Genome-wide Association Study Identified the Human Leukocyte Antigen (HLA) Region as a Novel Locus for Plasma Beta-2 Microglobulin (Supplementary Materials)

Supplementary Methods

Genotyping, Imputation, and Principal Component Generation

The genotyping in EA was supported by the National Institute of Health Gene Environment Association Studies (GENEVA) project 1; 2 and used the Affymetrix 6.0 platform. The initial quality filters included a call rate of 0.95, check for blind duplicates in each plate and gender mismatch. Of the 910,030 SNPs genotyped, 841,820 passed the initial quality filters. Of the SNPs that failed the initial quality filters, 96% were due to the call rate. After the genotype data of 9,713 individuals was received from GENEVA, additional quality filters were applied to exclude individuals who were more than 8 SD away from any of the 10 principal components or closely related based on identity by state distance (DST > 0.86) calculated by PLINK. 3 Altogether 658 individuals were excluded resulting in a genotype dataset of 9055 individuals. SNPs used for imputation were restricted to those with MAF >1%, missing data per SNP < 5%, and Hardy-Weinberg equilibrium (HWE) >0.00001. SNPs used for principal components generation are restricted to those with MAF ≥10%, missing data per SNP < 0.5%, and HWE p-value ≥ 0.001.

References

1. Cornelis, M.C., Agrawal, A., Cole, J.W., Hansel, N.N., Barnes, K.C., Beaty, T.H., Bennett, S.N., Bierut, L.J., Boerwinkle, E., Doheny, K.F., et al. (2010). The Gene, Environment Association Studies consortium (GENEVA): maximizing the knowledge obtained from GWAS by collaboration across studies of multiple conditions. Genet Epidemiol 34, 364-372.

2. Laurie, C.C., Doheny, K.F., Mirel, D.B., Pugh, E.W., Bierut, L.J., Bhangale, T., Boehm, F., Caporaso, N.E., Cornelis, M.C., Edenberg, H.J., et al. (2010). Quality control and quality assurance in genotypic data for genome-wide association studies. Genet Epidemiol 34, 591-602.

3. Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M.A., Bender, D., Maller, J., Sklar, P., de Bakker, P.I., Daly, M.J., et al. (2007). PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81, 559-575.

Supplementary Figures

Supplementary Figure 1. Quantile-quantile plot of the GWAS results of log (B2M).

Supplementary Figure 2. Regional association plots of the HLA Locus of log(B2M).

(3A) HLA-A region (r2 is with respect to rs9260489 and the other index SNP is highlighted as purple diamond.)

(3B) HLA-B and HLA-C region (r2 is with respect to rs9264638 and the other three index SNPs are highlighted as purple diamond.)

(3A)
/ (3B)

Supplementary Figure 3. Regional association plot of log(B2M) associations at SH2B3 locus.

The linkage disequilibrium measure, r2, was from HapMap II CEU.

Supplementary Table 1. Measures of Linkage Disequilibrium (r2, D') between Index SNPs of B2M in the HLA Region calculated from the most likely genotypes in the ARIC sample.

Imputation Quality / 0.94 / 0.91 / 0.92 / 0.95 / 1.00 / 0.99
MAF / 0.42 / 0.38 / 0.41 / 0.41 / 0.07 / 0.07
rs9260489 / rs2023472 / rs9264638 / rs2523608 / rs16899524 / rs2596466
rs9260489 / 0.08, 0.3 / 0.03, 0.18 / 0.0, 0.02 / 0.01, 0.11 / 0.02, 0.60
rs2023472 / 0.06, 0.25 / 0.0, 0.02 / 0.05, 0.31 / 0.0, 0.07
rs9264638 / 0.0, 0.02 / 0.02, 0.54 / 0.04, 0.90
rs2523608 / 0.01, 0.09 / 0.1, 0.97
rs16899524 / 0.01, 1.0
rs2596466

Note: Imputation quality was measured by the ratio of the observed vs. expected variance of the imputed genotype. Although rs2596466 had a D’ > 0.9 with three other index SNPs near HLA-B and HLA-C, it remained statistically significant (p=2.2x10-3 > 0.0083 = 0.05/6, Table 2) after controlled for the other five index SNPs. The addition of rs2596466 to a linear regression model already included the other 5 index SNPs increased the multiple R2 by 0.0017. Haplotype analysis using most likely genotype showed the minor allele at rs2596466 was more likely to be in phase with the major alleles at rs9264638 and rs16899524 and the minor allele of rs2523608 (data not shown). Therefore, the high D’ of rs2596466 with the three other index SNPs did not prevent it from making independent contribution in the linear regression model.

