THE HUMAN NRAMP2 GENE - CHARACTERIZATION OF THE GENE STRUCTURE, ALTERNATIVE SPLICING, PROMOTER REGION AND POLYMORPHISMS

Nramp2 is a gene encoding a transmembrane protein that is important in metal transport, in particular iron. Mutations in nramp2 have been sh own to be associated with microcytic anemia in mk/mk mice and defectiv e iron transport in Belgrade rats. Nramp2 contains a classical iron re sponsive element in the 3' untranslated region that confers iron depen dent mRNA stabilization. In this report, we describe a splice variant form of human nramp2 that has the carboxyl terminal 18 amino acids sub stituted with 25 novel amino acids and has a new 3' untranslated regio n lacking a classical iron-responsive element. This splice form of nra mp2, nramp2 non-IRE, was found to be derived from splicing of an addit ional exon into the terminal coding exon. The nramp2 gene is comprised of 17 exons and spans more than 36 kb. It contains an additional 5' e xon and intron (exon and intron 1) and an additional 3' exon (exon 17) and intron (intron 16) as compared to nramp1, a homologous gene. The additional exons and introns account for much of the difference in len gth between nramp2 (>36 kb) and nramp1 (12 kb). The exon-intron border s of nramp2 exons 3-15 are homologous to nramp1 exons 2-14. The nramp2 5' regulatory region contains two CCAAT boxes but lacks a TATA box. T he 5' regulatory region of nramp2 also contains five potential metal r esponse elements (MRE's) that are similar to the MRE's found in the me tallothionein-II, gene, three potential SP1 binding sites and a single gamma-interferon regulatory element. Five single nucleotide mutations or polymorphisms were identified within the nramp2 gene. One of these , 1303C-->A, occurs in the coding region of nramp2 and results in an a mino acid change from leucine to isolecine. A polymorphism, 1254T/C, a lso occurs in the coding region of nramp2 but does not cause an amino acid change. The other 3 polymorphisms are within introns (IVS2+11A/G, IVS4+44C/A, and IVS6+538G/Gdel). In addition, a polymorphic microsate llite A)(10-11)CCCCCTATA(TATC)(3)(TCTG)(5)(TCCG(TCTA)(6) was identifie d in intron 3. Analysis of cDNA derived by direct amplification of rev ersed transcribed RNA or cDNA clones isolated from a library provide e vidence of skipping of exons 10 and 12 of nramp2. Deletion of either o f these exons would result in a sequence that remains in frame yet wou ld generate a protein that would lack transmembrane spanning region 7 or 8 respectively. The deletion of a single transmembrane domain would have severe topological consequences. The coding region of the nramp2 gene of hemochromatosis patients with or without mutations in the hem ochromatosis gene, HFE, were examined and found to be normal. One hemo chromatosis patient, with a normal HFE genotype, was heterozygous for the 1303C-->A mutation. Furthermore, in an examination of hemochromato sis patients with mutant HFE and normal HFE genes, we did not observe a linkage disequilibrium of either group with a particular nramp2 hapl otype. These data suggest that mutations in nramp2 are not commonly as sociated with hemochromatosis. (C) 1998 Academic Press.