Age-associated increase of thyroid hormone receptor ß gene promoter methylation coexists with decreased gene expression.

PURPOSE: It is not established if healthy aging of the thyroid axis is associated with alterations other than changes in hormone secretion. METHODS: The expression of thyroid hormone receptor ß gene (THRB) was analyzed in peripheral blood mononuclear cells (PBMC) obtained from young, elderly, and long-lived individuals. The interaction between the 3'UTR of TRß1 mRNA and selected miRNAs was measured using pmirGLO reporter vector. Methylation of the THRB CpG island was analyzed using methylation-sensitive restriction/RT-PCR and bisulfite sequencing methods. RESULTS: Old age was associated with a significantly lower amount of total TRß mRNA (p = 0.033) and of TRß1 mRNA (p = 0.02). Older age was also associated with significantly higher methylation of the THRB promoter (restriction/RT-PCR: p = 0.0023, bisulfite sequencing: p = 0.0004). Higher methylation corresponded to a lower expression of the THRB mRNA, but this correlation did not reach the level of significance. miR-26a interacted with two sites in the 3'UTR of the TRß1 mRNA leading to the decrease of the reporter protein activity (p < 0.0001 and p = 0.0005), and miR-496 interacted with one of the two putative binding sites which also decreased the reporter protein activity (p < 0.0001). Analysis of the expression of miR-21, miR-26a, miR-146a, miR-181a, miR-221, and miR-496 showed that the expression of miR-26a was significantly decreased in old subjects (p = 0.017), while the levels of other miRNAs were unaffected. CONCLUSIONS: Age-related decrease of THRB expression in PBMC of elderly and long-lived humans might be, in part, a result of the increased methylation of its promoter, but is unrelated to the activity of the miRNAs analyzed here.

miR-96, miR-145 and miR-9 expression increases, and IGF-1R and FOXO1 expression decreases in peripheral blood mononuclear cells of aging humans.

BACKGROUND: In mammals, the IGF-1 pathway affects the phenotype of aging. Since the function of the immune system is modulated by IGF-1, it is plausible that immunosenescence might in part result from altered control by this pathway. We therefore examined whether the expression of IGF-1R, FOXO1, and FOXO3a in peripheral blood mononuclear cells (PBMC) changes with age and if this might be due to changes in the expression of select miRNAs. METHODS: The expression of IGF-1R, FOXO1, FOXO3a, as well as of miR-9, miR-96, miR-99a, miR-132, miR-145, and miR-182 was examined in PBMC of young (27.8 ± 3.7 years), elderly (65.6 ± 3.4 years), and long-lived (94.0 ± 3.7 years) Polish Caucasians using real-time PCR. mRNA/miRNA interactions were studied in HEK 293 cells using luciferase-expressing pmirGLO reporter vector. RESULTS: The median expression of IGF-1R decreased with age (p < 0.000001), as did the expression of FOXO1 (p < 0.000001), while the expression of FOXO3a remained stable. We also found an age-associated increase of the median expression of miR-96 (p = 0.002), miR-145 (p = 0.024) and miR-9 (p = 0.026), decrease of the expression of miR-99a (p = 0.037), and no changes regarding miR-132 and miR-182. Functional studies revealed that miR-96 and miR-182 interacted with human IGF-1R mRNA, and that miR-145 and miR-132 interacted with human FOXO1 mRNA. CONCLUSIONS: The age-associated higher expression of miR-96 and miR-145 might contribute to the lower expression of IGF-1R while the higher expression of miR-96, miR-145 and miR-9 might contribute to the lower expression of FOXO1 in peripheral blood mononuclear cells of aging humans. Sustained expression/function of FOXO3a but not of the other two genes might be important for the maintenance of the immune system function in these individuals.

Decreased expression and the Lys751Gln polymorphism of the XPD gene are associated with extreme longevity.

Aging is associated with progressing genomic instability. The XPD gene encodes a DNA helicase involved in nucleotide excision repair and in transcription. We analyzed the common XPD polymorphisms that were previously shown to affect protein's DNA repair efficiency and to increase the risk of developing various cancers. Analysis was performed in 149 centenarians (mean age 101.1 years old) and in 413 young subjects (mean age 27.1 years old). We showed that the distribution of the Lys751Gln genotypes differed significantly between these groups (P = 0.017). In centenarians, the homozygous genotypes AA and CC were found less frequently than in young controls (29 vs. 36%, OR = 0.71, and 14 vs. 20%, OR = 0.652, respectively). The Arg156Arg and Asp312Asn were not significantly associated with extreme longevity. Analysis of the XPD mRNA level in blood mononuclear cells of people divided into three age groups (mean ages 28.7, 65.8 and 92.7 years old) showed that extreme longevity is associated with the decrease of the mean level of the specific mRNA; the differences between young or middle-aged vs. extremely old group were significant (P < 0.0001, P < 0.0001, respectively). In addition, the methylation pattern of the XPD promoter was analyzed in 30 people divided into three age groups (29.5, 65.9, and 101.4 years old). We showed that overall methylation of the XPD promoter is a rare event; however, aging is associated with the increase of methylation level upstream of the transcription start site. In summary, we showed for the first time that both the XPD polymorphic variants and the decreased level of its expression might be associated with aging.

