A quantitative PCR method for measuring absolute telomere length.

We describe a simple and reproducible method to measure absolute telomere length (aTL) using quantitative real-time polymerase chain reaction (qPCR). This method is based on the Cawthon method for relative measurement of telomere length (TL) but modified by introducing an oligomer standard to measure aTL. The method describes the oligomer standards, the generation of the standard curve and the calculations required to calculate aTL from the qPCR data. The necessary controls and performance characteristics of the assay are described in detail and compared relative to other methods for measuring TL. Typical results for this assay for a variety of human tissue samples are provided as well as a troubleshooting schedule. This method allows high throughput measurement of aTL using small amounts of DNA making it amenable for molecular epidemiological studies. Compared to the traditional relative TL qPCR assays, the aTL method described in this protocol enables a more direct comparison of results between experiments within and between laboratories.

Grape seed polyphenols and curcumin reduce genomic instability events in a transgenic mouse model for Alzheimer's disease.

The study set out to determine (a) whether DNA damage is elevated in mice that carry mutations in the amyloid precursor protein (APP695swe) and presenilin 1 (PSEN1-dE9) that predispose to Alzheimer's disease (AD) relative to non-transgenic control mice, and (b) whether increasing the intake of dietary polyphenols from curcumin or grape seed extract could reduce genomic instability events in a transgenic mouse model for AD. DNA damage was measured using the micronucleus (MN) assay in both buccal mucosa and erythrocytes and an absolute telomere length assay for both buccal mucosa and olfactory bulb tissue. MN frequency tended to be higher in AD mice in both buccal mucosa (1.7-fold) and polychromatic erythrocytes (1.3-fold) relative to controls. Telomere length was significantly reduced by 91% (p=0.04) and non-significantly reduced by 50% in buccal mucosa and olfactory bulbs respectively in AD mice relative to controls. A significant 10-fold decrease in buccal MN frequency (p=0.01) was found for AD mice fed diets containing curcumin (CUR) or micro-encapsulated grape seed extract (MGSE) and a 7-fold decrease (p=0.02) for AD mice fed unencapsulated grape seed extract (GSE) compared to the AD group on control diet. Similarly, in polychromatic erythrocytes a significant reduction in MN frequency was found for the MGSE cohort (65.3%) (p<0.05), whereas the AD CUR and AD GSE groups were non-significantly reduced by 39.2 and 34.8% respectively compared to the AD Control. A non-significant 2-fold increase in buccal cell telomere length was evident for the CUR, GSE and MGSE groups compared to the AD control group. Olfactory bulb telomere length was found to be non-significantly 2-fold longer in mice fed on the CUR diet compared to controls. These results suggest potential protective effects of polyphenols against genomic instability events in different somatic tissues of a transgenic mouse model for AD.

Salivary α-amylase copy number is not associated with weight trajectories and glycemic improvements following clinical weight loss: results from a 2-phase dietary intervention study.

BACKGROUND: Several studies recently reported contradicting results regarding the link between amylase 1 (AMY1) copy numbers (CNs), obesity, and type 2 diabetes. OBJECTIVE: The aim of this study was to assess the impact of AMY1 CN on anthropometrics and glycemic outcomes in obese individuals following a 2-phase dietary weight loss intervention. METHODS: Using the paralog ratio test, AMY1 CNs were accurately measured in 761 obese individuals from the DiOGenes study. Subjects first underwent an 8-wk low-calorie diet (LCD, at 800 kcal/d) and then were randomly assigned to a 6-mo weight maintenance dietary (WMD) intervention with arms having different glycemic loads. RESULTS: At baseline, a modest association between AMY1 CN and BMI (P = 0.04) was observed. AMY1 CN was not associated with baseline glycemic variables. In addition, AMY1 CN was not associated with anthropometric or glycemic outcomes following either LCD or WMD. Interaction analyses between AMY1 CN and nutrient intake did not reveal any significant association with clinical parameters (at baseline and following LCD or WMD) or when testing gene × WMD interactions during the WMD phase. CONCLUSION: In the absence of association with weight trajectories or glycemic improvements, the AMY1 CN cannot be considered as an important biomarker for response to a clinical weight loss and weight maintenance programs in overweight/obese subjects. This trial was registered at www.clinicaltrials.gov as NCT00390637.

Effects of Intermittent Versus Continuous Energy Intakes on Insulin Sensitivity and Metabolic Risk in Women with Overweight.

