An in-depth analysis of the mitochondrial phylogenetic landscape of Cambodia.

Cambodia harbours a variety of human aboriginal populations that have scarcely been studied in terms of genetic diversity of entire mitochondrial genomes. Here we present the matrilineal gene pool of 299 Cambodian refugees from three different ethnic groups (Cham, Khmer, and Khmer Loeu) deriving from 16 Cambodian districts. After establishing a DNA-saving high-throughput strategy for mitochondrial whole-genome Sanger sequencing, a HaploGrep based workflow was used for quality control, haplogroup classification and phylogenetic reconstruction. The application of diverse phylogenetic algorithms revealed an exciting picture of the genetic diversity of Cambodia, especially in relation to populations from Southeast Asia and from the whole world. A total of 224 unique haplotypes were identified, which were mostly classified under haplogroups B5a1, F1a1, or categorized as newly defined basal haplogroups or basal sub-branches of R, N and M clades. The presence of autochthonous maternal lineages could be confirmed as reported in previous studies. The exceptional homogeneity observed between and within the three investigated Cambodian ethnic groups indicates genetic isolation of the whole population. Between ethnicities, genetic barriers were not detected. The mtDNA data presented here increases the phylogenetic resolution in Cambodia significantly, thereby highlighting the need for an update of the current human mtDNA phylogeny.

Results from the German Chronic Kidney Disease (GCKD) study support association of relative telomere length with mortality in a large cohort of patients with moderate chronic kidney disease.

Telomere length is known to be inversely associated with aging and has been proposed as a marker for aging-related diseases. Telomere attrition can be accelerated by oxidative stress and inflammation, both commonly present in patients with chronic kidney disease. Here, we investigated whether relative telomere length is associated with mortality in a large cohort of patients with chronic kidney disease stage G3 and A1-3 or G1-2 with overt proteinuria (A3) at enrollment. Relative telomere length was quantified in peripheral blood by a quantitative PCR method in 4,955 patients from the GCKD study, an ongoing prospective observational cohort. Complete four-year follow-up was available from 4,926 patients in whom we recorded 354 deaths. Relative telomere length was a strong and independent predictor of all-cause mortality. Each decrease of 0.1 relative telomere length unit was highly associated with a 14% increased risk of death (hazard ratio1.14 [95% confidence interval 1.06-1.22]) in a model adjusted for age, sex, baseline eGFR, urine albumin/creatinine ratio, diabetes mellitus, prevalent cardiovascular disease, LDL-cholesterol, HDL-cholesterol, smoking, body mass index, systolic and diastolic blood pressure, C-reactive protein and serum albumin. This translated to a 75% higher risk for those in the lowest compared to the highest quartile of relative telomere length. The association was mainly driven by 117 cardiovascular deaths (1.20 [1.05-1.35]) as well as 67 deaths due to infections (1.27 [1.07-1.50]). Thus, our findings support an association of shorter telomere length with all-cause mortality, cardiovascular mortality and death due to infections in patients with moderate chronic kidney disease.

Influence of DNA extraction methods on relative telomere length measurements and its impact on epidemiological studies.

Measurement of telomere length is widely used in epidemiologic studies. Insufficient standardization of the measurements processes has, however, complicated the comparison of results between studies. We aimed to investigate whether DNA extraction methods have an influence on measured values of relative telomere length (RTL) and whether this has consequences for epidemiological studies. We performed four experiments with RTL measurement in quadruplicate by qPCR using DNA extracted with different methods: 1) a standardized validation experiment including three extraction methods (magnetic-particle-method EZ1, salting-out-method INV, phenol-chloroform-isoamyl-alcohol PCI) each in the same 20 samples demonstrated pronounced differences in RTL with lowest values with EZ1 followed by INV and PCI-isolated DNA; 2) a comparison of 307 samples from an epidemiological study showing EZ1-measurements 40% lower than INV-measurements; 3) a matching-approach of two similar non-diseased control groups including 143 pairs of subjects revealed significantly shorter RTL in EZ1 than INV-extracted DNA (0.844 ± 0.157 vs. 1.357 ± 0.242); 4) an association analysis of RTL with prevalent cardiovascular disease detected a stronger association with INV than with EZ1-extracted DNA. In summary, DNA extraction methods have a pronounced influence on the measured RTL-values. This might result in spurious or lost associations in epidemiological studies under certain circumstances.

Do telomeres have a higher plasticity than thought? Results from the German Chronic Kidney Disease (GCKD) study as a high-risk population.