ESR2 gene G1730A variant is associated with triglycerides level and myocardial infarction in young men but not in women.

OBJECTIVES: The aim of the study was to investigate the role of estrogen receptor type 2 gene (ESR2) variant G1730A in myocardial infarction (MI) in young age. METHODS: Genotyping was performed with restriction fragments length polymorphism method in 158 patients (79.1% men) with MI aged <50 years (studied group) and in control groups: 150 healthy individuals aged <50 years (63.3% men) and 202 patients (64.3% men) with MI aged ≥50 years. RESULTS: The AA genotype of ESR2 G1730A variant was significantly more frequent in men with MI aged <50 comparing to men with MI aged ≥50 (21.6% vs. 8.4%, P = 0.004) and to healthy young men (21.6% vs. 11.6%, P = 0.048). There was statistically significant difference between AA genotype and GA + GG genotypes male carriers with MI aged <50 in median triglyceride (TG) level (2.0 vs. 1.7 mmol/l respectively, p = 0.023). CONCLUSIONS: Our findings suggest a possible role of ESR2 G1730A variant as the risk factor of MI in a young age not as an independent but a potential risk factor associated with TG level in men but not in women.

Age-related changes of leptin and leptin receptor variants in healthy elderly and long-lived adults.

AIM: Aging is usually associated with hyperleptinemia and leptin resistance, both increasing the risk of age-related diseases. It was relevant to establish if healthily aging, non-obese individuals develop changes in leptin, the soluble leptin receptor (OB-Re), free leptin index (FLI), in methylation of the leptin receptor gene (LEPR) promoter, and in the expression of long (OB-Rb) and short (OB-Ra) leptin receptor isoforms. METHODS: We analyzed these parameters in 38 young (aged 26.8 ± 3.6 years), 37 elderly (aged 64.7 ± 3.1 years) and 39 long-lived (aged 94.2 ± 3.7 years) healthy, non-obese Polish Caucasians. RESULTS: In elderly men, the median concentration of leptin and the median FLI were significantly higher than in young men (P = 0.009 and P = 0.007, respectively), which was probably partly due to a higher mean body mass index of the elderly study participants. In peripheral blood mononuclear cells, the expression of functionally active OB-Rb did not depend on age or sex, whereas the expression of OB-Ra was lower in the elderly and long-lived groups than in the young group (P < 0.0001 and P = 0.002, respectively), mostly due to changes observed in women. Most likely, this age-related decrease was not due to hypermethylation of the LEPR promoter, as methylation of the +20 to +281 fragment of the CpG island did not change with age. CONCLUSIONS: In healthy, non-obese individuals, only some elements of the leptin axis slightly change with age. On that basis, we suggest that proper function of this axis might be required for this particular phenotype of aging. The present results should, however, be replicated in prospective studies and in other ethnic groups.

Functional polymorphisms of the leptin and leptin receptor genes are associated with longevity and with the risk of myocardial infarction and of type 2 diabetes mellitus.

INTRODUCTION: Longevity is commonly associated with good health and with delayed onset of age-related diseases with usually benign course. Leptin (LEP) significantly affects metabolism and numerous functions of the organism. To find out if extreme longevity and its phenotype are associated with genetic variants of leptin and leptin receptor (LEPR) genes, we analysed the frequencies of the -2548 G/A and +19 G/A LEP, as well as the K109R, Q223R, and K656N LEPR polymorphisms in centenarians and in control groups. MATERIAL AND METHODS: The frequencies of the LEP and LEPR polymorphisms were tested by restriction fragment length polymorphism in 128 centenarians, 414 young controls (Y), 226 myocardial infarction (MI) patients, and 190 type 2 diabetes mellitus (DM2) patients. RESULTS: The GG genotype of the -2548 G/A LEP polymorphism was significantly more common in centenarians than in the Y, MI and DM2 groups (p = 0.048, p = 0.003, p = 0.049, respectively). In addition, the AA genotype of the K109R LEPR polymorphism was significantly less frequent in centenarians than in the Y, MI, and DM2 groups (p = 0.026, p = 0.013, and p = 0.001, respectively). CONCLUSIONS: We suggest that the leptin pathway plays a role in the regulation of longevity, possibly by modulating the risk of development of MI and of DM2.