Supplementary Table 2. Association between log(B2M) and Imputed Classical Alleles of HLA-A, HLA-B, and HLA-C with Frequency > 1%

Gene / allele / Beta / SE / P-Value / Allele Frequency
HLA-A / 101 / 0.03 / 0.01 / 1.04E-08 / 0.17
HLA-A / 1101 / 0.01 / 0.01 / 5.42E-01 / 0.07
HLA-A / 201 / -0.03 / 0.00 / 9.28E-10 / 0.27
HLA-A / 2301 / -0.05 / 0.02 / 3.52E-03 / 0.02
HLA-A / 2402 / 0.00 / 0.01 / 8.07E-01 / 0.08
HLA-A / 2501 / -0.02 / 0.02 / 2.39E-01 / 0.02
HLA-A / 2601 / -0.02 / 0.01 / 1.83E-01 / 0.02
HLA-A / 2902 / -0.02 / 0.01 / 4.36E-02 / 0.04
HLA-A / 3001 / 0.01 / 0.02 / 6.29E-01 / 0.01
HLA-A / 301 / 0.03 / 0.01 / 2.49E-06 / 0.16
HLA-A / 3101 / 0.00 / 0.01 / 9.43E-01 / 0.03
HLA-A / 3201 / 0.00 / 0.01 / 9.92E-01 / 0.04
HLA-A / 6801 / -0.02 / 0.01 / 1.15E-01 / 0.04
HLA-B / 1302 / 0.00 / 0.02 / 9.75E-01 / 0.02
HLA-B / 1402 / -0.01 / 0.01 / 3.72E-01 / 0.02
HLA-B / 1501 / -0.05 / 0.01 / 1.65E-10 / 0.07
HLA-B / 1801 / 0.02 / 0.01 / 6.85E-02 / 0.05
HLA-B / 2705 / -0.02 / 0.01 / 1.03E-01 / 0.04
HLA-B / 3501 / 0.01 / 0.01 / 3.04E-01 / 0.06
HLA-B / 3503 / 0.05 / 0.02 / 1.32E-02 / 0.01
HLA-B / 3701 / 0.06 / 0.02 / 3.14E-04 / 0.02
HLA-B / 3801 / 0.01 / 0.02 / 5.21E-01 / 0.02
HLA-B / 4001 / -0.03 / 0.01 / 9.44E-05 / 0.07
HLA-B / 4002 / 0.03 / 0.02 / 6.93E-02 / 0.01
HLA-B / 4402 / 0.02 / 0.01 / 1.52E-02 / 0.09
HLA-B / 4403 / -0.05 / 0.01 / 1.40E-05 / 0.05
HLA-B / 4901 / -0.04 / 0.02 / 4.04E-02 / 0.01
HLA-B / 5101 / -0.02 / 0.01 / 7.69E-02 / 0.05
HLA-B / 5501 / -0.03 / 0.02 / 1.12E-01 / 0.01
HLA-B / 5701 / -0.06 / 0.01 / 4.10E-07 / 0.04
HLA-B / 702 / 0.01 / 0.01 / 1.10E-01 / 0.14
HLA-B / 801 / 0.06 / 0.01 / 1.66E-20 / 0.13
HLA-C / 102 / -0.034 / 0.012 / 4.61E-03 / 0.04
HLA-C / 1203 / 0.011 / 0.011 / 3.31E-01 / 0.05
HLA-C / 1402 / 0.002 / 0.020 / 9.26E-01 / 0.01
HLA-C / 1502 / -0.030 / 0.014 / 3.17E-02 / 0.03
HLA-C / 1601 / -0.025 / 0.011 / 2.72E-02 / 0.04
HLA-C / 202 / -0.008 / 0.010 / 4.31E-01 / 0.05
HLA-C / 303 / -0.050 / 0.010 / 1.41E-06 / 0.05
HLA-C / 304 / -0.043 / 0.008 / 7.98E-08 / 0.08
HLA-C / 401 / -0.001 / 0.007 / 8.56E-01 / 0.11
HLA-C / 501 / 0.015 / 0.008 / 6.69E-02 / 0.08
HLA-C / 602 / -0.021 / 0.008 / 7.00E-03 / 0.09
HLA-C / 701 / 0.054 / 0.006 / 1.02E-18 / 0.16
HLA-C / 702 / 0.010 / 0.006 / 8.98E-02 / 0.15
HLA-C / 704 / 0.025 / 0.016 / 1.21E-01 / 0.02
HLA-C / 802 / -0.006 / 0.011 / 5.78E-01 / 0.04

Supplementary Table 3. Associations between log(B2M) and GWAS index SNPs in the HLA Class I region Controlled for imputed HLA classical alleles.