OBJECTIVE: This study aimed to compare intermittent fasting (IF) versus continuous energy intakes at 100% or 70% of calculated energy requirements on insulin sensitivity, cardiometabolic risk, body weight, and composition. METHODS: Women with overweight (n = 88; 50 ± 1 years, BMI 32.3 ± 0.5 kg/m2 ) were randomized to one of four diets (IF70, IF100, dietary restriction [DR70], or control) in a 2:2:2:1 ratio for 8 weeks. IF groups fasted for 24 hours after breakfast on three nonconsecutive days per week. All foods were provided and diets matched for macronutrient composition (35% fat, 15% protein, 50% carbohydrate). Insulin sensitivity by hyperinsulinemic-euglycemic clamp, weight, body composition, and plasma markers were assessed following a "fed" day (12-hour fast) and a 24-hour fast (IF only). RESULTS: IF70 displayed greater reductions in weight, fat mass, total- and low-density lipoprotein cholesterol, and nonesterified fatty acids compared with DR70 and IF100 (all P ≤ 0.05). IF100 lost more weight and fat than control. However, fasting insulin was increased. There were no group differences in insulin sensitivity by clamp; however, a 24-hour fast transiently reduced insulin sensitivity. CONCLUSIONS: When prescribed at matched energy restriction, IF reduced weight and fat mass and improved total and low-density lipoprotein cholesterol more than DR. IF prescribed in energy balance did not improve health compared with other groups, despite modest weight loss.

Telomere length in white blood cells, buccal cells and brain tissue and its variation with ageing and Alzheimer's disease.

Changes in telomere length have been associated with ageing and with certain age-related degenerative diseases. We report results using a quantitative RTm-PCR method to measure absolute telomere length (in kb per diploid genome) and show the age-related changes in white blood cells and buccal cell telomere length (in kb per diploid genome) in normal healthy individuals and Alzheimer's patients. We observed a significantly lower telomere length in white blood cells (P < 0.0001) and buccal cells (P < 0.01) in Alzheimer's patients relative to healthy age-matched controls (31.4% and 32.3%, respectively). However, there was a significantly greater telomere length in hippocampus cells of Alzheimer's brains (P = 0.01) compared to control samples (49.0%). We also observed that telomere length in buccal cells was 52.2-74.2% shorter than that observed in white blood cells (P < 0.0001). The odd's ratio of being diagnosed with Alzheimer's disease (AD) was 10.8 (95% CI 1.19-97.85) if white blood cell telomere length was less than 115 kb per diploid genome with a specificity of 46% and sensitivity of 92.9%. The odds ratio for AD diagnosis was 4.6 (95% CI 1.22-17.16) if buccal cell telomere length was less than 40 kb per diploid genome with a sensitivity of 72.7% and a specificity of 63.1%. These results suggest important differences in telomere maintenance in Alzheimer's cases compared to healthy controls depending on sampled tissue. These results need to be replicated in larger studies and in cohorts of other neurodegenerative disorders to determine specificity of changes to Alzheimer's patients.

CBFA2T3 (MTG16) is a putative breast tumor suppressor gene from the breast cancer loss of heterozygosity region at 16q24.3.

Numerous cytogenetic and molecular studies of breast cancer have identified frequent loss of heterozygosity (LOH) of the long arm of human chromosome 16. On the basis of these data, the likely locations of breast cancer tumor suppressor genes are bands 16q22.1 and 16q24.3. We have mapped the CBFA2T3 (MTG16) gene, previously cloned as a fusion partner of the AML1 protein from a rare (16;21) leukemia translocation, to the 16q24.3 breast cancer LOH region. The expression of CBFA2T3 was significantly reduced in a number of breast cancer cell lines and in primary breast tumors, including early ductal carcinomas in situ, when compared with nontransformed breast epithelial cell lines and normal breast tissue. Reintroduction of CBFA2T3 into different breast tumor derived cell lines with decreased expression of this gene reduced colony growth on plastic and in soft agar. CBFA2T3 was shown to function as a transcriptional repressor when tethered to the GAL4 DNA-binding domain in a reporter gene assay and, therefore, has the potential to be a transcriptional repressor in normal breast epithelial cells. Taken together, these findings suggest that CBFA2T3 is a likely candidate for the breast cancer tumor suppressor gene that is the target for the frequent 16q24 LOH in breast neoplasms.

Absolute qPCR for measuring telomere length in bone marrow samples of plasma cell disorders.

Telomere length (TL) is currently used as an emerging biomarker in understanding the development/progression of hematological malignancies. The absolute quantitative PCR (qPCR) methodology has allowed the study of TL from a variety of mammalian tissues, but it has not been tested for bone marrow (BM) samples. In this study, we have examined the relationship between TL data generated by absolute qPCR versus those obtained by terminal restriction fragments (TRF) in 102 BM samples from patients with plasma cell disorders. A significant linear correlation between both methodologies was observed (p < 0.0001; r (2) = 0.70). Results were also analyzed in relation to clinical characteristics and significant associations between telomere shortening and parameters of adverse prognosis were observed. Furthermore, another set of 47 BM samples from patients with low quantity of DNA for TRF assay were suitably analyzed by qPCR, indicating the usefulness of the absolute qPCR methodology for the inclusion of patients with scarce material to the study. Taken together, these findings are of interest considering the importance of telomere dysfunction in the pathogenesis of cancer and give a new alternative to measure TL in hematologic disorders with substantial time and cost savings.