Total and high molecular weight adiponectin and level-modifying polymorphisms of ADIPOQ in centenarians.

INTRODUCTION: Adiponectin demonstrates a protective role against the development of obesity, type 2 diabetes mellitus, and cardiovascular disease. The -11377C 〉 G, -11391G 〉 A, and -11426A 〉 G promoter polymorphisms of ADIPOQ gene influence the level of circulating adiponectin. We examined the level of total and high molecular weight (HMW) adiponectin in centenarians and associated it with biochemical parameters. We checked if the expression and concentration-modifying polymorphisms of ADIPOQ are associated with extreme longevity. MATERIAL AND METHODS: Total and HMW adiponectin were examined using ELISA in 40 female centenarians. The frequencies of the ADIPOQ polymorphisms were tested by restriction fragment length polymorphism in 148 centenarians, 414 young controls, in 207 myocardial infarction patients, and in 190 type 2 diabetes mellitus patients. RESULTS: The mean concentration of total adiponectin in centenarians was 13.19 ± 1.37 mg/mL and of HMW adiponectin it was 9.17 ± 1.15 mg/mL. They were positively correlated with HDL (r = 0.4696, p = 0.0025 and r = 0.3912, p = 0.015, respectively), and negatively with BMI (r = -0.3702, p = 0.034 and r = -0.3963, p = 0.025) and triglycerides (r = -0.346, p = 0.028 and r = -0.3227, p = 0.045). A very rare AA genotype of the -11391G 〉 A polymorphism was significantly more common in centenarians than in young controls (p = 0.026) and, while compared to the GG genotype, it was associated with a 2.4-fold higher mean concentration of total adiponectin (26.53 ± 13.29 mg/ mL v. 10.97 ± 4.28 mg/mL) and with an almost 3-fold higher mean HMW adiponectin (20.65 ± 12.72 mg/mL v. 7.36 ± 3.35 mg/mL). CONCLUSIONS: Serum adiponectin concentration in female centenarians is associated with biochemical parameters that are favourable for cardiovascular risk. We suggest that adiponectin might be of importance for extreme longevity.

Aging is accompanied by a progressive decrease of expression of the WRN gene in human blood mononuclear cells.

The WRN gene encodes DNA helicase participating in genome maintenance. We looked for associations of natural aging with expression and methylation of this gene in blood mononuclear cells and with its common polymorphisms. Analyses were performed in ethnically homogenous Polish Caucasians. The mean level of the WRN messenger RNA was significantly lower in long-living individuals than in young and middle-aged controls (p < .001 and p = .025, respectively). Analysis of the 361 bp WRN promoter CpG island showed that aging might be accompanied by a slight increase of its methylation status; however, it seems to be biologically insignificant. Finally, analysis of the WRN R834C, L1074F, and C1367R polymorphisms showed that the frequencies of the L1074F and C1367R polymorphisms were similar in all age groups tested, whereas the R834C polymorphism was absent from Polish Caucasians. We suggest that age-related decrease of the WRN expression but not its common genetic variants might contribute to human immunosenescence.

The -351A/G polymorphism of ESR1 is associated with risk of myocardial infarction but not with extreme longevity.

BACKGROUND: Women live longer than men. Some possible reasons for this advantage are the protection provided by high concentrations of 17ß-estradiol (E2) during the premenopausal period and polymorphic variants of the estrogen receptors (ERs), which mediate various cardiovascular functions of E2. METHODS: We tested whether the -351A/G and -397T/C polymorphisms of the ERα-encoding ESR1 were associated with extreme longevity. The genomic DNA of 148 centenarians (C), 414 young controls (Y), and 208 myocardial infarction patients (MI) was analyzed by RFLP-PCR. RESULTS: Both polymorphisms were equally distributed in the Y, C, and in centenarians never diagnosed with MI (HC). In centenarians, none of these polymorphisms was associated with a particular lipid profile. The AA genotype of the -351A/G polymorphism was less frequent in the C, HC and Y groups than in MI patients (p=0.058, p=0.021, and p=0.004, respectively). In MI patients, the GG genotype of the -351A/G polymorphism was associated with significantly lower mean total cholesterol, LDL, and HDL levels compared to the AG (p=0.0194, p=0.0213, and p=0.0367, respectively) and AA genotypes (p=0.0014, p=0.0078, and p=0.0448, respectively). CONCLUSIONS: The -351A/G ESR1 polymorphism might be associated with MI, but not with extreme longevity.