Polymerase Chain Reaction Allelic Discrimination Research Articles

Associations between single nucleotide polymorphisms of cytokines and hepatitis B virus-related liver cirrhosis: A case-controlstudy.

Various inflammatory and immune cytokines play key roles in the progression of hepatitis B virus (HBV)-related liver cirrhosis (LC). This study explored the relationship between single nucleotide polymorphisms (SNPs) in cytokines with the combined effect of polymorphisms and gender-polymorphisms interaction and LC risk. In this study, a case-control design was used, samples were selected from 45 patients with hepatitis B-related cirrhosis and 45 age-gender-matched chronic HBV-infected patients without cirrhosis attending the tumor hospital of Wuwei Academy of Medical Sciences. Fifteen SNPs were examined using a real-time polymerase chain reaction allelic discrimination system. Logistic regression was utilized to assess cytokine-associated SNPs and the association between SNPs and LC progression in HBV-infected patients. The multivariate-adjusted logistic model revealed that the GG/AG dominant model (OR, 16.38; 95% CI, 1.13-236.70) and G allele (OR, 5.93; 95% CI, 0.98-36.01) of rs1800896 were associated with an increased risk of cirrhosis in CHB patients. Instead, rs2227306 CT presented a reduced cirrhosis risk (OR, 0.22; 95% CI, 0.04-1.38). Rs2055979 AA/AC was negatively associated with the risk of cirrhosis, potentially reversed in males (p = 0.021). Rs1799964 CC/CT was positively related to the risk of cirrhosis but reduced the risk of cirrhosis in males (OR, 0.13; 95% CI, 0.022-0.808; p = 0.028). Both rs1799964 TT and rs1799724 CT/TT genotype showed a synergistic effect in reducing the risk of cirrhosis with rs1800896 AA (OR, 0.08; 95% CI, 0.01-1.43 and OR, 0.12; 95% CI, 0.01-2.21). Polymorphisms rs1800896 and rs2227306 are potentially associated with the risk of cirrhosis. For the first time, the study highlights that the rs2055979 AA/AC and rs1799964 CC/CT polymorphism interact with gender and its potential reversal of cirrhosis risk in males. Furthermore, rs1800896 AA showed a synergistic effect with rs1799964 TT and rs1799724 CT/TT to prevent the progression of HBV infection to cirrhosis.

Association of CIITA (rs8048002) and CLEC2D (rs2114870) gene variants and type 1 diabetes mellitus.

Type I diabetes mellitus (T1DM) is a significant health challenge, especially for children, owing to its chronic autoimmune nature. Although the exact etiology of T1DM remains elusive, the interplay of genetic predisposition, immune responses, and environmental factors are postulated. Genetic factors control immune reactivity against β-cells. Given the pivotal roles of CIITA and CLEC2D genes in modulating a variety of immune pathologies, we hypothesized that genetic variations in CIITA and CLEC2D genes may impact T1DM disease predisposition. This study was designed to explore the association between gene polymorphisms in CIITA (rs8048002) and CLEC2D (rs2114870) and type 1 diabetes (T1DM), with a focus on analyzing the functional consequence of those gene variants. The study enlisted 178 healthy controls and 148 individuals with type 1 diabetes (T1DM) from Suez Canal University Hospital. Genotyping for CIITA and CLEC2D was done using allelic-discrimination polymerase chain reaction (PCR). Levels of glycated hemoglobin (HbA1c) and lipid profiles were determined through automated analyzer, while fasting blood glucose and insulin serum levels were measured using the enzyme-linked immunosorbent assay (ELISA) technique. RegulomeDB was used to examine the regulatory functions of CIITA (rs8048002) and CLEC2D (rs2114870) gene variants. Analysis of the genotype distribution of the CIITA rs8048002 polymorphism revealed a significantly higher prevalence of the rare C allele in T1DM patients compared to the control group (OR = 1.77; P = 0.001). Both the CIITA rs8048002 heterozygote TC genotype (OR = 1.93; P = 0.005) and the rare homozygote CC genotype (OR = 3.62; P = 0.006) were significantly more frequent in children with T1DM when compared to the control group. Conversely, the rare A allele of CLEC2D rs2114870 was found to be significantly less frequent in T1DM children relative to the control group (OR = 0.58; P = 0.002). The heterozygote GA genotype (OR = 0.61; P = 0.033) and the rare homozygote AA genotype (OR = 0.25; P = 0.004) were also significantly less frequent in T1DM patients compared to the control group. Both CIITA (rs8048002) and CLEC2D (rs2114870) gene variants were predicted to have regulatory functions, indicated by a RegulomeDB score of (1f) for each. The rare C allele of CIITA rs8048002 genetic variant was associated with an increased risk of developing T1DM, while the less common A allele of CLEC2D rs2114870 was associated with a reduced risk of T1DM. The online version contains supplementary material available at 10.1007/s40200-024-01402-w.

