| Popis: |
Additional file 1: Supplemental 1. Development of MSPCR and BGS analysis for examination of FHIT methylation. (A) MSPCR was used to determine the FHIT methylation status in patient samples. A standard was derived by mixing bisulfite treated DNA from a patient with UM FHIT with bisulfite treated DNA from a patient with M FHIT. Ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 were used in MSPCR for UM and M PCR, respectively. (B) Example of BSG sequencing of an UM and M patient sample. Arrows point to distinct CpG islands in the same DNA sequence of FHIT; demonstrating UM (C to T) or M (C remains) in bisulfite treated DNA. Supplemental 2. Primers used in FHIT study. Accuprime (Invitrogen) or Q-solution (Qiagen) was used for FHIT, miR-124a, CDKN1A, and CDKN2A PCRs. Two PCRS were carried out for FHIT, SHP1, SYK, CDKN1A, and CDKN2A. BGS PCR conditions were as follow: FHIT BGS: 95–30″, TD: 61–51–1′, 72–1′ (Touchdown). SHP1 BGS: 95–30″, 64–1′, 72–1′ (35-40c). SYK BGS: 95–30″, 49–30″, 72–30″ (40c). CDKN1A BGS: 95–30″, TD: 55–45-1′, 72–1′ (Touchdown). CDKN2A BGS: 95–30″, TD: 61–51–1′, 72–1′ (Touchdown). miR-124a BGS: 95–30′, 54–40″, 72–40″ (40c). Supplemental 3. Statistical analysis of HTLV-I diseases for FHIT methylation. Odd risk ratios and chi-square statistics were determined for different HTLV-I diseases (Acute, chronic, smoldering, and lymphoma ATL) against HD, AC, or TSP patients for FHIT methylation. Odd risk ratios and chi-square statistics were determined for different HTLV-I diseases (Acute, chronic, smoldering, and lymphoma ATL) against HD, AC, or TSP patients for FHIT methylation. Chi-square results were determined using X2 (degrees of freedom, N = sample size) = chi-square statistic value, p = p value). Supplemental 4. Geographical distribution of patient samples. Pie diagrams were used to illustrate the geographical distribution of ATL (acute, chronic, smoldering, and lymphoma type), TSP/HAM, and ACs. Continent of origin (Asia, Africa, North America (N.Amer.), South America (S.Amer.), and Europe) was determined from providers. If the continent of origin was not known at the time, the samples are marked as “unknown”. Acute ATL (n = 124), chronic ATL (n = 44), smoldering ATL (n = 20), lymphoma ATL (n = 20), TSP/HAM (n = 136), and asymptomatic (AC), HTLV-I carriers (n = 89). Supplemental 5. FHIT methylation in viral lymphomas. (A) Representation of BGS of lymphoma-type ATL patients. U vs M alleles are noted by white and black boxes, respectively. Horizontal rows represent sequencing of a single colony. (B) FHIT methylation status in viral lymphomas. HTLV-I, ATL-lymphoma (n = 10), Kaposi’s sarcoma herpesvirus (human herpesevirus-8) (KSHV) associated lymphoma (n = 13), Epstein-barr (EBV) associated Hodgkin’s lymphoma (HL) (n = 18), and hepatitis C virus (HCV) associated lymphomas (n = 15). Results were determined by MSPCR of FHIT. Supplemental 6. Deletional Analysis of the FHIT gene in ATL patients. (A) Chart representing UM (or very weak M) ATL patients for deletional analysis of the FHIT gene. One patient carried a deletion in exon 5. (B) Representation of the PCR bands for exon 4, 5, and 8. In some patients, exon 4 was also amplified. Genomic DNA was used to amplify different FHIT exons with the following primers: FHIT Exon 4: F-GACTAGGAATCAGAAATGAATAATTA, R-GCATGTCAGTCAGGTAACAGGTAAGC; FHIT Exon 5: F-GCTGTTTTATTGTCCACGTGGAAGCT, R-CTCAGCTATGGTAGTGAAAAGGTCAA; and FHIT Exon 8: F-GATGCACTGTCATTTCAAAGCACTGG, R-CATATCTCCATGCAAATATTTACTGTC. |