Controlled for HLA-A classical allele genotype / Controlled for HLA-B classical allele genotype / Controlled for HLA-C classical allele genotype
SNP / Closest gene / beta / SE / p-value / beta / SE / p-value / beta / SE / p-value
rs9260489 / HLA-A / -0.017 / 0.008 / 3.4E-02 / -0.028 / 0.005 / 4.7E-09 / -0.031 / 0.005 / 2.9E-11
rs2023472 / TRIM31 / 0.019 / 0.005 / 3.2E-04 / 0.021 / 0.005 / 4.4E-05 / 0.024 / 0.005 / 1.5E-06
rs9264638 / HLA-C / -0.038 / 0.005 / 2.3E-15 / -0.015 / 0.006 / 7.9E-03 / -0.024 / 0.006 / 3.2E-05
rs2523608 / HLA-B / 0.027 / 0.005 / 1.0E-08 / 0.000 / 0.006 / 9.3E-01 / 0.002 / 0.005 / 6.4E-01
rs16899524 / HLA-B / 0.026 / 0.009 / 3.8E-03 / 0.031 / 0.012 / 8.3E-03 / 0.048 / 0.008 / 4.1E-09
rs2596466 / MICA / -0.046 / 0.009 / 1.7E-07 / -0.007 / 0.017 / 6.9E-01 / -0.029 / 0.011 / 9.2E-03

Supplementary Table 4. Associationsof B2M Index SNPs at the HLA Locus with Serum Creatinine based eGFR in ARIC and CKDGen Discovery Meta-analysis Results.

ARIC log(eGFRcr) / CKDGen log(eGFRcr)
SNPID / Chr / Position (B36) / Beta / SE / P-Value / Beta / SE / P-Value
rs9260489 / 6 / 30028311 / -5.62E-04 / 3.37E-03 / 8.68E-01 / -2.20E-03 / 1.30E-03 / 8.86E-02
rs2023472 / 6 / 30183843 / -7.30E-03 / 3.51E-03 / 3.77E-02 / -1.60E-03 / 1.30E-03 / 1.92E-01
rs9264638 / 6 / 31346297 / 4.49E-03 / 3.45E-03 / 1.93E-01 / 9.00E-04 / 1.30E-03 / 4.81E-01
rs2523608 / 6 / 31430538 / -1.78E-04 / 3.37E-03 / 9.58E-01 / 1.00E-04 / 1.20E-03 / 9.40E-01
rs16899524 / 6 / 31470289 / 3.80E-03 / 6.19E-03 / 5.39E-01 / 4.00E-04 / 2.30E-03 / 8.61E-01
rs2596466 / 6 / 31513596 / -5.63E-04 / 6.19E-03 / 9.27E-01 / 2.00E-04 / 2.30E-03 / 9.28E-01

Abbreviation: eGFRcr, estimated glomerular filtration rate calculated using the Modification of Diet in Renal Disease Study equation.

Supplementary Table 5. Associations Between B2M Index SNPs in the Novel HLA Locus and All-cause Mortality in ARIC Adjusted for Age, Gender, and Center using Visit 4 as Baseline.

SNP / Coded/non-coded Alleles / Beta / StdErr / P-Value / Hazard Ratio (95% CI)
rs9260489 / G/T / 0.11 / 0.05 / 0.02 / 1.11 (1.02, 1.22)
rs2023472 / A/G / 0.03 / 0.05 / 0.50 / 1.03 (0.94, 1.13)
rs9264638 / A/G / -0.01 / 0.05 / 0.84 / 0.99 (0.9, 1.09)
rs2523608 / A/G / -0.04 / 0.04 / 0.36 / 0.96 (0.88, 1.05)
rs16899524 / C/T / 0.07 / 0.08 / 0.38 / 1.08 (0.91, 1.27)
rs2596466 / C/T / 0.11 / 0.08 / 0.19 / 1.11 (0.95, 1.3)

Note: The number of dead events was 1070 among 6738 participants with a median follow-up time of 11.2 years. Among the six SNPs, rs9260489 has the lowest p-value but did not pass the Bonferroni corrected threshold of 0.0083 (=0.05/6). Moreover, the G allele was associated with increased hazard here but lower B2M levels in the genome-wide scan (Table 2). Therefore, the association of increased level of B2M with mortality found in other studies cannot possibly be attributed to this locus.