Angio-Long Noncoding RNA MALAT1 (rs3200401) and MIAT (rs1061540) Gene Variants in Ovarian Cancer.

The genotyping of long non-coding RNA (lncRNA)-related single-nucleotide polymorphisms (SNPs) could be associated with cancer risk and/or progression. This study aimed to analyze the angiogenesis-related lncRNAs MALAT1 (rs3200401) and MIAT (rs1061540) variants in patients with ovarian cancer (OC) using "Real-Time allelic discrimination polymerase chain reaction" in 182 formalin-fixed paraffin-embedded (FFPE) samples of benign, borderline, and primary malignant ovarian tissues. Differences in the genotype frequencies between low-grade ovarian epithelial tumors (benign/borderline) and malignant tumors and between high-grade malignant epithelial tumors and malignant epithelial tumors other than high-grade serous carcinomas were compared. Odds ratios (ORs)/95% confidence intervals were calculated as measures of the association strength. Additionally, associations of the genotypes with the available pathological data were analyzed. The heterozygosity of MALAT1 rs3200401 was the most common genotype (47.8%), followed by C/C (36.3%). Comparing the study groups, no significant differences were observed regarding this variant. In contrast, the malignant epithelial tumors had a higher frequency of the MIAT rs1061540 C/C genotype compared to the low-grade epithelial tumor cohorts (56.7% vs. 37.6, p = 0.031). The same genotype was significantly higher in high-grade serous carcinoma than its counterparts (69.4% vs. 43.8%, p = 0.038). Multivariate Cox regression analysis showed that the age at diagnosis was significantly associated with the risk of OC development. In contrast, the MIAT T/T genotype was associated with a low risk of malignant epithelial tumors under the homozygote comparison model (OR = 0.37 (0.16-0.83), p = 0.017). Also, MIAT T allele carriers were less likely to develop high-grade serous carcinoma under heterozygote (CT vs. CC; OR = 0.33 (0.12-0.88), p = 0.027) and homozygote (TT vs. CC; OR = 0.26 (0.07-0.90), p = 0.034) comparison models. In conclusion, our data provide novel evidence for a potential association between the lncRNA MIAT rs1061540 and the malignant condition of ovarian cancer, suggesting the involvement of such lncRNAs in OC development.

RS3480 Polymorphism of FNDC5/Irisin Is Associated with Type 2 Diabetes Mellitus in Maya-Mestizo Women.

Objective: To investigate the possible association between rs3480 and rs16835198 of the fibronectin type III domain containing 5 (FNDC5)/Irisin and their haplotypes with the presence of type 2 diabetes mellitus (T2DM) in Maya-Mestizo women. Methods: We studied 547 postmenopausal women of Maya-Mestizo origin. The diagnosis of T2DM was based on the criteria of the American Diabetes Association. DNA was obtained from blood leukocytes. rs3480 and rs16835198 of FNDC5/Irisin were studied using real-time polymerase chain reaction allelic discrimination. Deviations from Hardy-Weinberg equilibrium and alleles differences, as well as genotype frequencies between groups, were assessed by χ2 tests. Using logistic regression analysis, the odds ratio and 95% confidence intervals were calculated to estimate the association between both polymorphisms of FNDC5/Irisin and the presence of T2DM. Pairwise linkage disequilibrium between polymorphisms was calculated by direct correlation r2, and haplotype analysis was conducted. Results: We found that the G-allele of rs3480, as well as under a dominant model, this polymorphism was significantly associated with T2DM (P = 0.028 and P = 0.003, respectively). Besides, one haplotype was associated with T2DM (P = 0.035). Conclusions: Our results suggest that the FNDC5/Irisin rs3480, and one haplotype formed by rs3480 and rs16835198 were associated with the risk of presenting T2DM in Maya-Mestizo women.

Hair Follicle-Related MicroRNA-34a Serum Expression and rs2666433A/G Variant in Patients with Alopecia: A Cross-Sectional Analysis.

