Genotyping of SLC variants
The genomic DNA was extracted from 3-4 ml of blood using the phenol-chloroform extraction technique. The genotype of variations was determined using the PCR-PFLP method as previously mentioned.10,19,20 For amplifying a DNA fragment for the SLC19A1 (G80A , rs1051266) variant following sets of primers were used: forward primer 5’- AGTGTCACCTTCGTCCCCTC-3’ and reverse primer 5’- CTCCCGCGTGAAGTTCTT-3′. For SLCO1B1 A388G in exon 4 (rs2306283), the set of primers used was forward primer 5’-ATAATGGTGCAAATAAAGGGG-3′and reverse primer 5’-ACTATCTCAGGTGATGCTCTA-3’ and for SLCO1B1 T521C (rs4149056) variant, following sets of primers were used: forward primer 5’-TTGTCAAAGTTTGCAAAGTG-3’ and reverse primer 5’-GAAGCATATTACCCATGAGC-3’. For amplifying a DNA fragment for theSLCO1B3 (A1683-5676G , rs11045585) variant following sets of primers were used: forward primer 5’-GTGGGTAAAAGGCAGGTAAATG-3’ and reverse primer 5’-GAATTCAAACATCTC ACTGTGCTC-3’. The PCR mixture of 20 µl comprised of 1X PCR buffer, 0.5 µM of forward and reverse primer, 1.5 mM MgCl2, 100 µg/ml bovine serum albumin (BSA), 200 µM dNTPs, 1U Taq polymerase (DNAzyme, Thermo Scientific), and 200 ng of DNA. PCR conditions used for the mixture were: 95˚C for 5 min and 94˚C for 30s (denaturation), 59˚C (G80A /rs1051266), 55 ˚C (A388G /rs2306283), 60˚C (T521C /rs4149056) and 57 ˚C (A1683-5676G /rs11045585) for 45s (annealing) followed by 72˚C for 30s (extension) for the 29 cycles as well as the final extension for 5 min at 72˚C. For SLC19A1 G80A , a PCR product of 230 base pairs (bp) was digested with 5U of HhaI restriction enzyme (New England Biolabs) at 37˚C. The wild-type alleles were recognized as GG (125, 68, 37 bp), variant alleles were identified as AA (162,68 bp), and heterozygous alleles produced both bands (162,125,68,37 bp) (Figure2a ).10 ForA388G , the PCR product of 214 bp was checked on 2.0% agarose gel and then digested with 5U of TaqI restriction enzyme (New England Biolabs), respectively at 65˚C. The wild type allele (AA ) yielded bands of 151, 63 bp variant allele (GG ), were identified by 128,63,23 bp, and heterozygous displayed all the bands(Figure-2b) . For T521C , a PCR product of 209 bp was digested with 5U of HhaI restriction enzyme (New England Biolabs) at 37˚C. The wild-type alleles were recognized as TT (209 bp), and variant alleles were identified as CC (189, 20 bp) (Figure2c ).19 ForA1683-5676G , a PCR product of 286 bp was digested with 5U of Rsa I restriction enzyme (New England Biolabs) at 37˚C. The wild type allele (AA) yielded bands of 262, 24 bp, variant allele (GG) were identified by 155, 107, 24 bp, and heterozygous displayed all the bands (Figure-2d) .20Digested fragments were separated on 8 % Native- polyacrylamide gel electrophoresis (PAGE) and were stained with the ethidium bromide and visualized under UV trans-illuminator. Two separate people looked into the data to eliminate any potential biases, and 20% of the samples were chosen at random, ensuring that the outcomes were 100% reproducible.