br Materials and methods br Study patients and blood
2. Materials and methods
2.1. Study patients and blood sampling
The study included a total 1067 Saudi individuals comprising 507 patients with differentiated thyroid cancer (DTC) and 560 disease-free controls of Saudi origin. Candidate patients had undergone total thyroidectomy, received radioiodine ablation and were on L-thyroxine suppressive therapy (Euthyrox, Merck Pharmaceuticals, NJ, USA). This therapy aimed to attain either suppressed (TSH < 0.1 mU/L) or near-suppressed (0.1 TSH < 0.5 mU/L) serum TSH levels with FT4 in the normal range (12–22 pmol/L). The study cases comprised 97.8% papillary thyroid cancer (PTC), 88.9% classic subtype, 9.1% follicular variant PTC, 1.2% tall cell variant, 0.4% diffuse sclerosing subtype, 0.4% insular subtype and 2.2% follicular thyroid cancer. Furthermore, 29.6% of the PTC patients presented with pos-itive family history of the disease. Healthy controls were recruited from the general population and known not to have thyroid disor-ders, family history of thyroid cancer or to have been previously exposed to therapeutic levels of external radiation. The cases did not differ significantly from the healthy controls in the important confounding variables, such as age and body-mass index (Table 1).
Excluded from the study group were individuals on multiple drug treatment, or those who would have changed the thyroxine brand 3 months prior to the launching of the study. We also excluded patients who might have been on drugs that could poten-tially interfere with thyroxine treatment. These included (a) anti-epileptics and bile Herboxidiene resins, (b) thyroid suppressors and other drugs that may alter thyroid hormone metabolism or production, and (c) medicines that could affect the pituitary-thyroid axis. Addi-tionally, expectant females, individuals with mental illness, other types of cancer, as well as those having significant renal impair-ment (glomerular filtration rate < 60 ml/min) or chronic liver dis-ease were also excluded. Compliance was determined through a questionnaire targeting each subject’s medical history, medication use, smoking, as well as measuring the TSH and FT4 levels.
The dose requirement study was directed at the same patient population involved in the disease association study to find the
role of gene polymorphism in LT4 dose requirement. Accordingly, 453 out of the 507 DTC patients were included in the analysis. Fifty-four patients were excluded from the analysis because they failed to meet the targeted therapeutic range of TSH and/or FT4 levels [(TSH 4.3 mU/L); (FT4 = 12.0–22.0 pmol/L)] suggesting non-compliance with thyroxine intake or inappropriate dose pre-scription. The analysis was performed in two stages. First stage involved whole patient group (ALL) in order to test whether this was general phenomenon with respect to patient response to drug therapy. In the second part, the patients were grouped based on their TSH level into the near suppressed (NSG) (0.1mUI TSH < 0.5 mU/I) and suppressed (SG) (TSH < 0.1 mU/I) TSH groups to test whether such associations may be related to certain delineable levels of dose required. All participants signed an informed consent approved by the King Faisal Specialist Hospi-tal and Research Centre Ethics Committee, and the study was per-formed in accordance with the regulations laid down by the Institutional Research Advisory Council in compliance with the Helsinki Declaration principles (http://www.wma.net/en/30publi-cations/10policies/b3/index.html).
Genomic DNA was isolated according to the manufacturer’s protocol (Gentra Puregene, Qiagen Sciences, Maryland, USA) from 5 ml peripheral blood drawn from each study individual into 6 ml vacutainer tubes containing K2EDTA (1 Becton Drive, Franklin Lakes, NJ USA). Briefly, 3 ml of blood were added to 9 ml red blood cell lysis buffer and incubated under continuous mixing for 5 min. The mixture was centrifuged at 2000g for 2 min, the leucocyte pel-let re-suspended and vortexed in 3 ml cell lysis buffer solution and proteins were precipitated for 20 s by centrifuging at 2000g for 5 min. The supernatant was mixed with 3 ml isopropanol in a 15 ml tube and genomic DNA precipitated gently. The tube was then centrifuged at 2000g for 3 min, the supernatant discarded, and the DNA pellet washed twice in 3 ml of 75% ethanol. The pellet was air-dried, dissolved in 250 ll hydration solution at 65 LC for 1 h, quantified by Nanodrop ND-1000 spectrophotometer (Wilm-ington, DE, USA), aliquoted into 50 ll portions and stored at 20 LC.