Alopecia areata (AA) is a type of immune-mediated alopecia. Recent studies have suggested microRNAs’ (miRNAs) implication in several cellular processes, including epidermal and hair follicle biology. Single nucleotide polymorphisms (SNPs) can modify gene expression levels, which may induce an autoimmune response. This case–control study included 480 participants (240 for each case/control group). MicroRNA-34a gene (MIR-34A) rs2666433A/G variant was genotyped using real-time allelic discrimination polymerase chain reaction (PCR). Additionally, circulatory miR-34a levels were quantified by quantitative reverse transcription PCR (qRT-PCR). On comparing between alopecia and non-alopecia cohorts, a higher frequency of A variant was noted among patients when compared to controls—A allele: 28 versus 18% (p < 0.001); A/A genotype: 9 versus 2%; A/G genotype: 39 versus 32% (p < 0.001). A/A and A/G carriers were more likely to develop alopecia under heterozygote comparison (OR = 1.83, 95% CI = 1.14–2.93), homozygote comparison (OR = 4.19, 95% CI = 1.33–13.1), dominant (OR = 2.0, 95% CI = 1.27–3.15), recessive (OR = 3.36, 95% CI = 1.08–10.48), over-dominant (OR = 1.65, 95% CI = 1.04–32.63), and log additive (OR = 1.91, 95% CI = 1.3–2.82) models. Serum miR-34a expression levels were upregulated in alopecia patients with a median and quartile fold change of 27.3 (1.42–2430). Significantly higher levels were more pronounced in A/A genotype patients (p < 0.01). Patients carrying the heterozygote genotype (rs2666433 * A/G) were two times more likely to develop more severe disease grades. Stratified analysis by sex revealed the same results. A high expression level was associated with concomitant autoimmune comorbidities (p = 0.001), in particular SLE (p = 0.007) and vitiligo (p = 0.049). In conclusion, the MIR34A rs2666433 (A/G) variant is associated with AA risk and severity in the studied population. Furthermore, high miR-34a circulatory levels could play a role in disease pathogenesis.

Erythrocyte Complement Receptor 1 Gene Polymorphisms and Neonatal Respiratory Distress Syndrome.

To evaluate the role of erythrocyte complement receptor 1 ( ECR1 ) gene in the predisposition to respiratory distress syndrome (RDS), we studied 50 infants with RDS and 50 controls. Real-time polymerase chain reaction allelic discrimination analysis of A3650G (rs2274567) and genotyping of the alleles (HindIII) were performed. Allele L of HindIII restricted single nucleotide polymorphism (SNP) associated with the severity of RDS. Duration of oxygen and ventilation in genotypes AA and AG of A3650G SNP was longer than genotype GG (17.6 ± 19.4 and 8.6 ± 4.5 days, p = 0.01) and (8.9 ± 11.9 and 3.9 ± 3.53 days, p = 0.03), respectively. A3650G and HINDIII digested gene polymorphisms of ECR1 may be of little importance for RDS.

Lack of association between interleukin 13, interleukin 4 receptor alpha, andMS4A2 gene polymorphisms and asthma in adult Saudis

Objectives: Several genes, including interleukin-13 (IL13), MS4A2, and IL 4 receptor alpha (IL4Rα), have been shown to associate with the inflammatory process in asthma. We investigated the possible association between asthma and five single-nucleotide polymorphisms (SNPs) in the IL13, IL4Rα, and MS4A2 gene in comparison with the level of the IL-13 protein in the Saudi asthmatics and controls. Subjects and Methods: The study group included 50 adult asthmatic patients and 50 normal healthy controls. Blood was extracted, and DNA was prepared. Genotyping was performed on DNA using direct sequencing for four SNPs (rs20541, rs1800925, rs1801275, and rs569108), and real-time polymerase chain reaction allelic discrimination to study rs1805010 polymorphism. Levels of IL-13 were measured in plasma using (enzyme-linked immunosorbent. Genotype and allele frequencies were calculated for each SNP. Results: The results showed that GG genotype of rs1801275 in the IL4Rα gene was more common in the asthmatic patients compared to the control group, though the results did not show any statistical significance (P G transition nonsynonymous mutation, resulting in the substitution of glutamine by arginine showed an association, but it was statistically not significant. Interestingly, presence of the mutant allele of both SNPs of IL-13 gene significantly elevated IL-13 level. Comparison of the Saudi results with those of other populations, showed several significant differences. We conclude that further more detailed studies are necessary in Saudis to identify the contribution of SNPs in cytokine genes in asthma development.

Analysis of the autoimmune regulator (AIRE) gene variant rs2075876 (G/A) association with breast cancer susceptibility.

