Identification of two independent risk factors for lupus within the MHC in United Kingdom families

Abstract

The association of the major histocompatibility complex (MHC) with SLE is well established yet the causal variants arising from this region remain to be identified, largely due to inadequate study design and the strong linkage disequilibrium demonstrated by genes across this locus. The majority of studies thus far have identified strong association with classical class II alleles, in particular HLA-DRB1*0301 and HLA-DRB1*1501. Additional associations have been reported with class III alleles; specifically, complement C4 null alleles and a tumor necrosis factor promoter SNP (TNF-308G/A). However, the relative effects of these class II and class III variants have not been determined. We have thus used a family-based approach to map association signals across the MHC class II and class III regions in a cohort of 314 complete United Kingdom Caucasian SLE trios by typing tagging SNPs together with classical typing of the HLA-DRB1 locus. Using TDT and conditional regression analyses, we have demonstrated the presence of two distinct and independent association signals in SLE: HLA-DRB1*0301 (nominal p = 4.9 x 10(-8), permuted p < 0.0001, OR = 2.3) and the T allele of SNP rs419788 (nominal p = 4.3 x 10(-8), permuted p < 0.0001, OR = 2.0) in intron 6 of the class III region gene SKIV2L. Assessment of genotypic risk demonstrates a likely dominant model of inheritance for HLA-DRB1*0301, while rs419788-T confers susceptibility in an additive manner. Furthermore, by comparing transmitted and untransmitted parental chromosomes, we have delimited our class II signal to a 180 kb region encompassing the alleles HLA-DRB1*0301-HLA-DQA1*0501-HLA-DQB1*0201 alone. Our class III signal importantly excludes independent association at the TNF promoter polymorphism, TNF-308G/A, in our SLE cohort and provides a potentially novel locus for future genetic and functional studies.

Figure 1. LD Plot of All 69 MHC Markers Typed in 314 UK SLE Trios

The figure illustrates the correlation between all 69 MHC markers successfully typed across 2.4 Mb of genome encompassing MHC class II and class III in 314 UK SLE trios. The markers are identified by rs numbers for the 68 SNPs while HLA_DRB1 indicates all HLA-DRB1 alleles coded as a biallelic marker: HLA-DRB1*0301/non-HLA-DRB1*0301. The numbers shown below the marker names correspond to the marker numbering in Tables 1, S1, and S3 for ease of interpretation. The grey ticks indicate SNP location to scale. The triangular units designate haplotype blocks. The open rectangles represent the 14 markers that show significant association in the cohort after permutation testing; the green rectangle highlights our class III signal, SNP rs419788; and the red rectangle designates our class II signal, HLA-DRB1. The 53 markers used for the EHH analyses and haplotype bifurcation plots are designated by open squares. The degree of correlation between pairs of markers is indicated by the correlation coefficient r ² (where r ² = 0 = no correlation, white; 0 < r ² < 1, shades of grey; r ² = 1 = complete correlation, black).

Figure 2. Structure of Transmitted and Untransmitted HLA-DRB1*0301 and rs419788-T Allele Haplotypes

