Postula et al:New single nucleotide polymorphisms associated with differences in platelet reactivity in patients with type 2 diabetes treated with acetylsalicylic acid: genome-wide association approach and pooled DNA strategy

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Details of themicroarray method and SNPs selection process

The DNA pools analyzed on the Illumina Omni 2.5 microarray resulted in generation of the relative allelic frequency data for 2,443,177 genomic markers in each pooled group. The initial data filtration process (i.e., removal of non-HapMap listed markers labeled GA, kgp, VGXS in the Illumina microarray manifest, as well as mitochondrial and sex chromosome markers) resulted in the total of 698,809 markers. The remaining markers were subsequently filtered again in order[MP1] to remove all SNPs with minor allele frequency of less than 5%, which resulted in the final number of 200,521 markers for further statistical analysis by the Tcomb statistics. The summary results of this analysis are presented as Manhattan plot in Figure 1. The statistical significance of the differences between allele frequency in both investigated group was compared with corrected p for the total number of markers available for the final statistical analysis (p <0.05/200,521~2.5-7).

Supplemental Table 1. Summary of VEGAS testing results for 25 selected genes and its subsequent verification by individual genotyping for the most significant SNP within the gene.

Chromosome / Gene / MAF / nSNPs / Rank in VEGAS / VEGAS P value / SNP rs# / Verification P
6 / KIAA1244 / 0.125 / 7 / 4 / 1.00E-06 / rs1013054 / 0.478
8 / EYA1 / 0.328 / 25 / 10 / 2.00E-06 / rs13275654 / 0.251
10 / HELLS / 0.357 / 6 / 11 / 2.00E-06 / rs11188019 / 0.395
8 / GSR / 0.447 / 4 / 14 / 4.00E-06 / rs3779647 / 0.006
1 / RGS7 / 0.421 / 92 / 15 / 5.00E-06 / rs2502448 / 0.0001*
1 / AK5 / 0.156 / 31 / 21 / 1.00E-05 / rs4949803 / 0.044
7 / DPP6 / 0.276 / 104 / 50 / 8.50E-05 / rs1387180 / 0.003*
9 / GLIS3 / 0.364 / 103 / 51 / 0.00041 / rs10974395 / 0.203
9 / KCNV2 / 0.488 / 15 / 200 / 0.00082 / rs1006698 / 0.074
8 / KCNB2 / 0.288 / 51 / 193 / 0.00082 / rs7824350 / NA
11 / FOSL1 / 0.221 / 11 / 314 / 0.0014 / rs10791830 / 0.071
5 / ADRA1B / 0.196 / 9 / 326 / 0.00149 / rs13171967 / 0.246
8 / MMP16 / 0.356 / 15 / 327 / 0.00149 / rs2664369 / 0.051
1 / ALDH4A1 / 0.325 / 18 / 330 / 0.00151 / rs7537155 / 0.386
2 / HS6ST1 / 0.206 / 18 / 370 / 0.00174 / rs6711447 / 0.737
10 / ANK3 / 0.451 / 50 / 406 / 0.002 / rs2393602 / 0.023
2 / ERBB4 / 0.317 / 107 / 463 / 0.00239 / rs10195573 / 0.017
19 / CACNA1A / 0.224 / 40 / 641 / 0.00354 / rs8103699 / 0.007
10 / ALOX5 / 0.268 / 9 / 761 / 0.00447 / rs2291427 / 0.276
12 / SLC2A13 / 0.419 / 30 / 857 / 0.00518 / rs10784356 / 0.915
17 / ITGB3 / 0.320 / 15 / 867 / 0.00523 / rs8074094 / 0.5
17 / ALOX15 / 0.382 / 8 / 1333 / 0.00928 / rs4247362 / 0.147
13 / ABCC4 / 0.315 / 83 / 1764 / 0.01348 / rs7331142 / NA
15 / PLA2G4E / 0.399 / 13 / 3412 / 0.03487 / rs1704365 / 0.033
7 / TBXAS1 / 0.282 / 49 / 4061 / 0.0461 / rs6948035 / 0.491

MAF- minor allele frequency, nSNP – number of analyzed SNPs in the gene, Rank in VEGAS- order rank for particular gene in the final VEGAS analysis, P value in the VEGAS test for the analyzed gene; Rs# - identification number for the most significant SNP in the analyzed gene; verification P – p value for the most significant SNP in the analyzed gene in the individual genotyping analysis. Bold fonts indicate nominally statistical results, * indicates significance after multiple comparison testing multiple-comparisons indicate that 111 SNPs remained statistically significant.

