Data sources and study sample

Data came from the follow-up phase of a two-wave population-based longitudinal study of CFS and fatiguing illness in Georgia, USA, conducted in 2004 and 2009 [2, 20]. The study was approved by the Institutional Review Boards of the Centers for Disease Control & Prevention, Atlanta, GA and Abt Associates, Atlanta, GA. All participants gave written informed consent for participating in the study.

The current analysis focused on data from the clinical evaluation of the follow-up phase, which included a detailed medical history, physical exam, laboratory tests, and psychiatric evaluation via the Structured Clinical Interview for DSM Disorders (SCID). Participants also completed a number of questionnaires including a demographics form, the 20-item Multidimensional Fatigue Inventory (MFI-20) [21], the 36-item Short Form Health Survey (SF-36 v2) [22, 23], and the CDC Symptom Inventory (SI) [24]. Demographic information was collected during a phone interview and further confirmed at the in-person clinical evaluation. Vital signs including heart rate (bpm), systolic and diastolic blood pressure (mmHg), height, weight, and waist and hip circumferences were collected at the clinic as part of the physical exam. The derived measures, body mass index (BMI) and waist to hip ratio (WHR) were calculated [BMI = weight/height2 (kg/m2)]; [WHR = waist/hip (cm/cm)]. The analysis included biological measures from Quest Laboratory test results such as HDL cholesterol (mg/dL), triglycerides (mg/dL), fasting glucose (mg/dL), insulin (µIU/mL), C-reactive protein (CRP, mg/dL), and albumin (g/dL).

Participants were classified as CFS if they met the 1994 international research definition applied using previously described methods [2]. Those with unexplained chronic illness with insufficient symptoms/fatigue to meet all of the criteria for CFS were grouped as insufficient symptoms/fatigue (termed as ISF). Participants meeting none of the criteria for CFS were grouped as non-fatigued (NF) controls. Additionally, participants in each group were broken into those with and without exclusionary medical/psychiatric conditions [25]. Of the 751 participants who were clinically evaluated in the GA CFS surveillance study, 71 met all the criteria for CFS without exclusionary conditions (CFS group), 78 met all the criteria for CFS but with exclusionary conditions (CFS-X group), 340 met the criteria for ISF regardless of exclusionary conditions, 212 met the criteria for NF controls without exclusionary conditions, 47 met the criteria for NF with exclusions and 3 participants were classified as “indeterminate” due to incomplete information for case definition criteria. We excluded participants with indeterminate classification, NF with medical/psychiatric exclusion, those missing data on fasting glucose and those with insufficient DNA for analysis, leaving 639 participants in 4 illness groups: 64 CFS, 77 CFS-X, 302 ISF (with and without exclusionary conditions), and 196 NF without exclusionary conditions.

Determination of relative telomere length (T/S ratio)

Relative telomere length was measured based on a widely used real-time PCR protocol [26] with modifications as described below. The assay is based on telomere-specific primers’ ability to generate a signal proportional to the total summed length of all the telomeres in the cell. This telomere signal is normalized to the signal from primers that amplify the single copy gene, 36B4 that encodes the acidic ribosomal phosphoprotein P0 (also known as RPLP0). The ratio of telomere (T) signal to the single copy gene (S) signal (expressed as T/S ratio) is proportional to the average telomere length per cell. This ratio is expressed relative to a reference DNA (K562 DNA from Promega Corporation, Madison, WI) which is assigned a T/S ratio of 1.0 as it is always compared to itself.

DNA from whole blood collected in PAXgene tubes was extracted following the manufacture’s protocol (Qiagen, Valencia, CA). DNA quality and quantity were assessed by 0.8% agarose gel electrophoresis and Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) respectively. LightCycler 480 (Roche Life Science, Indianapolis, IN) was used for real time PCR that consisted of a 20 µL reaction containing 30 ng DNA, 1X SYBR Green I Master, and 1 µM of telomere specific or 36B4 specific primers reported previously [27]. The cycling conditions were as follows: initial denaturation at 95 °C 10 min; amplification for 35 cycles consisting of 95 °C 30 s, 58 °C 10 s, 72 °C 10 s; melting 95 °C 5 s, 70 °C 1 min, 95 °C continuous; cooling 40 °C 30 s.

