HAPLOTYPE MAPPING AND SEQUENCE-ANALYSIS OF THE MOUSE NRAMP GENE PREDICT SUSCEPTIBILITY TO INFECTION WITH INTRACELLULAR PARASITES

The mouse chromosome 1 locus Beg (Ity, Lsh) controls the capacity of the tissue macrophage to restrict the replication of antigenically unrelated intracellular parasites and therefore determines the natural resistance (BCG-R, dominant) or susceptibility (BCG-S, recessive) of inbred mouse strains to infection with diverse pathogens, including several Mycobacterium species, Salmonella typhimurium, and Leishmania donovani. We have used a positional cloning strategy based on genetic and physical mapping, YAC cloning, and exon trapping to isolate a candidate gene for Beg (Nramp) that encodes a predicted macrophage-specific transport protein. We have analyzed a total of 27 inbred mouse strains of BCG-R and BCG-S phenotypes for the presence of nucleotide sequence variations within the coding portion of Nramp and have carried out haplotype typing of the corresponding chromosome 1 region in these mice, using 11 additional polymorphic markers mapping in the immediate vicinity of Nramp. cDNA cloning and nucleotide sequencing identified 5 nucleotide sequence variations within Nramp in the inbred strains; while 4 of these represented silent sequence polymorphisms, one G to A substitution at nucleotide position 783 resulted in the nonconservative replacement of Gly(105) to Asp(105) within the second predicted transmembrane domain (TM2) of the Nramp protein. An absolute association of this allelic variation and Beg phenotype was observed in the 20 BCG-R strains (Gly(105)) and 7 BCG-S strains (Asp(105)) tested. Moreover, sequence analysis of the corresponding region of the Nramp gene from distantly related species indicated strong amino acid sequence conservation of TM2, including an invariant glycine at position 105. Haplotype mapping using sequence polymorphism identified within Nramp and additional RELPs and SSLPs from the region revealed that although the 20 BCG-R strains analyzed showed diverse allelic combinations for these markers, the 7 BCG-S strains tested share a conserved core haplotype of 2.2 Mb overlapping and including Nramp. Taken together, these results suggest that (1) Gly(105) is, the wildtype form of Nramp and that the nonconservative substitution to Asp(105) underlies the BCG-S phenotype, and (2) Bcg(s) alleles carry the same Gly(105) --> ASP(105) mutation and are identical by descent. (C) 1994 Academic Press, Inc.