Lupus risk variants in the PXK locus alter B-cell receptor internalization

Abstract

Genome wide association studies have identified variants in PXK that confer risk for humoral autoimmune diseases, including systemic lupus erythematosus (SLE or lupus), rheumatoid arthritis and more recently systemic sclerosis. While PXK is involved in trafficking of epidermal growth factor Receptor (EGFR) in COS-7 cells, mechanisms linking PXK to lupus pathophysiology have remained undefined. In an effort to uncover the mechanism at this locus that increases lupus-risk, we undertook a fine-mapping analysis in a large multi-ancestral study of lupus patients and controls. We define a large (257kb) common haplotype marking a single causal variant that confers lupus risk detected only in European ancestral populations and spans the promoter through the 3' UTR of PXK. The strongest association was found at rs6445972 with P < 4.62 × 10(-10), OR 0.81 (0.75-0.86). Using stepwise logistic regression analysis, we demonstrate that one signal drives the genetic association in the region. Bayesian analysis confirms our results, identifying a 95% credible set consisting of 172 variants spanning 202 kb. Functionally, we found that PXK operates on the B-cell antigen receptor (BCR); we confirmed that PXK influenced the rate of BCR internalization. Furthermore, we demonstrate that individuals carrying the risk haplotype exhibited a decreased rate of BCR internalization, a process known to impact B cell survival and cell fate. Taken together, these data define a new candidate mechanism for the genetic association of variants around PXK with lupus risk and highlight the regulation of intracellular trafficking as a genetically regulated pathway mediating human autoimmunity.

Keywords: B cells; BCR; PXK; fine-mapping; lupus.

Figure 1

Variants in the PXK locus are associated with lupus. (A) Plot of –log₁₀(p) values for directly genotyped (red) and imputed (blue) SNPs spanning the previously identified region of association with lupus. Gene locations are indicated above with arrows indicating direction of transcription. (B) Plot of –log₁₀(p) values for different ancestral cohorts. EA, European ancestry; AA, African American ancestry; HA, Hispanic and Amerindian ancestry; AS, Asian ancestry. While not the most highly associated SNP, rs4681677 (purple) was used to choose samples for biological studies because it was genotyped; this SNP is in high LD (r² > 0.9) with the imputed SNPs that are more highly associated.

Figure 2

Evaluation of linkage disequilibrium in the PXK locus and Bayesian association analysis. (A) Association plot of genotyped and imputed SNPs shaded to identify the significant LD in the region. Colors correspond to r²-values, with darker shade indicating higher r²-values. (B) Bayesian analysis of variants in the PXK locus. Plot of posterior probability calculated for each variant in the locus. Variants included in the 95% credible set based on posterior probabilities are colored green.

Figure 3

Step-wise logistic regression analysis reveals one independent genetic effect at the PXK locus. Plot of P values (–log₁₀) following adjustment for genotype at rs4681677. Adjustment for other SNPs with r² > 0.9 with rs4681677 can also account for the genetic association (P < 0.01) in the region.

Figure 4

PXK expression in cell lines derived from study participants. (A) Plot of ΔΔCt values of PXK mRNA expression using TaqMan probes. (n = 14 risk/14 protective) (B) PXK expression at the protein level using flow cytometetry. (n = 8 homozygous risk/5 homozygous protective). Data for both A and B are representative of 3 independent experiments using control cell lines. Statistical analysis using a Student's t-test was performed in PRISM.

Figure 5

PXK colocalizes with the BCR following receptor crosslinking. (A) Quantification of colocalization between PXK and the BCR following BCR crosslinking in primary B cells from subjects without lupus. (B) Representative images showing colocalization. Statistical analysis using a One-Way ANOVA was performed in PRISM.

Figure 6

B cells carrying PXK risk allele demonstrate decreased internalization of the BCR. (A) Representative flow cytometry histogram showing decreasing fluorescence following internalization of the BCR from the B cell surface using patient-derived LCLs. (B) Quantification of BCR internalization as measured by the percent internalization (decrease in gMFI) following receptor crosslinking. Results of linear mixed model analysis as performed in R are shown. This experiment was performed using 20 cell lines from subjects without lupus in three independent experiments. The results of these independent experiments were combined and presented in (B).

Figure 7

shRNA knockdown of PXK disrupts BCR colocalization. (A) PXK protein expression following shRNA knockdown in 5 LCL lines from non-lupus subjects (expression was assessed in triplicate for each cell line). (B) Summary of BCR internalization at 2 min in LCLs following viral transduction with either an shRNA against PXK or a scrambled control. Results from unpaired Student's t-test shown. (C) Correlation between PXK protein expression and BCR internalization following shRNA knockdown. The significance was assessed using a Pearson correlation. The graphs in (B,C) are representative of the results of three independent experiments.