Supplementary Table 6. Associations of the Index SNPs in Known GFR Loci with B2M and eGFR Calculated Using Traditional Biomarkers at Visit 4.

Only participants with all three biomarkers were included (N=6735). All regressions controlled for visit 4 age, gender, center, and significant principal components.

log(eGFRcr) at visit 4 / log(eGFRcys) / log(Scaled B2M) / log(Scaled B2M) adjusted for index SNPs in HLA locus
SNP / Chr / Gene / Beta / SE / P-Value / Beta / SE / P-Value / Beta / SE / P-Value / Beta / SE / P-Value
rs267734 / 1 / ANXA9 / 0.019 / 0.004 / 1.6E-05 / 0.011 / 0.005 / 3.0E-02 / 0.010 / 0.005 / 3.9E-02 / 0.010 / 0.005 / 3.8E-02
rs1260326 / 2 / GCKR / -0.014 / 0.004 / 1.3E-04 / -0.008 / 0.004 / 7.5E-02 / -0.002 / 0.004 / 6.2E-01 / -0.003 / 0.004 / 4.7E-01
rs13538 / 2 / NAT8 / -0.017 / 0.004 / 7.6E-05 / -0.010 / 0.005 / 4.4E-02 / -0.011 / 0.005 / 1.6E-02 / -0.011 / 0.005 / 1.4E-02
rs347685 / 3 / TFDP2 / -0.011 / 0.004 / 6.3E-03 / -0.010 / 0.005 / 3.6E-02 / -0.008 / 0.004 / 6.4E-02 / -0.009 / 0.004 / 4.3E-02
rs17319721 / 4 / SHROOM3 / -0.017 / 0.004 / 2.8E-06 / -0.016 / 0.004 / 2.4E-04 / -0.015 / 0.004 / 1.9E-04 / -0.016 / 0.004 / 5.4E-05
rs11959928 / 5 / DAB2 / -0.005 / 0.004 / 1.3E-01 / -0.008 / 0.004 / 7.8E-02 / -0.003 / 0.004 / 4.1E-01 / -0.004 / 0.004 / 3.0E-01
rs6420094 / 5 / SLC34A1 / 0.017 / 0.005 / 3.2E-04 / 0.009 / 0.006 / 1.1E-01 / 0.009 / 0.005 / 6.9E-02 / 0.011 / 0.005 / 3.1E-02
rs881858 / 6 / VEGFA / -0.011 / 0.004 / 7.2E-03 / -0.016 / 0.005 / 1.2E-03 / -0.007 / 0.004 / 1.3E-01 / -0.006 / 0.004 / 1.6E-01
rs7805747 / 7 / PRKAG2 / -0.014 / 0.006 / 2.3E-02 / -0.015 / 0.007 / 4.6E-02 / -0.018 / 0.007 / 7.6E-03 / -0.018 / 0.007 / 6.5E-03
rs10109414 / 8 / STC1 / 0.005 / 0.004 / 1.5E-01 / 0.013 / 0.004 / 2.2E-03 / 0.007 / 0.004 / 6.0E-02 / 0.008 / 0.004 / 4.7E-02
rs4744712 / 9 / PIP5K1B / -0.007 / 0.004 / 6.6E-02 / -0.002 / 0.004 / 6.3E-01 / -0.008 / 0.004 / 4.6E-02 / -0.007 / 0.004 / 5.9E-02
rs653178 / 12 / ATXN2 / -0.004 / 0.004 / 2.6E-01 / -0.010 / 0.004 / 2.8E-02 / -0.023 / 0.004 / 2.0E-08 / -0.022 / 0.004 / 3.5E-08
rs626277 / 13 / DACH1 / -0.004 / 0.004 / 3.2E-01 / -0.006 / 0.004 / 1.5E-01 / -0.011 / 0.004 / 6.2E-03 / -0.010 / 0.004 / 1.5E-02
rs1394125 / 15 / UBE2Q2 / -0.012 / 0.004 / 4.4E-03 / -0.004 / 0.005 / 3.8E-01 / -0.010 / 0.005 / 2.8E-02 / -0.010 / 0.005 / 3.0E-02
rs12917707 / 16 / UMOD / -0.024 / 0.005 / 8.0E-07 / -0.024 / 0.006 / 3.7E-05 / -0.015 / 0.005 / 4.1E-03 / -0.016 / 0.005 / 2.6E-03
rs12460876 / 19 / SLC7A9 / 0.012 / 0.004 / 8.7E-04 / 0.007 / 0.004 / 1.3E-01 / 0.008 / 0.004 / 6.2E-02 / 0.007 / 0.004 / 9.7E-02