BackgroundRecently, unexpected autoimmune regulator (AIRE) implication in the scenario of several cancers, including breast cancer (BC), has emerged. This study aims to explore for the first time the possible association between AIRE gene rs2075876 G>A variant and BC risk in a sample of the Middle East population.MethodIn this case‐control study, we genotyped AIRE rs2075876 G>A variant in 200 unrelated patients with BC and 340 cancer‐free controls using a real‐time allelic discrimination polymerase chain reaction. Odds ratios (ORs) and 95% confidence intervals (CIs) were applied to estimate the strength of association under several genetic models. In silico analysis of AIRE was also executed.ResultsThe minor allele (A) frequency of the specified variant accounted for 0.28 in the controls. G/G homozygote was significantly more frequent among patients (94%) compared to controls (66%) (P < .001). After adjusting confounding variables, individuals with A allele conferred protection against developing BC under allelic model (OR = 0.33, 95% CI = 0.20‐0.55), recessive model (OR = 0.25, 95% CI = 0.10‐0.60), dominant model (OR = 0.12, 95% CI = 0.05‐0.29), and homozygote comparison (OR = 0.20, 95% CI = 0.08‐0.50). In silico analysis revealed AIRE enrichment in several cancer‐related pathways. Kaplan‐Meier plotter for the cancer databases showed association of AIRE expression with prognosis in triple‐negative BC (HR = 2.44, 95% CI = 1.44‐4.15, log‐rank P‐value < .001).ConclusionThe AIRE rs2075876 G>A variant showed association with BC risk in the study population. Further large‐scale replication studies in different ethnicity are warranted to confirm the findings.

Abstract WMP56: Association of Mean Platelet Volume and Its Genetic Variants With Stroke Severity and One-year Mortality in a Polish Stroke Cohort

Background: Increased mean platelet volume (MPV) is a marker of worse outcomes in cardiovascular disorders, however its prognostic role in patents with ischemic stroke and possible genetic underpinnings remains uncertain. We aimed to determine whether MPV and selected single nucleotide polymorphisms (SNPs) that have been associated with MPV in genome-wide association studies (GWAS) relate to stroke severity, functional outcome on discharge, and one-year mortality in patients with ischemic stroke. Methods: We retrospectively analyzed 577 patients with first ever ischemic stroke. Genotyping of 3 SNPs (rs342293, rs1354034, rs7961894) was performed using a real-time polymerase chain reaction allelic discrimination assay. Multivariable regression was used to determine the association of MPV and MPV-associated SNPs with the National Institutes of Health Stroke Scale (NIHSS) score on admission, modified Rankin Scale (mRS) on discharge, and data on one-year mortality. Results: Rs7961894, but not rs342293 and rs1354034 SNP, was independently associated with MPV in the highest quartile (MPV Q4). MPV Q4 was associated with significantly greater admission NIHSS (p=0.004), poor discharge outcome (p=0.034), and worse one-year mortality (p=0.033). After adjustment for pertinent covariates, MPV Q4 remained independently associated with a greater admission NIHSS score (p=0.025). Patients carrying T&gt;C variant of rs7961894 SNP had a significantly lower one-year mortality as compared to patients with CC genotype (p=0.029; Log-Rank test; Fig.1) and T&gt;C variant of rs7961894 SNP remained an independent marker of a lower one-year mortality (HR=0.30; 95%CI:0.13-0.70; p=0.006) in the studied population. Conclusion: MPV is a marker of stroke severity and rs7961894 is independently associated with MPV in acute phase of ischemic stroke and relates to one-year mortality after stroke.

Association of cytokine gene polymorphisms with osteoarthritis susceptibility.

Osteoarthritis (OA) is a multifactorial disease characterized by low-grade inflammatory processes that are mediated initially by the cells and factors of the innate immune system. In addition to their key role in inflammation, cytokines contribute to the pathogenesis of OA through angiogenesis and chemotaxis. The purpose of the present case-control study was to investigate a possible association of four cytokine single nucleotide polymorphisms (SNPs), IL-4R -3223C>T (rs2057768), IL-8 -251T>A (rs4073), IL-10 -1082A>G (rs1800896) and TNF -A-308G>A (rs1800629) with OA susceptibility. Genomic DNA was isolated from blood samples collected from 305 Romanian subjects (90 patients with OA and 215 controls) and the genotyping of the SNPs was performed by TaqMan allelic discrimination polymerase chain reaction using predesigned assays. Our data indicated a significant association for IL-4R rs2057768 C>T SNP. The subjects that carried the CT genotype were at a higher risk for OA (OR 2.03, 95% CI: 1.21-3.42, P=0.007) compared with those that had the CC genotype. Furthermore, the carriers of the T allele were at a 1.9 fold higher risk for OA (OR 1.92; 95% CI, 1.17-3.17; P=0.009) in a dominant model. The association remained significant only for knee OA in the subgroups analysis. No correlations were observed between the other remaining SNPs and OA. The results suggested that the IL-4R rs2057768 SNP could contribute to OA susceptibility in the Romanian population, providing novel evidence for the involvement of IL-4/IL-4R pair in the pathogenesis of OA.

