Fine-Mapping and Genetic Analysis of the Loci Affecting Hepatic Iron Overload in Mice

abstract: ...The liver, as the major organ for iron storage and production of hepcidin, plays pivotal roles in maintaining mammalian iron homeostasis. A previous study showed that Quantitative Trait Loci (QTLs) on chromosome 7 (Chr7) and 16 (Chr16) may control hepatic non-heme iron overload in an F2 intercross derived from C57BL/6J (B6) and SWR/J (SWR) mice. In this study, we aimed to validate the existence of these loci and identify the genes responsible for the phenotypic variations by generating congenic mice carrying SWR chromosome segments expanding these QTLs (D7Mit68-D7Mit71 and D16Mit125-D16Mit185, respectively). We excluded involvement of Chr7 based on the lack of iron accumulation in congenic mice. In contrast, liver iron accumulation was observed in Chr16 congenic mice. Through use of a series of subcongenic murine lines the interval on Chr16 was further fine-mapped to a 0.8 Mb segment spanning 11 genes. We found that the mRNA expression pattern in the liver remained unchanged for all 11 genes tested. Most importantly, we detected 4 missense mutations in 3 candidate genes including Sidt1 (P172R), Spice1(R708S), Boc (Q1051R) and Boc (S450-insertion in B6 allele) in the liver of SWR homozygous congenic mice. To further delineate potential modifier gene(s), we reconstituted seven candidate genes, Sidt1 , Boc , Zdhhc23 , Gramd1c , Atp6v1a , Naa50 and Gtpbp8 , in mouse liver through hydrodynamic transfection. However, we were unable to detect significant changes in liver iron levels upon reconstitution of these candidate genes. Taken together, our work provides strong genetic evidence of the existence of iron modifiers on Chr16. Moreover, we were able to delineate the phenotypically responsible region to a 0.8 Mb region containing 11 coding genes, 3 of which harbor missense mutations, using a series of congenic mice.

... missense mutations in 3 candidate genes including Sidt1 (P172R), Spice1(R708S), Boc (Q1051R) and Boc (S450-insertion in B6 allele) in the liver of SWR homozygous congenic mice. To further delineate potential modifier gene(s), we reconstituted seven candidate genes, Sidt1 , Boc , Zdhhc23 , Gramd1c , Atp6v1a , Naa50 and Gtpbp8 , in mouse liver through hydrodynamic transfection. However, we were unable to detect significant changes in liver iron levels upon reconstitution of these candidate genes. Taken

... sequence analysis DNA segments of every exon and intron-exon boundary of the candidate genes were amplified using PCR and the sequences were aligned in comparison with B6 using Lasergene software (DNASTAR). All sequencing data has been deposited in GenBank and the Accession numbers are listed in Table 1 . 10.1371/journal.pone.0063280.t001 Table 1 Summary of genomic DNA sequencing results in the candidate interval. Gene Accession number Polymorphism Position in gene Protein Variation Sidt1 rs4179590 G/A

...), rabbit anti-flag(11000 dilution, cell signaling), rabbit anti-Sidt1(11000 dilution, Santa Cruz), rabbit anti--actin (12000 dilution, Sigma), rabbit anti-mouse ferroportin(11000).Quantification of western blots was performed using Quantity One (Bio-Rad) according to the manual. Constructs and plasmids Full-length mouse cDNA of candidates including Zdhhc23 , Gramd1c , Atp6v1a , Naa50 , Sidt1 , Boc and Gtpbp8 were obtained from Open Biosystems. We first cloned each gene's CDS into pCMV-3Tag-3A (Agilent Technologies) with a C-terminally expressed 3Flag tag. The 3flag fusion proteins were then subcloned into the final pLive Vector (Mirus, Madison, WI) for Hydrodynamic-based transfection. For cell-based iron uptake assays, CDS of mSidt1 (B6 and SWR allele) was cloned into pCMV-Sport6 (Open Biosystems). Mutagenesis: The pCMV-Sidt1-SWR clone containing the P172R mutation was constructed using the QuikChange II Site-Directed Mutagenesis Kit(Stratagene) following the manufacturer's instructions. Hydrodynamic

... either have a coding sequence variation that changes the function of the protein or a regulatory sequence variation that could cause differences in gene expression levels between the two parental strains. Exon sequencing all of 11 genes in the critical interval ( Figure 4B ) identified 27 SNPs across four genes ( Table 1 ). Among these, three genes have coding sequence changes: Sidt1 (P172R), Spice1 (R708S), Boc (Q1051R and S450-insertion in B6 allele). 10.1371/journal.pone.0063280.g004 Figure 4 Fine

...] ) and high liver iron strains (FVB/J, DBA/2J and Blab/cJ) ( Figure 4C ). Boc has an insertion of a Ser residue in C57BL strains that makes it different from all other mouse strains and species tested. Moreover, Boc also contained a Q1051R variation, which is conserved in some other mammalian species ( Figure S4A, S4B ). The P172R amino acid substitution in Sidt1 is unique to C57BL strains, altering a residue that is conserved between mouse strains and primates ( Figure S4C ). While Spice1 has an

... counter and normalized using protein concentrations. Similar results were obtained in three independent experiments with triplicates. Data represent mean SEM. N.S: no significance, * P<0.05, ** P<0.01. Among the candidates, Sidt1 encodes a mouse gene homologous with Systemic RNAi Deficient-1 in C. elegans , a multi-span transmembrane protein necessary for systemic RNAi activity [32] . A recent study identified Sidt1 as an active dsRNA-gated dsRNA channel [33] . In mice, Sidt1 protein is located on

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Gene: SIDT1 - SID1 transmembrane family, member 1 - (NCBI)

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