Gene hypermethylation in blood leukocytes in humans long term after radiation exposure - Validation set.

Hypermethylation of СpG islands in the promoter regions of several genes with basic protective function in blood leukocytes of individuals exposed to ionizing radiation long time ago (2-46 years), and differential effects of age and radiation exposure on hypermethylation was reported in our previous work. To validate these results, epigenetic modifications were assessed in an independent series of 49 nuclear industry workers from the "Mayak" facility (67-84 years old at sampling) with documented individual accumulated doses from the prolonged external γ-radiation exposure (95.9-409.5 cGy, end of work with radiation:0.3-39 years ago), and in 50 non-exposed persons matched by age. In addition to the genes analyzed before (RASSF1A, p16/INK4A, p14/ARF, GSTP1), four additional loci were analyzed: TP53, ATM, SOD3, ESR1. The frequency of individuals displaying promoter methylation of at least one of the 8 genes (71.4%) was significantly higher in exposed group as compared to the control group (40%), p = .002, OR = 3.75. A significantly elevated frequency of individuals with hypermethylated СpG islands in GSTP1, TP53, SOD3 promoters was revealed among exposed subjects as compared to the control group (p = .012, OR = 8.41; p = .041, OR = 4.02 and p = .009, OR = 3.42, respectively). A similar trend (p = .12, OR = 3.06) was observed for the p16/INK4A gene. As a whole, p16/INK4A and GSTP1 promoter hypermethylation in irradiated subjects from both previously and currently analyzed groups was pronounced. Thus, the direction of the effects was fully confirmed, suggesting the result reproducibility. No statistically significant correlation between promoter methylation and individual radiation dose was found. Further studies are required to create an array of blood epigenetic markers of radiation exposure associating with premature aging and age-related diseases and to accurately evaluate radiation-added effect across the range of doses. SYNTHESIS: The results of studies of epigenetic changes in two independent samples of irradiated subjects indicated the significance of radiation factor in the induction of hypermethylation of CpG islands in gene promoters that is revealed in blood cells years and decades after exposure.

Genotype Analyses in the Japanese and Belarusian Populations Reveal Independent Effects of rs965513 and rs1867277 but Do Not Support the Role of FOXE1 Polyalanine Tract Length in Conferring Risk for Papillary Thyroid Carcinoma.

BACKGROUND: Several functional single-nucleotide polymorphisms (SNPs) at the FOXE1 locus on chromosome 9q22.33 have been associated with the risk for papillary thyroid carcinoma (PTC). This study set out to elucidate whether their effects are independent, using genotyping results in populations of Asian and European descent. METHODS: SNPs rs965513 and rs1867277 and a polymorphic region determining the length of the FOXE1 polyalanine (poly-Ala) tract were genotyped in 501 patients with PTC and 748 healthy individuals from Japan, and in 660 patients and 820 population controls from Belarus. Functional analysis of transactivation activities of FOXE1 isoforms with varying number of alanine repeats was performed by a Dual-Luciferase® Assay. RESULTS: All three polymorphisms were significantly associated with PTC in both populations on univariate analysis. However, conditional analysis revealed independent effects of rs965513 and rs1867277 SNPs but not of the FOXE1 poly-Ala polymorphism. The independent effect of the lead rs965513 SNP was observed in both populations, while that of rs1867277 was only identified in the Japanese population, in which linkage disequilibrium between the three polymorphisms is markedly weaker. Despite the strong decrease in transcriptional activity with increasing FOXE1 poly-Ala tract length, no difference in transactivation potential of the FOXE1 poly-Ala isoforms could be seen after adjustment for the minimal promoter activity in the reporter vectors. Plasmids encoding FOXE1 isoforms of increasing poly-Ala tract length were also found to produce less FOXE1 protein after cell transfection. CONCLUSIONS: The functional variants rs965513 and rs1867277 independently contribute to genetic predisposition to PTC, while a contributing role of the FOXE1 poly-Ala polymorphism could not be confirmed.

Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure.

Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24-77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19-77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2-51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62-11.76, p=3.9×10(-7)). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (ß=0.242; p=1.7×10(-5)). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (ß=0.290; p=1.7×10(-7)). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging and/or with the development of age-associated cancer and non-cancer diseases.

The association of PLA2G2A single nucleotide polymorphisms with type IIa secretory phospholipase A2 level but not its activity in patients with stable coronary heart disease.

