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Genome wide association study in Immune TTP
We performed a genome-wide association study (GWAS) in UK and French iTTP cohorts and identified association of alleles both within and beyond the HLA locus.
Methods
Cohorts
As part of the UK TTP registry, patients were consented for DNA analysis (MREC: 08/H0810/54) (see the Online Supplementary Appendix). Patients on the UK TTP registry were screened for the clinical diagnosis, and confirmed with an ADAMTS13 level <10 IU/dL at diagnosis (utilizing FRETS methodology)8 and the presence of an anti-ADAMTS13 autoan- tibody.2,3 The French replication cohort TTP samples were obtained from the French Reference Center for TMA (CNR- MAT) and informed consent was obtained from each patient with confirmed iTTP (see above criteria) (Institutional Review Board of Pitié Salpêtrière Hospital; clinicaltrials gov. Identifier: NCT00426686). The European control genotypes were obtained from the Wellcome Trust Case Control Consortium (WTCCC), both the 1958 British Birth Cohort and National Blood Service control samples.9 In addition, controls were used from the Illumina reference panel10 and Oxford controls.11,12
Genotyping, quality control and imputation
TTP samples were genotyped on the Illumina Human Omni Express single-nucleotide polymorphisms (SNP) chips and con- trols were genotyped on different SNP chips (see the Online Supplementary Appendix). Pre-imputation quality control was per- formed in all data sets separately, and then in a combined cohort (Online Supplementary Figure S1). Quality control (QC) was per- formed for individuals and SNP. Individuals were selected for further analysis by European ancestry principal component analysis (PCA) (see the Online Supplementary Figure S2). Only SNP present in all data sets were subsequently analyzed.
Genome-wide imputation was performed on markers that had passed quality control, and were present in all datasets using Beagle (version 5.0) utilizing the 1.000 Genome Project Phase 3 as a reference panel.13 In addition to standardized QC, only SNP with a dosage R2 (DR2) >0.8 were included.
Genome-wide association study and loci characterization
GWAS was performed using SNP & Variation Suite v8, using logistic regression with principal component correction.14,15 The logistic regression P-values, odds ratios (OR) were calculated in addition to l inflation factors, and QQ plots are shown (Online Supplementary Figure S3). A standardized genome wide signifi- cance level of 5x10-8 was applied.15 For discovery and replication analysis meta-data please contact the authors.
Conditional analyses were undertaken using a full versus reduced regression model. Lead SNP at each locus were used as conditional inputs to determine independence, with results plot- ted using Locus Zoom software.16
Imputation of HLA types was performed utilizing SNP2HLA with previously genotyped markers. 17 Imputed HLA types were excluded if the R2 (confidence) was <0.80. Conditional analyses were subsequently performed as described above.
Expression quantitative trait locus (eQTL) analysis was per- formed to associate identified SNP with differential gene expres- sion.18 Additional markers in linkage disequilibrium with the lead SNP at the chromosome 3 locus were identified by LD-link (https://ldlink.nci.nih.gov).19 Functional annotation of the hap-
loblock was performed using ChipSeq data via the UCSC genome browser (https://genome.ucsc.edu). Binding sites of transcription factors (highlighted through genome annotation) were obtained from FactorBook,20 and position weight matrix (PWM) binding motifs generated. Binding motifs were generated using Mast-Meme.21
Results
Discovery cohort
Following quality control as outlined in the methods (Online Supplementary Figure S1) there were 241 TTP cases and 3,200 controls in the UK discovery cohort. Following imputation and quality control 3,649,347 SNP were avail- able for analysis. Association testing was performed using a logistic regression model with PCA correction, and the genomic inflation factor (l) was 1.0239 (Online Supplementary Figure S3).
In the UK discovery cohort two peaks were identified (Figure 1) (Online Supplementary Figure S4) (lead SNP are summarized in Table 1). The peak with the strongest association corresponded to the class II HLA region on chromosome 6, with 1,017 SNP reaching genome wide significance. The lead SNP rs28383233 located in the intergenic region between HLA-DRB1 and HLA-DQA1 (P=2.20x10-23, OR 3.12, 95% Confidence Interval [CI]: 2.49-3.93) (Table 1; Figure 2).
Conditional analysis was performed on rs28383233 and the lead SNP following this was rs1064994 (within HLA- DQA1), with a P-value of 1.13x10-10 (OR 2.20, 95% CI: 2.06-3.37). Following conditioning on both rs28383233 and rs1064994 no further markers reached significance within the class II HLA region, indicating that there are two detectable independent genetic associations with iTTP within the HLA region.
HLA imputation was performed on the UK discovery cohort, and following quality control, 95 imputed HLA alleles remained. HLA-DRB1*11:01 was the allele most strongly associated with iTTP, with a P-value of 3.25x10- 17 (OR 2.79, 95% CI: 2.23-3.50). Following conditional analysis of HLA-DRB1*1101, no other HLA types reached genome wide significance, but HLA-DQA1*03:01 remained significant (with a HLA-only Bonferroni correc- tion, P<5.26x10-4) at 1.49x10-6 (OR 0.47, 95% CI: 0.33- 0.65) suggesting that the protective effect of this allele is independent of HLA-DRB1*11:01.
In addition to the class II HLA peak on chromosome 6, a novel association was observed on chromosome 3. Sixteen markers reached genome wide significance, with the lead SNP, rs9884090(A), having a P-value of 5.22x10-10 (OR 0.40, 95% CI: 0.29-0.56) (Table 1; Figure 3). Upon conditional analysis of the lead SNP no markers reached genome wide significance indicating one detectable signal at this locus. No statistical epistasis was seen between the chromosome 3 and chromosome 6 associations, with each association being independent. Five genes are anno- tated within this chromosome 3 haploblock: ARHGAP31, TMEM39A, POGLUT1, TIMMDC1, and CD80.
Replication cohort
Within the French replication cohort there were 112 cases and 2,603 controls following quality control as out- lined in the methods (Online Supplementary Figures S1 and S2). 3,649,546 SNP were available for analysis, and asso-
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