Genetic and functional analysis of common MRC1 exon 7 polymorphisms in leprosy susceptibility

Abstract

The chromosomal region 10p13 has been linked to paucibacillary leprosy in two independent studies. The MRC1 gene, encoding the human mannose receptor (MR), is located in the 10p13 region and non-synonymous SNPs in exon 7 of the gene have been suggested as leprosy susceptibility factors. We determined that G396S is the only non-synonymous exon 7-encoded polymorphism in 396 unrelated Vietnamese subjects. This SNP was genotyped in 490 simplex and 90 multiplex leprosy families comprising 704 patients (47% paucibacillary; 53% multibacillary). We observed significant under-transmission of the serine allele of the G396S polymorphism with leprosy per se (P = 0.036) and multibacillary leprosy (P = 0.034). In a sample of 384 Brazilian leprosy cases (51% paucibacillary; 49% multibacillary) and 399 healthy controls, we observed significant association of the glycine allele of the G396S polymorphism with leprosy per se (P = 0.016) and multibacillary leprosy (P = 0.023). In addition, we observed a significant association of exon 7 encoded amino acid haplotypes with leprosy per se (P = 0.012) and multibacillary leprosy (P = 0.004). Next, we tested HEK293 cells over-expressing MR constructs (293-MR) with three exon 7 haplotypes of MRC1 for their ability to bind and internalize ovalbumin and zymosan, two classical MR ligands. No difference in uptake was measured between the variants. In addition, 293-MR failed to bind and internalize viable Mycobacterium leprae and BCG. We propose that the MR-M. leprae interaction is modulated by an accessory host molecule of unknown identity.

Fig. 1

Pairwise linkage disequilibrium (LD) as defined by r² across the MRC1 gene. The chromosome 10p location of 47 SNPs from rs2947600 (MRC1 intron 1) to rs941 (MRC1 3′-UTR) is indicated above the LD plot. The r² value for each SNP pair is indicated within the corresponding diamond. Increasing depth of black color indicates higher r² values. D′-defined haplotype blocks are also indicated at the top of the LD plot. There is no evidence for long range LD extending over the entire MRC1 gene

Fig. 2

Human MRC1 exon 7 haplotypes. In the Brazilian sample, four common MRC1 exon7 polymorphisms were found, three of which were non-synonymous (boxed). Due to strong LD between SNPs two (rs2478577), three (rs2437256) and four (rs2437257), only three amino acid haplotypes were observed and termed MR (GAF), MR (SAF) and MR (GTL). SNP 1 (rs1926736) gives rise to a G/S amino acid polymorphism that was only observed on the MR(AF) background. Among these three haplotypes, only two–MR(GAF) and MR(SAF)–were observed in the Vietnamese sample

Fig. 3

Ectopic MR is functional in HEK293 cells. A MR cDNA was transduced into HEK293 cells. The resulting cells were compared to vector-transduced cells and microvascular endothelial cells (HDMEC) which express endogenous MR. a Cells were treated with Alexa-488-conjugated ovalbumin (an MR ligand, shown in green) for 1 h then immunostained with monoclonal anti-MR followed by Cy3-conjugated anti-mouse (red). b Cells were treated with fluorescein-conjugated zymosan for 16 h, then washed and stained with rabbit anti-fluorescein followed by Cy3 anti-rabbit antibodies. Ingested zymosans are green while non ingested particles are stained red and appear yellow in the merged images. Nuclei are stained blue with DAPI

Fig. 4

Absence of biological differences among MR variants. HEK293 transductants were analyzed for MR function by flow cytometry. a Transduced stable populations of HEK293 cells were immunostained for MR. b Populations were incubated for 1 h with 5 μg/ml Alexa488-conjugated ovalbumin and uptake was measured by FACS. Percentage of positive cells is given as well as median fluorescence value (MFV) of the positive population. c Populations were incubated with 10⁶ zymosan-FITC particles (ratio zymosan:cell 5:1) for 16 h. Extracellular particles were then stained with anti-FITC and Cy5-conjugated secondary antibodies. Cells were lysed and intracellular and extracellular zymosans were gated in FL1 and counted in FL4 channels. Data is expressed as percent of ingested zymosans over total bound, and as number of bound zymosan per cell. Results shown here are representative of three independent experiments

Fig. 5

Absence of impact of ectopic MR expression on binding to mycobacteria. HEK293 transductants were incubated for 16 h with PKH67-stained fluorescent Mycobacterium leprae (a) and GFP-expressing Mycobacterium bovis BCG strain Pasteur (b) at a MOI of 5. Measurement of bacterial binding was performed by microscopic examination, counting at least 500 cells in four different fields. Results per field were averaged per cell and are given on the left of the graph. Results are given for HEK293 control cells (PURO) and HEK293 cells over-expressing the MR(GAF), MR(SAF) and MR(GTL) expression constructs