Abbreviations:eGFRcr, eGFR calculated using the MDRD equation;eGFRcys calculated from cystatin C levels using the equation with age and sex covariates from Stevens et al. 2008.
Supplementary Table 7. Associations between log(B2M) and Reported Index SNPsfor Autoimmune Disorders in the HLA Class I Region.

Reported Index SNP / PUBMED ID / Reported Disease / Trait / Reported Risk Allele / Reported Risk Allele Freq. / Reported P-Value / Reported OR / Beta / Position (B36) / B2M Coded/ Noncoded Allele / B2M Coded Allele Freq. / B2M Beta / B2M SE / B2M P-Value
rs1521 / 21841780 / Graves disease / T / 0.79 / 2.0E-65 / 1.92 / 31458683 / C/T / 0.24 / -0.01 / 0.00 / 7.8E-02
rs2281388 / 21841780 / Graves disease / T / 0.32 / 2.0E-65 / 1.64 / 33168096 / A/G / 0.02 / 0.00 / 0.01 / 1.0E+00
rs6457617 / 21841780 / Graves disease / T / 0.45 / 7.0E-33 / 1.4 / 32771829 / C/T / 0.50 / 0.01 / 0.00 / 1.3E-03
rs3135338 / 20159113 / Multiple sclerosis / A / 0.13 / 2.0E-25 / 3.43 / 32509195 / C/T / 0.33 / -0.01 / 0.00 / 2.1E-01
rs2523393 / 19525953 / Multiple sclerosis / A / 0.59 / 1.0E-17 / 1.28 / 29813638 / A/G / 0.54 / 0.03 / 0.00 / 3.9E-12
rs9260489 / 22190364 / Multiple sclerosis / T / 0.45 / 1.0E-11 / 1.21 / 30028311 / G/T / 0.58 / -0.04 / 0.00 / 5.9E-23
rs10484554 / 20953190 / Psoriasis / NR / NR / 4.0E-214 / 4.66 / 31382534 / C/T / 0.87 / 0.01 / 0.01 / 9.8E-02
rs10484554 / 18369459 / Psoriasis / T / 0.15 / 2.0E-39 / 2.8 / 31382534 / C/T / 0.87 / 0.01 / 0.01 / 9.8E-02
rs12191877 / 19169254 / Psoriasis / T / 0.15 / 1.0E-100 / 2.64 / 31360904 / C/T / 0.87 / 0.01 / 0.01 / 1.1E-01
rs12191877 / 20953188 / Psoriasis / NR / NR / 4.0E-32 / 2.79 / 31360904 / C/T / 0.87 / 0.01 / 0.01 / 1.1E-01
rs2395029 / 18369459 / Psoriasis / C / 0.03 / 2.0E-26 / 4.1 / 31539759 / G/T / 0.04 / -0.05 / 0.01 / 2.6E-06
rs3134792 / 18364390 / Psoriasis / NR / NR / 1.0E-09 / NR / 31420305 / G/T / 0.12 / 0.06 / 0.01 / 3.2E-19
rs13191343 / 20953186 / Psoriatic arthritis / T / 0.13 / 2.0E-72 / 2.37 / 31349088 / C/T / 0.86 / 0.00 / 0.01 / 4.2E-01
rs9296015 / 21505073 / Rheumatoid arthritis / NR / NR / 2.0E-38 / NR / 32326967 / A/G / 0.18 / -0.01 / 0.01 / 2.5E-02
rs3131379 / 18204446 / SLE / A / 0.1 / 2.0E-52 / 2.36 / 31829012 / A/G / 0.11 / 0.05 / 0.01 / 1.6E-15
rs6457617 / 21750679 / Systemic sclerosis / T / 0.5 / 2.0E-37 / 1.61 / 32771829 / C/T / 0.50 / 0.01 / 0.00 / 1.3E-03
rs9272346 / 18978792 / Type 1 diabetes / G / NR / 6.0E-129 / NR / 32712350 / A/G / 0.58 / 0.00 / 0.00 / 8.9E-01

Abbreviation: SLE, Systemic lupus erythematosus; NR, not reported.