Hypoxia and renal cell carcinoma: The influence of HIF1A+1772C/T functional genetic polymorphism on prognosis

ObjectivesHypoxia-inducible factor (HIF-1) is a key regulator of the genes involved in the cellular response to hypoxia. Overexpression of HIF-1 has been implicated in the pathogenesis of renal cell carcinoma (RCC), and functional polymorphisms of the HIF1A gene may confer susceptibility to RCC. Our purpose was to assess the influence of HIF1A+1772C/T (rs11549465) polymorphism on RCC prognosis. Material and methodsThis study evaluated the associations of the HIF1A+1772C/T (rs11549465) polymorphism with clinicopathologic prognostic factors, recurrence/progression, and survival in a cohort of 179 patients with RCC treated at Portuguese Oncology Institute of Porto. Genotyping analysis, using DNA extracted from peripheral blood, was performed by real-time polymerase chain reaction allelic discrimination. The genotype associations with clinicopathologic parameters and recurrence/progression were analyzed by the chi-square or Fisher tests. Genotypes influencing cancer-specific survival were compared using Cox proportional hazard regression, Kaplan-Meier curves, and Breslow test. ResultsNone of the genotypes (CC, CT, or TT) were significantly associated with clinicopathologic prognostic factors. The TT genotype and T allele were associated with recurrence/progression (P = 0.042 and P = 0.02, respectively). Patients with CT and CT+TT genotypes tend to have an increased risk to RCC-related death (hazard ratio = 2.79; 95% CI: 0.88–8.82; P = 0.08 and hazard ratio = 2.76; 95% CI: 0.93–8.22; P = 0.07, respectively) and showed worse cancer-specific survival curves than those with the CC genotype (P = 0.012 and P = 0.018, respectively). ConclusionsThese results suggest that HIF1A+1772C/T (rs11549465) polymorphism may have effects on RCC recurrence/progression and survival.

Functional polymorphism rs1024611 in the MCP1 gene is associated with the risk of varicose veins of lower extremities.

Monocyte chemoattractant protein 1 (MCP-1) is a chemokine responsible for monocyte, basophil, and T-lymphocyte attraction. Polymorphism rs1024611 located in the regulatory region of the MCP1 gene has previously been shown to be associated with increased MCP-1 production. In our study, we aimed to examine the association of rs1024611 with the risk of primary varicose veins (PVVs) of lower extremities. The case group comprised 470 patients with PVVs, and the control group included 269 individuals without a history of chronic venous disease. All cases and controls were ethnic Russians. Genotypes were determined by real-time polymerase chain reaction allelic discrimination. Association was studied by logistic regression analysis. We revealed the association of genotype G/G with the increased risk of PVVs (odds ratio [OR], 1.87; 95% confidence interval [CI], 1.02-3.44; P= .04). In the subgroup analysis, association was revealed only in patients with C2 Clinical, Etiology, Anatomy, and Pathophysiology class (allele G: OR, 1.62 [95% CI, 1.13-2.33; P= .008]; genotype G/G: OR, 3.22 [95% CI, 1.43-7.27; P= .005]), in patients with age at onset of PVVs before 30 years (allele G: OR, 1.41 [95% CI, 1.08-1.85; P= .01]; genotype G/G: OR, 2.35 [95% CI, 1.22-4.55; P= .01]), and in patients who declared no family history (allele G: OR, 1.46 [95% CI, 1.02-2.09; P= .04]; genotype G/G: OR, 2.50 [95% CI, 1.11-5.63; P= .03]). Our results provide evidence for MCP-1 involvement in the development of PVVs and indicate thatinflammation could be implicated in the pathogenesis of this condition.

Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV-infected Zulu population of South Africa.

