All patients were Brazilian and were recruited from the HTLV-1 outpatient clinic at the University of Sao Paulo, Brazil. We were unable to recruit more subjects, which would have enhanced the power of the study, and the total number of subjects was thus limited to 112. Thus, our study has an 80% power to detect a minimum odds ratio of 1.5 for the SNP, assuming α = 0.05 and the two tailed test.
Peripheral blood samples (5 ml) were collected from the 112 HTLV-1 positive individuals. Infected individuals were identified by the HTLV enzyme immunoassays Murex HTLV I + II (Abbott/Murex, Wiesbaden, Germany) and Vironostika HTLVI/II (bioMérieux bv, Boxtel, Netherlands), and infection was confirmed by HTLV BLOT 2.4 (HTLV blot 2.4, Genelabs Diagnostics, Science Park,. Singapore). Of these, 81 (72.3%) were asymptomatic carriers (ACs), 24 (21.4%) had HAM/TSP and 7 (6.3%) had ATLL. All ACs were diagnosed as HTLV-1 carriers at the time of blood donation. The clinical status of HAM/TSP was determined based on WHO criteria for HTLV-1 associated diseases . Diagnostic criteria for ATLL included serologic evidence of HTLV-1 infection and cytologically or histologically proven T cell malignancy. Written informed consent was obtained from each participant. The study was approved by the local review board (Comissão de Ética para Análise de Projetos de Pesquisa, CAPPesq).
Laboratory measurements included peripheral blood lymphocyte counts, quantitative measurement of HTLV-1 PvLs with the use of sensitive SYBR Green Real-Time PCR with a detection limit of 1 copy per 103 PBMC cells, and HTLV-1 genotypes . The results were obtained from the patients' medical records.
Based on the published human chromosome 19q13 sequence (NCBI Reference Sequence: NT_011109.16), a 293 base pair segment upstream of the IL-28 gene, flanking the rs12979860 polymorphism between nucleotides 12006835 and 120071103, relative to the chromosome 19 translational start codon, was analyzed. This region was PCR-amplified from genomic DNA using the forward primer 5'-GCTCAGCGCCTCTTCCTCCTGCG-3' and the reverse primer 5'-GGCAGGGCTCCCTTCTGTGATTGACC-3'. The PCR conditions consisted of an initial denaturation step at 95°C for 3 minutes, followed by 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds and 68°C for 1 minute, and a final extension at 68°C for 5 minutes. Negative controls without the DNA template were included in each experiment. After termination of the PCR cycle, the products were purified using a QIA quick PCR purification kit (Qiagen). Complementary DNA strands from each amplicon were directly sequenced by cycle sequencing using the same primers used for the PCR, BigDye terminator chemistry and Taq polymerase on an automated sequencer (ABI 3130, Applied Biosystems Inc., Foster City, CA), according to the protocols recommended by the manufacturer.
Continuous variables, including PvLs, were presented as the median (range) or mean ± standard deviation, while allelic frequencies were estimated by direct allele counting and were expressed as frequencies (%). Deviation from Hardy-Weinberg equilibrium was tested by a standard χ2 test with 1 degree of freedom. Differences between PvL variables were assessed by means of Student’s t test with the non-parametric U-Mann–Whitney test, while those between allelic frequencies variables were evaluated using the Pearson χ2 test with Yate's correction or Fisher’s exact test, when appropriate. A p value < 0.05 was considered significant. The data were analyzed with Stata statistical software (StataCorp, release 5.0, 1997; Stata, College Station, TX).