rs2298849 demonstrated higher infection/neoplasm mortality than major allele homozygotes also in multivariate analysis (HR 2.116, 95% CI 1.096-4.087, P = 0.026). Cardiovascular mortality was associated with major homozygosity (CC) in VDR rs2228570 (HR 1.896, 95% CI 1.163-3.091, P = 0.010). CC genotype patients were more often dyslipidemic than TT genotype subjects (46.1% versus 31.9%, P = 0.047). Dyslipidemics showed higher frequency of rs1544410_rs2228570 haplotype AC than nondyslipidemics (26 versus 18%, P corr = 0.005), whereas TT genotype patients were at lower risk of dyslipidemia compared with CC/CT genotype patients (HR 0.59, 95% CI 0.37-0.96, P = 0.04). In conclusion, GC rs2298849 and VDR rs2228570 SNPs are associated with survival on HD. VDR-related cardiovascular mortality may occur due to connections of rs2228570 with dyslipidemia.

Antibodies to hepatitis B virus surface antigen and survival of hemodialysis patients - a prospective study.

BACKGROUND: Antibodies to hepatitis B virus (HBV) surface antigen (anti-HBs) may develop in response to HBV vaccination or infection. We investigated whether anti-HBs are an independent predictor of survival in hemodialysis (HD) patients. METHODS: A 6-year prospective study was conducted in 532 HD patients. Survival analyses were performed using the Kaplan-Meier method and the Cox proportional hazard model. RESULTS: In HBV non-infected patients, age (P = 0.005), coronary artery disease (P = 0.002), and non-response to HBV vaccine (P = 0.008) were the independent risk factors of all-cause mortality. In HBV infected patients, the only independent predictor of all-cause mortality was coronary artery disease (P = 0.002). CONCLUSION: The ability to produce the protective anti-HBs titer in response to HBV vaccine is a positive predictor of survival in HBV non-infected HD patients.