Background. Mitochondrial toxicity, particularly symptomatic hyperlactataemia or lactic acidosis (SHL/LA), has been attributed to the use of nucleoside reverse transcriptase inhibitors (NRTIs), possibly because of their capacity to impede human mitochondrial DNA polymerase-γ (POLG), which is responsible for the replication of mitochondrial DNA. Objective. To determine whether known monogenic POLG1 polymorphisms could be linked with the unexpectedly high incidence of SHL/LA observed in HIV-infected Zulu-speaking patients exposed to the NRTIs stavudine or zidovudine in their antiretroviral therapy. Methods. One hundred and sixteen patients from Edendale Hospital, Pietermaritzburg, South Africa, participated in the study between March and August 2014. Fifty-nine symptomatic cases were compared with 57 non-symptomatic controls on stavudine for ≥24 months. Among the symptomatic patients, 13 had SHL with measured lactate between 3.0 and 4.99 mmol/L, and 46 had LA with a lactate level ≥5 mmol/L. Genomic DNA from 113 samples was used for subsequent allelic discrimination polymerase chain reaction screening for the R964C and E1143G single-nucleotide polymorphisms of POLG1. Sequencing was performed for 40/113 randomly selected samples for confirmation of the genotyping results. Results. Neither of the two known POLG1 mutations was observed. The cases presented with SHL/LA between 4 and 18 months on stavudine. Females (70.4%) were significantly (p<0.001) more likely to be cases (adjusted odds ratio 24.24, 95% CI 5.14 - 114.25) compared with males. Conclusion. This study has shown that our sample of the Zulu-speaking population does not exhibit a genetic predisposition to SHL/LA associated with known monogenic POLG1 mutations, indicating another possible predisposing factor for increased risk of SHL/LA.

Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV-infected Zulu population of South Africa

Background. Mitochondrial toxicity, particularly symptomatic hyperlactataemia or lactic acidosis (SHL/LA), has been attributed to the use of nucleoside reverse transcriptase inhibitors (NRTIs), possibly because of their capacity to impede human mitochondrial DNA polymerase-γ (POLG), which is responsible for the replication of mitochondrial DNA. Objective. To determine whether known monogenic POLG1 polymorphisms could be linked with the unexpectedly high incidence of SHL/LA observed in HIV-infected Zulu-speaking patients exposed to the NRTIs stavudine or zidovudine in their antiretroviral therapy. Methods. One hundred and sixteen patients from Edendale Hospital, Pietermaritzburg, South Africa, participated in the study between March and August 2014. Fifty-nine symptomatic cases were compared with 57 non-symptomatic controls on stavudine for ≥24 months. Among the symptomatic patients, 13 had SHL with measured lactate between 3.0 and 4.99 mmol/L, and 46 had LA with a lactate level ≥5 mmol/L. Genomic DNA from 113 samples was used for subsequent allelic discrimination polymerase chain reaction screening for the R964C and E1143G single-nucleotide polymorphisms of POLG1. Sequencing was performed for 40/113 randomly selected samples for confirmation of the genotyping results. Results. Neither of the two known POLG1 mutations was observed. The cases presented with SHL/LA between 4 and 18 months on stavudine. Females (70.4%) were significantly (p&lt;0.001) more likely to be cases (adjusted odds ratio 24.24, 95% CI 5.14 - 114.25) compared with males. Conclusion. This study has shown that our sample of the Zulu-speaking population does not exhibit a genetic predisposition to SHL/LA associated with known monogenic POLG1 mutations, indicating another possible predisposing factor for increased risk of SHL/LA.

Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV-infected Zulu population of South Africa.

Mitochondrial toxicity, particularly symptomatic hyperlactataemia or lactic acidosis (SHL/LA), has been attributed to the use of nucleoside reverse transcriptase inhibitors (NRTIs), possibly because of their capacity to impede human mitochondrial DNA polymerase-γ (POLG), which is responsible for the replication of mitochondrial DNA. To determine whether known monogenic POLG1 polymorphisms could be linked with the unexpectedly high incidence of SHL/LA observed in HIV-infected Zulu-speaking patients exposed to the NRTIs stavudine or zidovudine in their antiretroviral therapy. One hundred and sixteen patients from Edendale Hospital, Pietermaritzburg, South Africa, participated in the study between March and August 2014. Fifty-nine symptomatic cases were compared with 57 non-symptomatic controls on stavudine for ≥24 months. Among the symptomatic patients, 13 had SHL with measured lactate between 3.0 and 4.99 mmol/L, and 46 had LA with a lactate level ≥5 mmol/L. Genomic DNA from 113 samples was used for subsequent allelic discrimination polymerase chain reaction screening for the R964C and E1143G single-nucleotide polymorphisms of POLG1. Sequencing was performed for 40/113 randomly selected samples for confirmation of the genotyping results. Neither of the two known POLG1 mutations was observed. The cases presented with SHL/LA between 4 and 18 months on stavudine. Females (70.4%) were significantly (p&lt;0.001) more likely to be cases (adjusted odds ratio 24.24, 95% CI 5.14 - 114.25) compared with males. This study has shown that our sample of the Zulu-speaking population does not exhibit a genetic predisposition to SHL/LA associated with known monogenic POLG1 mutations, indicating another possible predisposing factor for increased risk of SHL/LA.