Supplemental Table 2. Summary of multiple statistical tests evaluating the results of individual genotyping (validation) for most significant SNPs from GWAS.

Gene / SNP rs# / CEPI-CT
Logreg<193 / CEPI-CT
Logreg<165 / CEPI-CT
Logreg<300 / CEPI-CT
Chi2 / VerifyNow
Chi2 / CEPI-CT
Median test / CEPI-CT
Tobitreg / CADP-CT
Tobitreg
ITGB3 / rs8074094 / 0.2a / 0.185a / 0.923a / 0.5 / 0.236 / 0.306a / >0.2
HS6ST1 / rs6711447 / 0.4r / 0.465d / 0.022r / 0.737 / 0.847 / 0.052 / 0.011r / >0.2
GLIS3 / rs10974395 / 0.08d / 0.208d / 0.578r / 0.203 / 0.066 / 0.267 / 0.226d / >0.2
KIAA1244 / rs1013054 / 0.3a / 0.834a / 0.836a / 0.478 / 0.576 / 0.326 / 0.361a / >0.2
FOSL1 / rs10791830 / 0.02a / 0.201a / 0.155d / 0.071 / 0.277 / 0.711 / 0.099d / >0.2
CACNA1A / rs8103699 / 0.03d / 0.017d / 0.117d / 0.007 / 0.455 / 0.076 / 0.034d / >0.2
PLA2G4E / rs1704365 / 0.01d / 0.284d / 0.201a / 0.033 / 0.716 / 0.367 / 0.088d / >0.2
ALDH4A1 / rs7537155 / 0.1r / 0.231a / 0.310r / 0.386 / 0.958 / 0.166 / 0.090r / >0.2
ADRA1B / rs13171967 / 0.09d / 0.263d / 0.065d / 0.246 / 0.483 / 0.201 / 0.069d / >0.2
GRS / rs3779647 / 0.002d / 0.031d / 0.078d / 0.006 / 0.026 / 0.052 / 0.008d / 0.0001a
AK5 / rs4949803 / 0.01a / 0.056a / 0.013d / 0.044 / 0.219 / 0.006 / 0.006d / >0.2
EYA1 / rs13275654 / 0.1a / 0.064a / 0.210a / 0.251 / 0.665 / 0.459 / 0.115r / >0.2
KCNV2 / rs1006698 / 0.02a / 0.020a / 0.010a / 0.074 / 0.056 / 0.017 / 0.007d / >0.2
DPP6 / rs1387180 / 0.001a / 0.0001d / 0.114d / 0.003 / 0.692 / 0.075 / 0.006d / >0.2
ANK3 / rs2393602 / 0.009r / 0.009r / 0.002r / 0.023 / 0.191 / 0.025 / 0.002r / >0.2
RGS7 / rs2502448 / 0.0001r / 0.0001r / 0.008r / 0.0001 / 0.210 / 0.014 / 0.001r / >0.2
MMP16 / rs2664369 / 0.02a / 0.152d / 0.137d / 0.051 / 0.477 / 0.073 / 0.015d / >0.2
ERBB4 / rs10195573 / 0.005d / 0.007d / 0.177a / 0.017 / 0.219 / 0.328 / 0.045d / >0.2
SLC2A13 / rs10784356 / 0.6d / 0.614a / 0.715r / 0.915 / 0.879 / 0.607 / 0.664a / >0.2
ALOX5 / rs2291427 / 0.1a / 0.141a / 0.137a / 0.276 / 0.342 / 0.066 / 0.055a / >0.2
HELLS / rs11188019 / 0.1a / 0.179a / 0.433a / 0.395 / 0.531 / 0.417 / 0.181a / 0.023d
TBXAS1 / rs6948035 / 0.2d / 0.149a / 0.713a / 0.491 / 0.347 / 0.745 / 0.352r / >0.2
ALOX15 / rs4247362 / 0.05d / 0.105d / 0.077d / 0.147 / 0.063 / 0.382 / 0.051d / 0.044a

Data expressed as p- value calculated using one of the statistical method (logreg - logistic regression, Ch2 - Chi square, median test and Tobit regression). a - additive genetic model, r - recessive genetic model, d - dominant genetic model.Nominally statistically significant results (p<0.05 before multiple tests correction) are shown in bold. The final statistically significant results (after applying multiple test correction, p < 0.05/23 or < 0.0022) are presented in bold italics.

[MP1]Please note that correction was made here.