The standard curve consisted of twofold dilution series from 60 to 0.94 ng, and was done in quintuplicate each for the telomere (PCR efficiency 1.91) and 36B4 (PCR efficiency 1.98) assays using K562 reference DNA. The standard curves were saved externally and imported into each PCR run at the time of analysis. The 30 ng point in the standard curve was carried in each PCR run and marked as the “standard point” to link with the external standard curve. The T/S ratio can be calculated using two methods: the ΔΔCt method using the Cp (crossing point) values or the standard curve nanogram (ng) method. T/S ratios calculated by both methods in this study were highly concordant based on the strength of linear relationship (R2 = 99%). We used the T/S ratios calculated by the standard curve (ng) method for the analyses in this study [28].

Prior to using the T/S ratio assay with study samples, we evaluated its reproducibility and validated it in comparison to southern blot hybridization-based terminal restriction fragment (TRF) length method using TeloTAGGG Telomere Length Assay kit (Roche Life Science). For both reproducibility and validation tests, PAXgene DNAs from 20 volunteer samples, extracted and evaluated similar to study samples were used. T/S ratios determined on 2 different days on the same set of samples were highly reproducible based on the measure of linear relationship (R2 = 94%) and coefficient of variation (CV, 3.30%). TRF length expressed in base pairs (bp) and T/S ratio showed a strong linear relationship (R2 = 0.78%) slightly higher than that previously reported (R2 = 68%) [26].

Quality control of the assay was done at multiple levels including evaluation of assay reproducibility and validation. Further, each reaction was run in triplicate, with both telomere and 36B4 reactions for each sample run in the same 96-well plate to minimize variability. With each PCR run, there were 11 study samples along with a representative dilution of the standard curve point (30 ng dilution), 3 volunteer DNA samples to assess plate-to-plate variability, and a negative water control. Over the 87 runs required to complete the study, the CV for the standard curve 30 ng point (reference DNA) and the 3 control volunteer DNA samples ranged from 6.15 to 8.19%. Based on this range of CV, we used a cutoff of CV ≤ 10% within sample T/S ratio to identify samples for repeat testing. In addition, a cutoff CV of ≤ 2% was used for the Cp values within sample triplicates in either the telomere or 36B4 assays. Sixty-eight samples were repeated in order to bring the T/S ratio CV to ≤ 10%. An additional 82 samples were diluted and repeated in order to fit the Cp values within the dynamic range of the standard curve. After all quality control measures, the mean CV of all tested samples (n = 705) was 0.50 and 0.23% for the Cp values of telomere and 36B4 respectively, and 4.39% for T/S ratio (median: 4.04%; range: 0.07–9.84%).

Statistical analyses

Data were explored to assess frequency distribution and normality. Normality test results and quantile–quantile plots showed that a transformation of T/S ratios did not significantly improve normality. As previous studies have found [29], log-transformation did make T/S ratios distribution more normal-like, we compared the results using log-transformed and untransformed data in preliminary analyses and did not find any significant difference. Therefore, the results from the analysis of the untransformed T/S ratio are presented. We used general linear models to estimate the association of CFS or T/S ratio with demographics and clinical measures. We adjusted the association of T/S ratio and CFS for covariates that showed significant association with T/S ratio in this study. We summarized associations, unadjusted and adjusted for covariates, and considered p-values below 0.05 to be statistically significant. Age- and sex-stratified analyses were also performed to investigate how these demographics moderate the association between CFS status and T/S ratio. The relationship of T/S ratio to mean TRF length (y = 4235x + 5303.8) determined from the 20 volunteer subjects was used to convert T/S ratio of a sample to mean TRF length in base pairs (bp) where x represents the T/S ratio. We estimated years of additional aging based on literature value for telomere attrition of 31–63 bp/year across 20–95 years of adulthood [5].