Association of a TFAM haplotype with aggressive prostate cancer in overweight or obese Mexican Mestizo men

BackgroundMitochondrial dysfunction has been associated with the development of cancer and obesity, being prostate cancer more aggressive in obese men. It has been suggested that the mitochondrial transcription factor A (TFAM) plays a central role in these events. ObjectiveThe aim of this study was to analyze the possible association of 3 TFAM polymorphisms, as well as their haplotypes, with the development of aggressive prostate cancer in overweight or obese Mexican Mestizo men. Subjects and methodsA total of 257 unrelated men with histologically confirmed prostate cancer, of Mexican Mestizo ethnic origin, were included. Body mass index was determined and the degree of prostate cancer aggressiveness was demarcated by the D’Amico classification. DNA was obtained from blood leukocytes. The rs1937, rs1049432, and rs11006132, as well as their haplotypes, were studied by real-time polymerase chain reaction allelic discrimination. Deviations from Hardy-Weinberg equilibrium were tested. Pairwise linkage disequilibrium between single nucleotide polymorphisms was calculated; haplotype analysis was performed. ResultsA higher risk (D’Amico classification) was documented in 56 patients (21.8%). We did not find a significant association among those polymorphisms analyzed; however, one haplotype was significantly associated with cancer aggressiveness. ConclusionsTo our knowledge, this constitutes the first study regarding the relationship of 3 TFAM polymorphisms, as well as their haplotypes, and the aggressiveness of prostate cancer in overweight or obese men; the most frequent haplotype was associated with cancer aggressiveness.

PNPLA3 rs738409 and TM6SF2 rs58542926 variants increase the risk of hepatocellular carcinoma in alcoholic cirrhosis

BackgroundPNPLA3 rs738409 polymorphism is associated with fatty liver disease, alcoholic or non-alcoholic (NAFLD) and hepatocellular carcinoma (HCC). TM6SF2 rs58542926 is clearly associated with NAFLD, but it is not clearly associated with HCC. The relationship between TM6SF2 rs58542926 and HCC and the potential synergistic effect of TM6SF2 and PNPLA3 variants in modifying the risk of HCC are not known. AimThis study assessed the interaction between PNPLA3 rs738409 and TM6SF2 rs58542926 variants in the conditioning of HCC development. MethodsA total of 511 cirrhotic patients (44% alcohol-related, 56% viral, 57.5% liver transplanted) were retrospectively investigated for HCC occurrence. PNPLA3 rs734809 and TM6SF2 rs58542926 were genotyped using restriction fragment length polymorphism and real-time allelic discrimination polymerase chain reaction methods. ResultsPatients with HCC were more likely to be PNPLA3 rs734809 G/G homozygotes (41/150 vs. 60/361, p=0.009) or TM6SF2 rs58542926 C/T-T/T (27/150 vs. 41/361, p=0.044). The presence of either PNPLA3 G/G or TM6SF2*/T identified high-risk genotypes for HCC, which were strongly associated with HCC (64/150 vs. 93/361, p=0.0002). This association was evident in alcohol-related (p=0.0007) but not in viral cirrhosis. ConclusionTM6SF2 C/T or T/T in conjunction with PNPLA3 G/G variants may be potential genetic risk factors for developing HCC in alcohol-related cirrhosis.

Association of RAMP1 rs7590387 with the risk of migraine transformation into medication overuse headache.