Telomere length is considered as a biological marker for aging. It is expected that telomeres shorten with age and with conditions associated with oxidative stress and inflammation. Both are present in patients with chronic kidney disease (CKD) who have a very high cardiovascular risk. We investigated whether CKD duration is associated with relative telomere length (RTL) in 4802 patients from the German Chronic Kidney Disease (GCKD) study. We measured RTL in each sample in quadruplicates using a quantitative polymerase chain reaction (qPCR). We observed a U-shaped association of RTL with CKD duration: the longest RTL was found in those 339 patients who reported the shortest disease duration (<6 months) and shorter RTL in 2108 patients with duration between 6 months and less than 5 years. Most importantly, those 2331 patients who reported a CKD duration of 5 years and more had significantly longer RTL compared to those with intermediate CKD duration (6 months to less than 5 years): mean 0.954, 95%CI 0.946-0.961 versus 0.937, 95%CI 0.929-0.944, p=0.002). Due to the cross-sectional nature of the study these surprising results have to be considered with caution and as hypothesis-generating. Whether the longer RTL in patients with long-lasting disease is caused by an activation of telomerase to counteract the shortening of RTL due to oxidative stress and inflammation or whether they are caused by a survival bias needs to be investigated in longitudinal studies. Our data are in support of a higher plasticity of shortening and elongations of RTL as until recently anticipated.

Association of relative telomere length with cardiovascular disease in a large chronic kidney disease cohort: the GCKD study.

BACKGROUND: Chronic kidney disease (CKD) affects 10-15% of the general population and affected individuals are at an increased risk for cardiovascular disease (CVD). Since telomere length is considered to be involved in biological aging, we tested whether relative telomere length (RTL) might be a marker for these two diseases. METHODS: The German Chronic Kidney Disease (GCKD) study is an ongoing prospective cohort study including patients with CKD of moderate severity. RTL was measured by qPCR in 4955 out of 5217 GCKD patients at baseline. RESULTS: RTL was distributed in the cohort with a mean ± SD of 0.95 ± 0.19. CVD was present in 1266 patients. Each decrease of RTL by 0.1 unit was associated with a higher probability for prevalent CVD: OR = 1.06, 95% CI 1.02-1.11, p = 0.007 (adjusted for age, sex, eGFR, BMI, ln-CRP, smoking, hypertension, diabetes, and lipids). Similar findings were observed for history of specific CVD entities, such as coronary artery disease (OR = 1.05, p = 0.025), myocardial infarction (OR = 1.08, p = 0.013) and percutaneous transluminal coronary angioplasty (OR = 1.06, p = 0.032). The strongest associations were found for interventions at the carotid arteries (OR = 1.25, p = 0.001) as well as aortic aneurysms (OR = 1.22, p = 0.001). CONCLUSIONS: In the presence of CKD there is a significant association between shorter RTL and CVD manifestations. RTL appears to be a marker reflecting changes in homeostasis associated with CKD that may contribute to the excess CVD risk.

Association of relative telomere length with progression of chronic kidney disease in two cohorts: effect modification by smoking and diabetes.

Chronic kidney disease (CKD) is a highly progressive disease. We studied the association between relative telomere length (RTL) and CKD progression and tested whether this association is modified by smoking and diabetes mellitus. RTL was measured by qPCR in two prospective cohort studies, the MMKD-Study (n = 166) and the CRISIS-Study (n = 889) with a median follow-up of 4.5 and 2.8 years, respectively. Progression was defined as doubling of baseline serum creatinine (MMKD-Study) and/or end stage renal disease (both studies). 59 and 105 of the patients from MMKD and CRISIS experienced a progression of CKD. Mean standardized pooled RTL was 0.74 ± 0.29. In the meta-analysis shorter RTL at baseline showed a borderline association with CKD progression (HR = 1.07 [95%CI 1.00-1.15]; p = 0.06). We observed an effect modification of RTL and CKD progression by smoking and diabetes (p-values of interaction p = 0.02 and p = 0.09, respectively). Each 0.1 unit shorter RTL was significantly associated with an increased hazard for CKD progression in active-smokers by 44% (HR = 1.44 [1.16-1.81]; p = 0.001) and in patients with diabetes mellitus by 16% (HR = 1.16 [1.01-1.34]; p = 0.03). Estimates were adjusted for baseline age, sex, proteinuria and GFR. This study in two independent cohorts reinforces that RTL is a marker and potentially a pathogenetic factor for CKD progression.

Leucocyte telomere length and risk of type 2 diabetes mellitus: new prospective cohort study and literature-based meta-analysis.

