Experiment
In previous studies (J:114755) C57BL/6J (B6) mice, low in liver and spleen iron content, were crossed with SWR mice that are higher in iron content in both the liver and spleen. Results revealed that QTL on Chrs 2, 7, and 16 control liver non-heme iron content in (C57BL/6J x SWR)F2 male mice. For two of the QTL, Nhil2 on Chr 16 and Nhil3 on Chr 7, the SWR alleles were associated with higher iron levels in male mice. In contrast, for the QTL on Chr 2, Nhil1, the higher iron levels in both liver and spleen were associated with the C57BL/6J allele. The aim of the current study was to identify the genes accountable for the variability of hepatic iron accumulation based on the previous QTL analysis.
A series of congenic strains were developed by continuous backcrossing of (C57BL/6J x SWR)F2 progeny to B6 mice with MIT SSLP marker-assisted tracing of the SWR chromosome 16 segments of interest. During backcrossing mice containing 20 Mb of SWR on Chr 16 were selected creating congenic strain B6.SWR-(D6Mit125-D16Mit185); backcrossing continued N>9.
Heterozygotes from the initial Line1 were backcrossed to B6 to generate subcongenic lines using the same crossing strategy creating B6.SWR-(D7Mit68 and D7Mit71). All iron phenotype analyses were performed using 8 wk old males unless otherwise indicated.
Chromosome 16 congenics that were homozygous for the SWR allele, containing the SWR haplotype, between D16Mit125 and D16Mit30 displayed significantly higher liver non-heme iron content, higher serum iron levels and transferrin (Tf)-saturation levels compared to the Chr 16 controls homozygous with B6 alleles. These results confirmed that the previously detected Nhil2 QTL fell in this region.
In contrast the Chr 7 congenics, homozygous for the SWR alleles did not show significant differences in liver non-heme iron content compared to controls homozygous with B6 alleles.
To fine map the Nhil2 QTL on Chr 16 congenic Line1 mice were continuouly backcrossed to generate subcongenic lines carrying various SWR chromosomal segments between D16Mit125 and D16Mit185. Six subcongenic lines were developed and the iron phenotype was examined in each. Congenic line 3, B6.SWR-(D16Mit379-D16Mf1) contained the smallest critical region. The SWR homozygotes of line 3 showed significantly higher serum iron and transferrin saturation levels as well as the significant increase in liver non-heme iron content compared with the B6 homozygous controls.
The iron phenotype analysis of the congenic lines refined the candidate gene interval of Chr 16 to an 830kb region. Eleven candidate genes were located within the critical region:
Zdhhc23, Gramd1c, Atp6v1a, Naa50, Gm608, Sidt1, Spice1, Wdr52, Boc, Bc027231 and Gtpbp8. Exon sequencing of the 11 genes in the critcal region identified 27 SNPs across 4 genes. Among those, 3 genes had coding sequence changes: Sidt1 (P172R), Spice1 (R708S) and Boc (Q1051R and S450-insertion in the B6 allele).
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