BACKGROUND: Single nucleotide polymorphisms (SNPs) of the secretory phospholipase A2 type IIa (sPLA-IIa) gene (PLA2G2A) affect sPLA2-IIa level and activity in patients with diabetes mellitus, acute coronary syndrome or recent cardiovascular surgical interventions. Our study examined the effects of PLA2G2A SNPs on sPLA2-IIa levels and activity in patients with stable CHD. METHODS AND RESULTS: The study included a total of 396 patients (30% women). Six SNPs of PLA2G2A: rs1774131, rs11573156, rs3753827, rs2236771, rs876018, and rs3767221, sPLA2-IIa level and activity were determined for all patients. Four SNPs (rs1774131, rs11573156, rs3753827, rs3767221) correlated with sPLA2-IIa level but not activity with the strongest correlation observed for rs11573156 (r=0.49, p=3.7·10(-13)). All partial correlations controlling for rs11573156 became insignificant, whereas, the partial correlation of rs11573156 with sPLA2-IIa level controlling for other SNPs remained significant. Only rs11573156 showed association with sPLA2-IIa level in multiple regression analysis. Haplotype CGGGTT was associated with a significantly higher sPLA2-IIa level but not activity compared with all other haplotypes after adjustment for gender, age, diabetes mellitus and statin use (p=0.0023). CONCLUSIONS: According to our results the examined SNPs affect the sPLA2-IIa level to a greater extent than its activity in patients with stable CHD. It seems that, the impact of these SNPs on sPLA2-IIa level is caused by their linkage to rs11573156 whose minor alleles were associated with higher sPLA2-IIa level. At the same time haplotype CGGGTT, which includes the minor allele of rs11573156, was the dominant haplotype and was associated with the highest sPLA2-IIa level.

CYP1A1, GCLC, AGT, AGTR1 gene-gene interactions in community-acquired pneumonia pulmonary complications.

This study was conducted to establish the possible contribution of functional gene polymorphisms in detoxification/oxidative stress and vascular remodeling pathways to community-acquired pneumonia (CAP) susceptibility in the case-control study (350 CAP patients, 432 control subjects) and to predisposition to the development of CAP complications in the prospective study. All subjects were genotyped for 16 polymorphic variants in the 14 genes of xenobiotics detoxification CYP1A1, AhR, GSTM1, GSTT1, ABCB1, redox-status SOD2, CAT, GCLC, and vascular homeostasis ACE, AGT, AGTR1, NOS3, MTHFR, VEGFα. Risk of pulmonary complications (PC) in the single locus analysis was associated with CYP1A1, GCLC and AGTR1 genes. Extra PC (toxic shock syndrome and myocarditis) were not associated with these genes. We evaluated gene-gene interactions using multi-factor dimensionality reduction, and cumulative gene risk score approaches. The final model which included >5 risk alleles in the CYP1A1 (rs2606345, rs4646903, rs1048943), GCLC, AGT, and AGTR1 genes was associated with pleuritis, empyema, acute respiratory distress syndrome, all PC and acute respiratory failure (ARF). We considered CYP1A1, GCLC, AGT, AGTR1 gene set using Set Distiller mode implemented in GeneDecks for discovering gene-set relations via the degree of sharing descriptors within a given gene set. N-acetylcysteine and oxygen were defined by Set Distiller as the best descriptors for the gene set associated in the present study with PC and ARF. Results of the study are in line with literature data and suggest that genetically determined oxidative stress exacerbation may contribute to the progression of lung inflammation.

The potential effect of gender in CYP1A1 and GSTM1 genotype-specific associations with pediatric brain tumor.

Brain tumors are the common site for solid tumors in childhood. Very few studies have investigated genes with low penetrance in relation to pediatric brain tumor (pBT) development. Brain tumors do occur more frequently in males compared to females regardless of age, tumor histology, or region of the world. Taken into account these facts, we have designed a study aimed to analyse the contribution of some genetic factors to pBP in males and females. Patients with glial and embryonic brain tumors (160 males, 124 females) and healthy controls (277 males, 187 females) were included in the study. All subjects were genotyped for eight polymorphic variants in the genes of xenobiotics detoxification CYP1A1 (rs2606345, rs4646903, rs1048943), GSTM1 (Ins/del), GSTT1 (Ins/del), repair ERCC2 (rs1799793, rs13181), and folate pathway MTHFR (rs1801133). Genotype-specific risks of pBT were sex-dependent. GSTM1 deletion and dual deletions in GSTM1-GSTT1 loci were associated with brain tumor in males (P = 1.2 × 10(-5); odds ratio (OR) = 2.56; 95 % confidence interval (CI), 1.45-3.85 and P = 4.9 × 10(-4); OR = 3.09; 95 % CI, 1.63-5.89, relatively). The increased risk of brain tumors was evident for CYP1A1 rs2606345 (P = 0.0028; OR = 2.06; 95 % CI, 1.27-3.34) and minor haplotypes rs2606345-rs1048943-rs4646903 in females (global haplotype association P value, 0.0011). This study provides first evidence for the different pronounced pBT associations in males and females. This phenomenon possibly reflects the sexual dimorphism as an important determinant of brain tumor biology.