Blood micronutrients and DNA damage in children.

SCOPE: Maintenance of normal cellular phenotype depends largely on accurate DNA replication and repair. DNA damage causes gene mutations and predisposes to cancer and other chronic diseases. Growing evidence indicates that nutritional factors are associated with DNA damage in adults; here, we investigate these associations in children. METHODS AND RESULTS: We conducted a cross-sectional study among 462 healthy children 3, 6, and 9 years of age. Blood was collected and micronutrient levels were measured. The cytokinesis-block micronucleus cytome assay was used to measure chromosomal DNA damage (micronuclei, nucleoplasmic bridges, and nuclear buds) in lymphocytes. Cell apoptosis, necrosis, and the nuclear division index were also measured. Nine loci in genes involved in folate metabolism and DNA repair were genotyped. Data were analyzed using linear regression with adjustment for potential confounders. Plasma calcium was positively associated with micronuclei and necrosis, and α-tocopherol negatively associated with apoptosis, nuclear division index, and nucleoplasmic bridges; lutein was positively associated with nucleoplasmic bridges. α-tocopherol was positively associated with necrosis. CONCLUSION: DNA damage in healthy children may be influenced by blood micronutrient levels and certain genotypes. Further investigation of associations between nutritional status and genomic integrity in children is needed to shed additional light on potential mechanisms.

Folate deficiency induces dysfunctional long and short telomeres; both states are associated with hypomethylation and DNA damage in human WIL2-NS cells.

The essential role of dietary micronutrients for genome stability is well documented, yet the effect of folate deficiency or excess on telomeres is not known. Accordingly, human WIL2-NS cells were maintained in medium containing 30, 300, or 3,000 nmol/L folic acid (FA) for 42 days to test the hypothesis that chronic folate deficiency would cause telomere shortening and dysfunction. After 14 days, telomere length (TL) in FA-deficient (30 nmol/L) cultures was 26% longer than that of 3,000 nmol/L FA cultures; however, this was followed by rapid telomere attrition over the subsequent 28 days (P trend, P < 0.0001); both long and short telomere status was positively correlated with biomarkers of chromosome instability (P ≤ 0.003) and mitotic dysfunction (P = 0.01), measured by the cytokinesis-block micronucleus cytome (CBMN-cyt) assay. The early increase in TL was associated with FA-deficiency-induced global DNA hypomethylation (P = 0.05), with an effect size similar to that induced by the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. Quantitative PCR analysis indicated a negative association between FA concentration and uracil incorporation into telomeric DNA (r = -0.47, P = 0.1), suggesting a possible plausible mechanism for uracil as a cause of folate deficiency-induced telomere dysfunction or deletion. Peptide nucleic acid-FISH (PNA-FISH) analysis showed that FA deficiency resulted in 60% of micronuclei containing acentric terminal fragments, an observation consistent with the 3-fold increase in terminal deletions (P = 0.0001). Together, these results demonstrate the impact of folate deficiency on biomarkers of telomere maintenance and integrity, and provide evidence that dysfunctional long telomeres may be as important as critically short telomeres as a cause of chromosomal instability.

Colonocyte telomere shortening is greater with dietary red meat than white meat and is attenuated by resistant starch.

BACKGROUND & AIMS: Population studies indicate that greater red meat consumption increases colorectal cancer risk while dietary fibre is protective. Previous work in rats showed that diets high in protein, including red meat, increase colonocyte DNA strand breaks and that this effect is attenuated by resistant starches (RS). Telomeres are long hexamer repeats that protect against spontaneous DNA damage which would lead to chromosomal instability. Telomere shortening is associated with greater risk of colorectal cancer. The current study aimed to determine the effects of cooked red and white meat intake on colonocyte telomere length in rats and whether dietary RS modified their effects. METHODS: After four weeks of feeding cooked beef or chicken at 15, 25 and 35% of diet with or without RS, colonocyte telomere length was measured. RESULTS: Telomere length decreased in proportion to red meat content of the diet. A similar trend was observed in the white meat group. Colonocyte telomere shortening due to increased dietary meat was attenuated by the inclusion of RS. CONCLUSION: These data support previous findings of increased colonocyte DNA damage with greater red and white meat intake and also the protective effect of dietary fibre.

Telomere dynamics: the influence of folate and DNA methylation.