Haplotype bifurcation plots were constructed using 120 randomly selected parental chromosomes from each of the four datasets for comparative purposes except (B),where there were only 90 chromosomes in the entire dataset: (A) T HLA-DRB1*0301 haplotypes, (B) UT HLA-DRB1*0301 haplotypes, (C) T rs419788-T allele haplotypes, and (D) UT rs419788-T allele haplotypes. The allelic composition of the most common haplotype in each subset is shown: the core allele is represented as a dark blue double bar indicating haplotypes to the right and to the left of the core; otherwise, the common haplotype is depicted by dark grey bars. In parts (A) and (B), the rs419788-T allele in class III that shows association independent of HLA-DRB1*0301 in our cohort is indicated in green, while in parts (C) and (D), the allele HLA-DRB1*0301 is shown in green. The key difference between HLA-DRB1*0301 T and UT haplotypes lies within the class II region of the MHC. All HLA-DRB1*0301 T haplotypes are identical across a 180 kb region defined by eight SNPs (light blue), whereas the corresponding region within UT HLA-DRB1*0301 haplotypes exhibits significant recombination. This conserved class II interval encompasses only three expressed genes: HLA-DRB1, HLA-DQA1, and HLA-DQB1. Given the strong LD exhibited by HLA-DRB1*0301 haplotypes, the allelic composition of this risk region is known to be HLA-DRB1*0301-HLA-DQA1*0501-HLA-DQB1*0201. Both T and UT rs419788-T allele haplotypes show similar structure overall. The rs419788-T allele is clearly present on HLA-DRB1*0301 and non-HLA-DRB1*0301-containing haplotypes, lending credence to our observation that rs419788-T or another variant in LD with it constitutes an association signal independent of HLA-DRB1*0301 in our UK SLE cohort. (A) 120 from a total of 176 parental chromosomes, (B) 90 from a total of 90 parental chromosomes, (C) 120 from a total of 284 parental chromosomes, and (D) 120 from a total of 182 parental chromosomes.

Figure 3. REHH versus Frequency Analysis and Comparison of HLA-DRB1*0301 Haplotype Bifurcation Plots in UK SLE, CEPH, and Yoruba Populations

(A) Composite REHH versus frequency analysis comparing CEPH with UK SLE and Yoruba populations generated in SWEEP. REHH is shown on the vertical axis and haplotype frequency on the horizontal axis. Background CEPH genotype data for Chromosome 6 is indicated by grey data points; the 53 SNPs common to the UK SLE, CEPH, and Yoruba datasets are shown as red, blue, and turquoise data points, respectively. The 95th percentile for background variation in CEPH is indicated. The position of the HLA-DRB1*0301 allele is shown in all three cohorts (a, b, and c). The alleles rs2187668-T (HLA-DRB1*0301 tag SNP in UK SLE) (d) and HLA-DRB1*0301 in UK SLE are the only core markers observed above the 95th percentile. The associated class III SNP, rs419788-T, is also indicated (e). We can only examine evidence for positive selection in CEPH, as these are the data we have used to assess background variation. There is no evidence for positive selection of the HLA-DRB1*0301 allele in CEPH. This allele is enriched in our lupus cohort (21% of parental chromosomes), and displays greater extended homozygosity when compared with HLA-DRB1*0301-bearing haplotypes in CEPH and Yoruba. (B) Comparison of HLA-DRB1*0301 haplotype bifurcation plots for (i) UK SLE, (ii) CEPH, and (iii) Yoruba populations. We show preservation of the common HLA-DRB1*0301 haplotype in CEPH and UK SLE, while that seen in the Yoruba is significantly different (differences indicated in yellow, core allele shown in dark blue). The class II regions of all three populations are essentially identical across our chosen SNPs; the main differences lie in class III. 120 (of 1,256) randomly selected transmitted and untransmitted parental UK SLE chromosomes used for haplotype bifurcation plot for comparison with 120 total CEPH and 120 total Yoruba chromosomes.

Figure 4. LD Plot of HapMap CEPH Data Centered on the Associated Class III SNP rs419788

One hundred and twenty kilobases of the MHC class III region centered on rs419788 (boxed) in HapMap CEPH families is illustrated. The grey ticks indicate SNP location with gene location shown above and rs numbers below. The triangular units designate haplotype blocks. The degree of correlation (LD) between pairs of markers is indicated by the correlation coefficient r ² (where r ² = 0 = no correlation, white; 0 < r ² < 1, shades of grey; r ² = 1 = complete correlation, black). The dashed arrows depict LD between rs419788 and surrounding SNPs. Significant LD (r ² > 0.8) is observed with rs419788 and three further SNPs within SKIV2L, two SNPs in STK19, and one SNP in CFB.