Functional polymorphisms in the CYP1A1, ACE, and IL-6 genes contribute to susceptibility to community-acquired and nosocomial pneumonia.

OBJECTIVES: To establish the contribution of genetic host factors to the risk of community-acquired pneumonia (CAP) and nosocomial pneumonia (NP) in the population of the Russian Federation. METHODS: A total of 796 subjects (CAP: 334 patients, 134 controls; NP: 216 critically ill patients with NP, 105 critically ill patients without NP) were included in two case-control studies. We analyzed 13 polymorphisms in 11 genes (IL-6, TNF-α, MBL2, CCR5, NOS3, CYP1A1 (three sites), GSTM1, GSTT1, ABCB1, ACE, and MTHFR) using a tetra-primer allele-specific PCR method. RESULTS: Individual single nucleotide polymorphism (SNP) analysis revealed a strong association between CYP1A1 rs2606345 and CAP (p=3.9 × 10(-5), odds ratio (OR) 0.42, 95% confidence interval (CI) 0.27-0.63). Three genes (CYP1A1, ACE, and IL-6) were identified that account for part of the increase in vulnerability to both diseases, CAP and NP. The carriage of three predisposing genotypes versus protective genotypes increased the CAP risk (p=0.001, OR 7.01, 95% CI 1.99-24.70) and NP risk (p=0.028, OR 4.34, 95% CI 1.15-16.45). CONCLUSIONS: Genetic predisposition to CAP and NP is attributed to the cumulative contribution of polymorphisms at the CYP1A1, IL-6, and ACE genes, independently of age, gender, causative pathogen, and the use of mechanical ventilation, in patients in the Russian Federation.

Host genetic risk factors for community-acquired pneumonia.

This study was conducted to establish the contribution of genetic host factors in the susceptibility to community acquired pneumonia (CAP) in the Russian population. Patients with CAP (n=334), volunteers without a previous history of CAP, constantly exposed to infectious agents, control A group (n=141) and a second control group B consisted of healthy persons (n=314) were included in the study. All subjects were genotyped for 13 polymorphic variants in the genes of xenobiotics detoxification CYP1A1 (rs2606345, rs4646903, and rs1048943), GSTM1 (Ins/del), GSTT1 (Ins/del), ABCB1 rs1045642); immune and inflammation response IL-6 (rs1800795), TNF-a (rs1800629), MBL2 (rs7096206), CCR5 (rs333), NOS3 (rs1799983), angiotensin-converting enzyme ACE (rs4340), and occlusive vascular disease/hyperhomocysteinemia MTHFR (rs1801133). Seven polymorphic variants in genes CYP1A1, GSTM1, ABCB1, NOS3, IL6, CCR5 and ACE were associated with CAP. For two genes CYP1A1 and GSTM1 associations remained significant after correction for multiple comparisons. Multiple analysis by the number of all risk genotypes showed a highly significant association with CAP (P=2.4×10(-7), OR=3.03, 95% CI 1.98-4.64) with the threshold for three risk genotypes. Using the ROC-analysis, the AUC value for multi-locus model was estimated as 68.38.

The dose and dose-rate effects of paternal irradiation on transgenerational instability in mice: a radiotherapy connection.

The non-targeted effects of human exposure to ionising radiation, including transgenerational instability manifesting in the children of irradiated parents, remains poorly understood. Employing a mouse model, we have analysed whether low-dose acute or low-dose-rate chronic paternal γ-irradiation can destabilise the genomes of their first-generation offspring. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm was established in DNA samples extracted from sperm of directly exposed BALB/c male mice, as well as from sperm and the brain of their first-generation offspring. For acute γ-irradiation from 10-100 cGy a linear dose-response for ESTR mutation induction was found in the germ line of directly exposed mice, with a doubling dose of 57 cGy. The mutagenicity of acute exposure to 100 cGy was more pronounced than that for chronic low-dose-rate irradiation. The analysis of transgenerational effects of paternal irradiation revealed that ESTR mutation frequencies were equally elevated in the germ line (sperm) and brain of the offspring of fathers exposed to 50 and 100 cGy of acute γ-rays. In contrast, neither paternal acute irradiation at lower doses (10-25 cGy), nor low-dose-rate exposure to 100 cGy affected stability of their offspring. Our data imply that the manifestation of transgenerational instability is triggered by a threshold dose of acute paternal irradiation. The results of our study also suggest that most doses of human exposure to ionising radiation, including radiotherapy regimens, may be unlikely to result in transgenerational instability in the offspring children of irradiated fathers.