Experiment
In the current study, basal liver iron levels in 18 inbred mouse strains were measured. Substantial variation was found between the strains. Secondary phenotypes measured included the level of circulating diferric transferrin as another measure of iron status, and liver zinc and copper levels due to the known interactions between iron and these metals. Genome-wide haplotype association mapping (HAM) analysis of these traits was performed in a subset of 14 inbred strains for which genotype information was available.
Four-week-old mice were obtained from The Jackson Laboratory (Bar Harbor, ME).At least six male and six female animals were used from 17 inbred strains: 129S1/SvImJ, A/HeJ, A/J, AKR/J, B10.D2-Hc0H2dH2-T18c/oSnJ, BALB/cByJ, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, LG/J, LP/J, MRL/MpJ, NZB/BlNJ, SM/J, and SPRET/EiJ. For the 18th strain, NZW/LacJ, only female animals were available. Mice were fed ad libitum an AIN93G purified diet containing ~35 ppm iron (Dyets) to 8 wk of age. Mice were euthanized, blood was collected and liver tissue was collected. Liver tissue was vacuum dried overnight, weighed and samples were diluted with metal-free water to give a final nitric acid concentration of ~1 M of nitric acid and were used to assess the levels of iron, copper, and zinc. Levels of plasma diferric transferrin were determined by urea polyacrylamide gel electrophoresis.
The 'SNPster' genotype file contained genotypic information for 16 of the phenotyped strains (A/HeJ and B10.D2-Hc0H2dH2-T18c/oSnJ had no genotype data available). Wild-derived strains, CAST/EiJ and SPRET/EiJ, were omitted for in silico analysis since the presence of unique haplotypes in these strains compromises characterization of haplotype blocks. The dataset from the remaining 14 strains contained allele calls for 137,868 SNPs (including high-confidence imputed calls) distributed over 20 chromosomes. SNP density averaged ~19 kb per SNP across the genome. All SNP locations were mapped to the National Center for Biotechnology Information mouse genome map build 36.1.
The F-statistic was calculated for a given phenotype and inferred haplotype groups in one-factor ANOVA. The significance of the F-statistic was estimated from background distribution simulated nonparametrically using 1 x 10(6) bootstraps of the phenotypic values. The resulting P value was -log(10) transformed to produce an association score (AS). Results are reported for putative QTL above a threshold set at the fifth percentile of the best 1,000 results for each trait. For every putative QTL corresponding QTL locations (Mb) are calculated by expanding each QTL to the left and right for as long as the association score remained >2 (or by at least 100 kb). For the comparison of these findings with those from linkage studies, centimorgan (cM) to mega-base (Mb) conversions were performed using Mouse Map Converter at http://cgd.jax.org. 'Gene Relationships Across Implicated Loci' (GRAIL) software was used to examine relationships between genes in putative QTL and known genes influencing a given trait.
Since relaxed significance criteria were used the discussion focused only on the six putative QTL for which corroborating data have been obtained [in the form of previously reported QTL influencing that specific trait, literature reports on the function of specific gene(s) in the putative QTL, or similarity to genes known to influence that trait, as predicted by GRAIL analysis].
Curator Note: If previously reported QTL were mapped using a different mapping population than the population used in the current study, we have assigned the QTL mapped here unique QTL nomenclature.
Results:
Putative QTL Lvil1 (liver iron level 1) was mapped in male mice to Chromosome 7 between 33.92-34.21 Mb. The QTL size was reported as 0.29 Mb and contained the genes Gpi1 and Lsm14a. [Table 2]. Livl1 overlapped with the previously reported Hfem1 QTL on Chr 7 (D7Mit246) identified in J:90494 using Hfetm1Gfn knockout animals from a (C57BL/6-Hfetm1Gfn x DBA/2-Hfetm1Gfn)F2 intercross to identify loci associated with susceptibility to iron loading. [Table 3].
Putative QTL Lvil2 (liver iron level 2) was mapped in female mice to Chromosome 7 between 133.93-134.15 Mb. The QTL size was reported as 0.22 Mb and contained the gene Adam12 previously identified as a candidate gene for fat mass associated with atherogenic diet. [Table 2]. Livl2 overlapped with the previously reported Nhil3 QTL (D7Mit71) identified in J:114755 using mice from a (C57BL/6JOla x SWR/Ola)F2 intercross to identify QTL associated with non-heme iron levels in the liver and spleen. [Table 3].
Putative QTL Lvil3 (liver iron level 3) was mapped in male mice to Chromosome 8 between 89.22-89.86 Mb. The QTL size was reported as 0.64 Mb and contained the genes Abcc12, Lonp2, Siah1a, Gm10638 and N4bp1. [Table 2]. Livl3 overlapped with a previously reported suggestive QTL mapping to D8Mit195 linked to liver iron content in female mice in map study J:114755. [Table 3].
Putative QTL Lvil4 (liver iron level 4) was mapped in female mice to Chromosome 11 between 56.70-57.02 Mb. The QTL was reported as 0.32 Mb in size and contained the Gria1 gene. [Table 2]. Lvil4 overlapped with a previously reported suggestive QTL mapping to D11Mit36 linked to liver iron content in female mice in map study J:114755. [Table 3].
The mapping study also identified an additional 22 novel putative QTL influencing iron levels in the liver [Table 2]. Two of the novel QTL contain genes reported to play a role in iron homeostatis:
Putative QTL Lvil5 (liver iron level 5) was mapped in male mice to Chromosome 5 between 144.01-144.36 Mb. The QTL was reported as 0.35 Mb in size and contained genes Eif2ak1, Aimp2, Pms2, Ocm, and Lmtk2. Eif2ak1 is known to play a role in hemoglobin production, the maturation of macrophages and the inflammatory response.
Putative QTL Lvil6 (liver iron level 6) was mapped in female mice to Chromosome 17 between 12.16-12.64 Mb. The QTL was reported as 0.48 Mb in size and contained the genes Map3k4, Plg, Slc22a3, Slc22a2, Igf2r and Airn. Igf2r has a known role in iron sensing.
Additional putative QTL were identified for diferric transferrin levels [Table 4], and for liver copper and zinc levels [Table 5].
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