BACKGROUND: Short telomeres have been linked to various age-related diseases. We aimed to assess the association of telomere length with incident type 2 diabetes mellitus (T2DM) in prospective cohort studies. METHODS: Leucocyte relative telomere length (RTL) was measured using quantitative polymerase chain reaction in 684 participants of the prospective population-based Bruneck Study (1995 baseline), with repeat RTL measurements performed in 2005 (n = 558) and 2010 (n = 479). Hazard ratios for T2DM were calculated across quartiles of baseline RTL using Cox regression models adjusted for age, sex, body-mass index, smoking, socio-economic status, physical activity, alcohol consumption, high-density lipoprotein cholesterol, log high-sensitivity C-reactive protein, and waist-hip ratio. Separate analyses corrected hazard ratios for within-person variability using multivariate regression calibration of repeated measurements. To contextualise findings, we systematically sought PubMed, Web of Science and EMBASE for relevant articles and pooled results using random-effects meta-analysis. RESULTS: Over 15 years of follow-up, 44 out of 606 participants free of diabetes at baseline developed incident T2DM. The adjusted hazard ratio for T2DM comparing the bottom vs. the top quartile of baseline RTL (i.e. shortest vs. longest) was 2.00 (95% confidence interval: 0.90 to 4.49; P = 0.091), and 2.31 comparing the bottom quartile vs. the remainder (1.21 to 4.41; P = 0.011). The corresponding hazard ratios corrected for within-person RTL variability were 3.22 (1.27 to 8.14; P = 0.014) and 2.86 (1.45 to 5.65; P = 0.003). In a random-effects meta-analysis of three prospective cohort studies involving 6,991 participants and 2,011 incident T2DM events, the pooled relative risk was 1.31 (1.07 to 1.60; P = 0.010; I2 = 69%). CONCLUSIONS/INTERPRETATION: Low RTL is independently associated with the risk of incident T2DM. To avoid regression dilution biases in observed associations of RTL with disease risk, future studies should implement methods correcting for within-person variability in RTL. The causal role of short telomeres in T2DM development remains to be determined.

Association of adiponectin and leptin with relative telomere length in seven independent cohorts including 11,448 participants.

Oxidative stress and inflammation are major contributors to accelerated age-related relative telomere length (RTL) shortening. Both conditions are strongly linked to leptin and adiponectin, the most prominent adipocyte-derived protein hormones. As high leptin levels and low levels of adiponectin have been implicated in inflammation, one expects adiponectin to be positively associated with RTL while leptin should be negatively associated. Within the ENGAGE consortium, we investigated the association of RTL with adiponectin and leptin in seven independent cohorts with a total of 11,448 participants. We performed partial correlation analysis on Z-transformed RTL and LN-transformed leptin/adiponectin, adjusting for age and sex. In extended models we adjusted for body mass index (BMI) and C-reactive protein (CRP). Adiponectin showed a borderline significant association with RTL. This appeared to be determined by a single study and when the outlier study was removed, this association disappeared. The association between RTL and leptin was highly significant (r = -0.05; p = 1.81 × 10(-7)). Additional adjustment for BMI or CRP did not change the results. Sex-stratified analysis revealed no difference between men and women. Our study suggests that high leptin levels are associated with short RTL.

The association of relative telomere length with symptomatic peripheral arterial disease: results from the CAVASIC study.

BACKGROUND AND OBJECTIVES: Short telomere length has been described to be associated with biological aging including atherosclerosis phenotypes. However, information in patients with symptomatic peripheral arterial disease (PAD) is sparse. We therefore aimed to investigate whether inter-individual differences in relative telomere length (RTL) are associated with symptomatic PAD. DESIGN: We measured RTL by a quantitative PCR method in the CAVASIC Study, a cohort of 241 male Caucasian patients diagnosed with intermittent claudication and 249 age- and diabetes-matched controls. RESULTS: We observed significantly shorter mean RTL in patients than in controls (1.24 ± 0.19 vs. 1.32 ± 0.23, p < 0.001). Each shortening of RTL by one standard deviation significantly increased the odds for PAD by 44%: age-adjusted OR = 1.44 (95%CI 1.19-1.75, p < 0.001). This association remained significant after additional adjustment for log-C-reactive protein, glomerular filtration rate, HDL cholesterol, current smoking and log N-terminal pro-B-type natriuretic peptide (NT-proBNP). Excluding patients with prevalent cardiovascular disease revealed very similar results. When we compared the model fit of the various adjustment models including cardiac risk factors and/or NT-proBNP the addition of RTL significantly improved discrimination between patients and controls. CONCLUSION: This study in a male cohort of patients with intermittent claudication and age- and diabetes-matched controls indicates a significant association of shorter relative telomere length with PAD. Our results reinforce RTL as a marker for PAD that reflects the influence of genetic and environmental risk factors. Moreover, the association remains significant after excluding patients and controls free from prevalent cardiovascular disease.