A Missense LRRK2 Variant Is a Risk Factor for Excessive Inflammatory Responses in Leprosy

Abstract

Background: Depending on the epidemiological setting, a variable proportion of leprosy patients will suffer from excessive pro-inflammatory responses, termed type-1 reactions (T1R). The LRRK2 gene encodes a multi-functional protein that has been shown to modulate pro-inflammatory responses. Variants near the LRRK2 gene have been associated with leprosy in some but not in other studies. We hypothesized that LRRK2 was a T1R susceptibility gene and that inconsistent association results might reflect different proportions of patients with T1R in the different sample settings. Hence, we evaluated the association of LRRK2 variants with T1R susceptibility.

Methodology: An association scan of the LRRK2 locus was performed using 156 single-nucleotide polymorphisms (SNPs). Evidence of association was evaluated in two family-based samples: A set of T1R-affected and a second set of T1R-free families. Only SNPs significant for T1R-affected families with significant evidence of heterogeneity relative to T1R-free families were considered T1R-specific. An expression quantitative trait locus (eQTL) analysis was applied to evaluate the impact of T1R-specific SNPs on LRRK2 gene transcriptional levels.

Principal findings: A total of 18 T1R-specific variants organized in four bins were detected. The core SNP capturing the T1R association was the LRRK2 missense variant M2397T (rs3761863) that affects LRRK2 protein turnover. Additionally, a bin of nine SNPs associated with T1R were eQTLs for LRRK2 in unstimulated whole blood cells but not after exposure to Mycobacterium leprae antigen.

Significance: The results support a preferential association of LRRK2 variants with T1R. LRRK2 involvement in T1R is likely due to a pathological pro-inflammatory loop modulated by LRRK2 availability. Interestingly, the M2397T variant was reported in association with Crohn's disease with the same risk allele as in T1R suggesting common inflammatory mechanism in these two distinct diseases.

Fig 1. Family based sample and study design.

Two sets of families were employed: those with T1R-affected offspring and those with leprosy but T1R-free offspring. The T1R-affected subset comprised 229 offspring belonging to 221 families while the T1R-free subset comprised 229 offspring in 209 families. Offspring were matched by clinical leprosy subtype in the two family sets. In a first analysis stage, the transmission disequilibrium test (TDT) was used to estimate significance of association of LRRK2 variants with disease in each subset. In a second stage, a formal heterogeneity test was performed to identify LRRK2 variants preferentially associated with T1R.

Fig 2. Association fine mapping of the LRRK2 locus in the T1R-affected and T1R-free family subsets.

Association results for 156 SNPs mapping to a 500 kb genomic interval encompassing the LRRK2/MUC19 genes are shown at the top (T1R-affected) and at the center (T1R-free) of the graph. The SNPs are plotted according to their chromosomal position (GRCh37) on the x-axis against their negative log¹⁰ of association P-values (NLP) on the left y-axis. The blue line indicates the corresponding recombination rate in centimorgan per mega-base (cM/Mb) according to the 1000 genomes project (right y-axis). The red line symbolizes the 0.05 threshold for significant associations. SNPs that displayed significant evidence for heterogeneity of association with disease in the two family sets are indicated in black. At the bottom, the diamond plot represents the linkage disequilibrium pattern among the 18 SNPs preferential associated with T1R. The strength of pairwise linkage disequilibrium (r²) is indicated by color with yellow indicating weak LD and red strong LD.

Fig 3. Host versus pathogen control of LRRK2 expression levels.

LRRK2 expression levels for 53 unrelated subjects are indicated on the y-axis and stratified according to rs2404580 genotypes on the x-axis. The left panel represents baseline expression while the right panel indicates gene expression levels following stimulation with M. leprae antigen.

Fig 4. Proposed mechanism for LRRK2 in T1R.

The LRRK2 M2397T amino acid substitution affects protein turnover. The methionine variant of LRRK2 displays a half-life of approximately 8 hours while the half-life of the threonine variant is 18 hours [34]. LRRK2 arrests the NFAT transcription factor in the cytoplasm through a complex mechanism mediated by Ca²⁺ [36]. This prevents NFAT to migrate to the nucleus and trigger the expression of pro-inflammatory cytokines [35]. The M2397 allele is in tight linkage disequilibrium with alleles of SNPs that promote an increase in LRRK2 expression creating a compensatory mechanism to counterbalance the shorter LRRK2-M2397 half-life. This compensatory mechanism is abrogated in the presence of M. leprae antigen. Hence, the effect of the M2397T amino acid substitution is most pronounced in the presence of M. leprae antigen.