We herein investigated the role of polymorphisms in calcitonin gene-related peptide (CGRP)-related genes looking at the association of rs3781719 (T > C) in the calcitonin gene-related polypeptide-alpha (CALCA) gene and of rs3754701 (T > A) and rs7590387 (C > G) at the receptor activity modifying 1 (RAMP1) locus with triptan response in patients with migraine without aura (MwoA). In addition, their role was evaluated as risk factors for transformation of episodic migraine into medication overuse headache (MOH). The CGRP has a central role in the pathogenesis of migraine; however, little information is currently available concerning the role of polymorphisms in CGRP-related genes as determinants of clinical response to anti-migraine drugs or as risk factors for migraine chronification. Genotyping was conducted retrospectively by real-time polymerase chain reaction allelic discrimination assay in 219 patients with MwoA and 130 with MOH in whom migraine was the primary headache type. Gene variants association was evaluated by logistic regression analysis adjusted by confounding factors. The threshold of statistical significance was set according to the total number of polymorphisms analyzed in the current study and in previous publications arising from overlapping datasets. No evidence of association was found between the three polymorphisms tested and triptan response in MwoA patients. Conversely, carriers of RAMP1 rs7590387GG displayed a lower risk of episodic migraine transformation into MOH (vs C allele carriers, odds ratio [OR]: 0.27, 95% confidence interval [CI]: 0.13-0.57, P = 0.0002; threshold of significance set at P < 0.0029). When genotype distribution for RAMP1 rs7590387 was compared between healthy controls (n = 209) and MOH patients, carriers of rs7590387GG were found at lower risk of developing MOH (OR: 0.43, 95%CI: 0.22-0.85, P = 0.011). These results suggest that RAMP1 rs7590387 may have a role in the transformation of episodic migraine into MOH.

Functional polymorphisms in COMT and SLC6A4 genes influence the prognosis of patients with medication overuse headache after withdrawal therapy

It is currently unknown if common genetic variants influence the prognosis of patients with medication overuse headache (MOH). Here the role of two common single nucleotide polymorphisms in the COMT gene (rs4680 and rs6269), as well as the STin2 variable number tandem repeat (VNTR) polymorphism in the SLC6A4 gene, were evaluated as predictors for long-term outcomes of MOH patients after withdrawal therapy. Genotyping was conducted by polymerase chain reaction (PCR), PCR restriction fragment length polymorphism analysis or real-time PCR allelic discrimination assay on genomic DNA extracted from peripheral blood. Gene variants association was evaluated by logistic regression analysis adjusted for clinical confounding factors, and the threshold of statistical significance for multiple testing was set at P<0.012. Sixty-five MOH patients with unsuccessful detoxification and 83 MOH patients with effective drug withdrawal therapy were available for the analysis. rs4680G allele carriers or the COMT rs6269G-rs4680G haplotype were found to be associated with a lower risk of relapse within the first year after successful detoxification therapy, in comparison with homozygous rs4680A allele carriers [odds ratio (OR) 0.17, 95% confidence interval (CI) 0.05-0.61, P=0.007] or with the COMT rs6269A-rs4680A haplotype (OR 0.19, 95% CI 0.06-0.54, P=0.003), respectively. In addition, carriers of the STin2 VNTR short allele were found at higher odds for the composite poor outcome including unsuccessful withdrawal therapy and relapse within 12months of follow-up after successful detoxification (OR 2.81, 95%CI 1.26-6.25, P=0.009). Our results indicate that genotyping for COMT rs4680 and SLC6A4 STin2 VNTR could be useful for the identification of MOH patients at higher risk of poor prognosis after drug withdrawal.

Modulation of human longevity by SIRT3 single nucleotide polymorphisms in the prospective study “Treviso Longeva (TRELONG)”

Human sirtuins are seven proteins with deacetylase activity that are emerging as key modulators of basic physiological functions. Some evidence links SIRT3 to longevity in mammals. This study aimed to investigate whether variants within SIRT3 gene were associated to human longevity. We analyzed 549 genomic DNA collected during the prospective study "Treviso Longeva," including elderly over 70 years of age from the municipality of Treviso, a small city in the northeast of Italy. We genotyped SIRT3 rs3825075, rs4980329, and rs11555236 single nucleotide polymorphisms (SNPs) by real-time polymerase chain reaction allelic discrimination assay. A cross-sectional analysis performed by comparing people over and under 85 years of age did not evidence association among the SIRT3 SNPs and longevity. However, when we performed a longitudinal analysis considering mortality as a dependent variable, we observed an association of SIRT3 rs11555236 and rs4980329 with longevity in the whole population (p values corrected for potential confounders = 0.04 and 0.03, respectively). After stratification according to gender, the same SNPs were associated to female longevity only (p values corrected for potential confounders = 0.03 and 0.02, respectively). Finally, as rs11555236 was reported to be in linkage disequilibrium with a putative functional enhancer within the SIRT3 gene, we assessed whether rs11555236 genotypes correlated with a different level of SIRT3 protein in peripheral blood mononuclear cells. We found an increased level of SIRT3 in subjects homozygous for the (T) allele. We suggest that SIRT3 genetic variability might be relevant for the modulation of human longevity in the Italian population.