Study population and definition
We conducted a cross-sectional study in patients with TDT, aged 18 years and up, who attended Adult Hematology clinic at Chiang Mai University Hospital. TDT was defined as a patient with thalassemia who required red cell transfusion at least 3 times per year.
Clinical and laboratory measurement
We collected clinical and laboratory parameters at the time of enrollment to identify factors associated with telomere length including age, sex, history of splenectomy, iron chelation, and factors associated with clinical severity including: pre-transfusion Hb level (mean steady-state Hb level in the previous 10 visits), units of red blood cell transfusion per month, serum ferritin (maximum, minimum, mean of serum ferritin at multiple times every year). All patients were investigated for telomere length, liver function test (LFT), non-transferrin bound iron (NTBI), hemolysis parameters, MRI for cardiac T2 star (T2*) and liver iron concentration (LIC). Thirty healthy individuals, age and sex-matched, were included as controls and were tested for telomere length.
Blood Samples and Genomic DNA Extraction
Blood samples (10 mL) were collected from the patients and healthy individuals in ethylenediaminetetraacetic acid (EDTA) tubes. Peripheral blood leukocytes were collected from blood samples by gradient density centrifugation through HiSep (HIMEDIA, Mumbai, India) and stored at −80 °C before they were used. In our study, we used the Blood DNA Extraction Kit (OMEGA, Norcross, GA, USA) to extract genomic DNA from the peripheral blood leukocytes.
Telomere length measurement
Telomere length was measured by using a real-time quantitative PCR as described by Cawthon, RM et al. . The telomere/single copy gene (T/S) ratio was calculated as the ratio of telomere repeat copies (T) to single-copy gene copy number and albumin gene (S) to represent the average length of telomere, which by definition is 1.00. The specificity of all amplifications was determined by a melting curve analysis. DNA from healthy individuals which have a T/S ratio of 1.00 were used as a reference DNA sample (the ‘standard DNA’). Four concentrations of the standard human genomic DNA samples were prepared by 3-fold serial dilutions (70 ng, 23.33 ng, 7.77 ng, and 2.59 ng per well), and aliquoted in duplicate to a 96-well PCR plate. The primers for amplification of the scg albumin are albumin forward primer, albu: 5’-CGGCGGCGGGCGGCGCGGGCTGGGCGGAAATGCTGCACAGAATCCTTG-3’ and albumin reverse primer, albd: 5’-GCCCGGCCCGCCGCGCCCGTCCCGCCGGAAAAGCATG
GTCGCCTGTT-3’; telomere forward primer, telg, 5’-ACACTAAGGTTTGGGTTTGGGTTTG
GGTTTGGGTTAGTGT-3’ and telomere reverse primer, telc, 5’-TGTTAGGTATCCCTATCCC
TATCCCTATCCCTATCCCTAACA-3’. The thermal cycling profile was Stage 1: 15 min at 95°C; Stage 2: 2 cycles of 15 s at 94°C, 15 s at 49°C; and Stage 3: 32 cycles of 15 s at 94°C, 10 s at 62°C, 15 s at 74°C with signal acquisition, 10 s at 84°C, 15 s at 88°C with signal acquisition. All samples were assayed in triplicates in order to minimize the sample-to-sample variation. Once PCR has completed, the Applied Biosystems QuantStudio™ 6 Flex Real-Time PCR analysis software was used to determine the T and S values for each experimental sample based on the standard curve method (Applied Biosystems, Foster City, CA, USA). The results of telomere length from PCR were expressed as T/S ratio and we converted the results to mean TRFL (kb) from a standard curve equation (y=3330x+3730) . The coefficient of variation in repeated measurements of the samples was 4.6%.
Mean telomere length of patients was compared with controls matched by age and sex using a Student’s T-test. We examined the association of clinical factors and the telomere length using ANOVA (F-test). Multivariable regression analysis was performed using a forward selection technique. We proposed to examine age, pre-transfusion hemoglobin level and type of iron chelator. The correlation coefficient between the telomere length and each parameter value was calculated using Pearson’s correlation. The significance level was set at a P value < 0.05.
Compliance with ethical standards
Informed consents were completed by all patients before enrollment. This study was approved by the Human Research Ethics Committee of Faculty of Medicine, Chiang Mai University. (study code: MED-2559-03967)