Since the suggestion of their existence, a wealth of literature on telomere biology has emerged aimed at solving the DNA end-underreplication problem identified by Olovnikov in 1971. Telomere shortening/dysfunction is now recognized as increasing degenerative disease risk. Recent studies have suggested that both dietary patterns and individual micronutrients--including folate--can influence telomere length and function. Folate is an important dietary vitamin required for DNA synthesis, repair, and one-carbon metabolism within the cell. However, the potential mechanisms by which folate deficiency directly or indirectly affects telomere biology has not yet been reviewed comprehensively. The present review summarizes recent published knowledge and identifies the residual knowledge gaps. Specifically, this review addresses whether it is plausible that folate deficiency may (1) cause accelerated telomere shortening, (2) intrinsically affect telomere function, and/or (3) cause increased telomere-end fusions and subsequent breakage-fusion-bridge cycles in the cell.

Weight loss in obese men is associated with increased telomere length and decreased abasic sites in rectal mucosa.

Telomere shortening may cause genome instability and is an initiating event in colorectal cancer (CRC). Obesity is associated with reduced telomere length in lymphocytes and is a risk factor for CRC, but the impact of obesity on telomere length in the rectal mucosa is unknown. The purpose of this study was to investigate the effect of weight loss, induced by calorie-restricted diets, on telomere length in the rectal mucosa of obese men. Midrectal biopsies were collected by sigmoidoscopy at three time points (at weeks 0, 12, and 52) during a programmed weight loss intervention. Weight was reduced by an average of 10.6 kg across the study. Telomere length, measured by quantitative real-time PCR (qPCR), was negatively correlated with body mass index (BMI) (r = -0.13, p = 0.05) at baseline (n = 54) and increased at week 12 (four-fold increase) and week 52 (10-fold increase) (analysis of covariance [ANCOVA] p = 0.01, n = 12). Abasic sites in DNA decreased at week 12 (30% decrease) and week 52 (65% decrease) (analysis of variance [ANOVA] p = 0.02). Furthermore, gain of telomere length appeared to be greater if more weight and body fat was lost (r = -0.65, p = 0.01 and r = -0.56, p = 0.01, respectively). These results suggest that weight loss by caloric-restricted diets may contribute to the prevention of telomere shortening and DNA base damage, which are important initiating events in carcinogenesis.

Telomere length in lymphocytes of older South Australian men may be inversely associated with plasma homocysteine.

Deficiencies in folate (FOL) and vitamin B12 (B12) result in increased chromosomal aberrations, a validated biomarker of cancer risk. Telomeres, the regions of DNA that cap the ends of each chromosome, are critical for maintaining chromosomal stability but the impact of micronutrients on telomere structure and function remains unclear. We hypothesized that telomere length maintenance might be compromised if the status of FOL and B12 was inadequate and plasma homocysteine (HCY) was increased. We investigated the relationship between telomere length in peripheral blood lymphocytes and plasma FOL, B12, and HCY status, and tested whether any such relationship was dependent on age, gender, body mass index, and common polymorphisms in folate metabolism genes. A single blood sample was collected from 43 younger (18-32 years) and 47 older (65-83 years) adults in South Australia. The younger cohort consisted of 18 males and 25 females, whereas the older group included 24 males and 23 females. Telomere length was determined in lymphocytes by flow cytometry. Telomere length in the younger cohort was 11.52% greater than in the older cohort (p = 0.015). In the older cohort, telomere length in females was 12.5% greater than in males (p = 0.028). In older males, there was a significant inverse correlation between telomere length and HCY (r = -0.57, p = 0.004), but this effect was not observed in the younger cohort or in the older female group. These results provide evidence that telomere length of lymphocytes in older men may be adversely affected by HCY in vivo.

Lack of association of the CD14 promoter polymorphism--159C/T with Caucasian inflammatory bowel disease.

OBJECTIVE: The inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), are multifactorial diseases resulting from a complex interaction of genetic and environmental factors. The recently described CARD15 and TNF-alpha risk alleles are believed to be contributors to disease by disrupting inflammatory pathways via impaired response to bacteria. Other bacterial receptors, such as CD14, may also have a role in disease. A promoter polymorphism (-159C/T) in CD14 has been implicated in IBD in a number of studies. MATERIAL AND METHODS: We have analysed this CD14 promoter polymorphism in probands from 206 multiplex IBD families, 110 sporadic IBD individuals and 189 healthy controls from the Australian population, all of whom are Caucasian. RESULTS: We could not replicate the described association between the CD14-159T allele and CD or UC, nor did we find any evidence for an interaction between the CARD15 or TNF-alpha risk alleles and the CD14-159T allele. CONCLUSIONS: It is possible that the association seen in other studies may be due to population stratification or to the CD14 polymorphism being in linkage with the real disease-causing variant(s).