Supplementary Table-1(a): Reagent Volumes for TAQMAN Genotyping assays, per

plate using TAQMAN buffer.

Reagent Volume (ul)

TAQMAN Mastermix (Life

technologies)

410

Nuclease Free Water (Sigma) 389.5 / 399.8

40x/80x SNP Specific Assay (Life

Technologies)

20.5 / 10.3

The volume of SNP specific assay (which contains primers and probes) depends on

whether it is supplied as 40 or 80x.

Supplementary Table-1(b): Reagent Volumes for KASPar genotyping assays per plate.

Reagent Volume (ul) for 1.8ul per

well

Volume (ul) for 3.6ul per

well

KASP Master mix 422.4 844.8

Nuclease free Water

(Sigma)

422.4 844.8

KASP Assay Mix 11.7 23.4

Supplementary Table-2(a):PCR conditions for TAQMAN Genotyping assays

Temperature Time

500C 2 mins

950C 10 mins

950C 15 sec x 40 cycles

600C 1 min x 40 cycles

Supplementary Table-2(b) PCR conditions for KASPar genotyping assays

Temperature

Decreasing 0.80C per

cycle

Time

940C 15mins

940C 20 sec x 10 cycles

650C-570C 60 sec x 10 cycles

940C 20 sec x 26 cycles

570C 60 sec x 26 cycles

Supplementary Table-2(c) PCR conditions when further cycling is required for KASPar

Genotyping Assays

Temperature Time

940C 20sec x 3 cycles

570C 60sec x 3 cycles

Supplementary Table-3(a): Sequencing pre-amplification reaction

Reagent Volume (ul)

Multiplex PCR Mastermix (Qiagen) 10

Nuclease Free Water (Sigma) 6

Forward Sequencing primer 1

Reverse sequencing primer 1

Template DNA (5ng/ul) 2

Supplementary Table-3 (b): PCR cycling conditions for sequencing pre-amplification

reactions

Temperature Time

950C 15 mins

940C 1 min x 35 cycles

65.50C 1 min x 35 cycles

720C 1 min x 35 cycles

720C 5 mins

Supplementary Table-3(c) : Sequencing Primers

Primer Sequence

MIA3_F 5’-ATCCAATCACCTTCCACCAG-3’

MIA3_R 5’-CCCAAATGTATCAGCAGCAAA-3’

CDKN2A(9p21)_F 5’-GTTTCTGCACATGGTGATGG-3’

CDKN2A(9p21)_R 5’-CATTCCCCAACATTTGTCCT-3’

APOA5_F 5’-GCAGGGTGAAGATGAGATGG-3’

APOA5_R 5’-TAGACGGAGTGGGTGTGTCA-3’

LPArs379_F 5’-GAAGGGGCTGGACCATATTT-3’

LPArs379_R 5’-AAGACCACAGGTGAGCGAGT-3’

LPLrs328_F 5’-CTTCCACAGGGTGATCTTCTG-3’

LPLrs328_R 5’-CATGAAGCTGCCTCCCTTTAG-3’

PCSK9_F 5’GACTACGAGGAGCTGGTGCT-3’

PCSK9_R 5’-CCTGCACTCCACTTCCTCTC-3’

MRAS_F 5’-TCTTGCTGCGTTTTCACATC-3’

MRAS_R 5’-TTGACTCCAAGGGAAGATGG-3’

APOB_F 5’-GCCCAGAATCTGTACCAGGA-3’

APOB_R 5’-TGGAATCTGGGGAAGTTCAG-3’

CXCL12_F 5’-GTCCAGATGAGGCCATCAAG-3’

CXCL12_R 5’-TGCCAAGAAAATGACACAGC-3’

LPArs104_F 5’-GCATAGCCAGACATGGGTTT-3’

LPArs104_R 5’-TGCCATGTTTGTCTTGGGTA-3’

APOE158_F 5’-CTGCGTAAGCGGCTCCTC-3’

APOE158_R 5’-CTGCCCATCTCCTCCATC-3’

APOE112_F 5’-GCCTACAAATCGGAACTGGA-3’

APOE112_R 5’CAGCTCCTCGGTGCTCTG-3’

F= forward; R=reverse

Supplementary Table-4: Selected CAD SNPs (Beaney et al.,2015)

Gene SNP SNP location Risk Allele Odds Ratio ReferenceMIA3 rs17465637 Intergenic C 1.14 Samani et al. CXCL12 rs1746048 Intergenic C 1.17 Samani et al. APOB rs1042031 E4181K A 1.73 Casas et al. LPA rs10455872 Intergenic G 1.70 Clarke et al. LPL rs328 S447X C 1.25 Casas et al. 9p21 rs10757274 Intergenic G 1.29 Samani, ,et al.PCSK9 rs11591147 R46L G 1.43 Benn et al. APOA5 rs662799 Promoter G 1.19 Sarwar et al. MRAS rs9818870 Intergenic T 1.15 Erdmann et al. LPA rs3798220 I1891M C 1.92 Clarke et al. APOE rs429358 C112R C 1.06 Bennet et al.APOE rs7412 C158R T 0.80 Bennet et al.SORT1 rs646776 Intergenic A 1.19 Samani et al.

Supplementary Table-5:- Multivariate analyses showing odds ratio of cytokines and

cytokine ratios for the prediction of premature atherosclerosis.

B S.E Exp(B) (95%CI) Sig.

IL-6(ng/dl) 1.867 0.648 6.470 (1.819-23.0)** 0.004

IL-18(pg/ml) 0.010 0.004 1.010 (1.003-1.018)** 0.006

IL-10 (pg/ml) 0.316 0.287 0.729 (0.415-1.281) 0.272

TNF-alpha(pg/

ml)

0.606 0.182 1.832 (1.282-2.618)** 0.001

TNF-alpha/IL-10 0.495 0.135 1.641 (1.261-2.137)** 0.000

IL-18/IL-10 ratio 0.003 0.003 0.997 (0.992-1.002) 0.270

IL-18 = Interleukin-18; IL-10 = Interleukin-10; TNF-alpha = Tumor Necrosis Factor-alpha; SNP = Single Nucleotide Polymorphism.

** p<0.01

* p<0.01

Supplementary Fig 1(a) Electropherogram showing the sequencing results for LPA

rs3798220. The genotypes for the samples are TT and CT respectively highlighted in

blue.

Sequencing primers used were:-

Forward: 5’-GAAGGGGCTGGACCATATTT-3’

Reverse: 5’-AAGACCACAGGTGAGCGAGT-3’

Supplementary Fig 1(b) Electropherogram showing the sequencing results for LPA

rs10455872. The genotype for both the samples is TT.

Sequencing primers used were:-

Forward: 5’-GCATAGCCAGACATGGGTTT-3’

Reverse: 5’-TGCCATGTTTGTCTTGGGTA-3’

Supplementary Fig 1(c) Electropherogram showing the sequencing results for PCSK9

rs11591147. The genotype for both the samples is GT.

Sequencing primers used were:-

Forward: 5’GACTACGAGGAGCTGGTGCT-3’

Reverse: 5’-CCTGCACTCCACTTCCTCTC-3’