Experiment
This study used a systems genetics analysis of fibrosis in the BXD murine reference panel that provided for the integration of multiple traits. BXD mice are the inbred progeny of F2 intercrosses of the fibrosis-susceptible inbred strain DBA/2J (D2) and the more resistant inbred strain C57BL/6J (B6).
In addition to the parental strains and the F1 hybrids liver fibrosis phenotypes and gene expression profiles were determined in 35 BXD lines with an average of 6 mice per sex and line, resulting in a total of 581 mice. Liver fibrosis was induced at 8 wks of age; administered by intraperitonal injections of CCL4 twice weekly.
The quantitative CCL4 -induced phenotypes measured were hepatic collagen contents (hydroxyproline levels and collagen areas) and fibrosis stage (F-score) was measured as a semi-quantitative measure in histological liver sections. Trait values of each BXD line were analyzed for each sex separately, as well as for combined data sets of female and male mice. All phenotypic data was integrated into the GeneNetwork database. For the identification of single QTL, interval mapping analyses were performed across all chromosomes, including the parental strains (NCBI Build 37). Composite interval mapping was performed for every phenotype, choosing genetic markers with the highest likelihood ratio statistics (LRS) at each single QTL region or interacting loci from pairwise interaction scans.
Significance was estimated from the LRS; genome wide significance was evaluated by testing 2000 permutations, which specified a significant threshold corresponding to a genome wide p-value of 0.05 and a suggestive threshold of 0.63. Confidence intervals of chromosomal regions spanning QTL positions were specified as 1.5 logarithm of the odds (LOD) support intervals.
Single QTL genome scans identified 28 trait associated loci with LRS scores above the suggestive threshold (LRS= or > 12) affecting liver fibrosis phenotypes. Among those 28, significantly linked loci were detected by composite interval mapping (p<0.05) on Chromosomes 4 (Lfibq13), 5 (Lfibq4, Lfibq5), 7 (Lfibq14), 12 (Lfibq16) and 17 (Lfibq28).
QTL Lfibq1 (liver fibrosis QTL 1) mapped to Chromosome 2 in the single QTL scan, LRS= 14.0 between SNPs rs638488 and rs3664044. The 1.5 LOD support interval mapped from 167.7 to 179.3 Mb. Lfibq1 influenced collagen area in male mice.
QTL Lfibq2 (liver fibrosis QTL 2) mapped to Chromosome 3 in the single QTL scan, LRS=12.5 between SNPs rs13476999 and mCV24211562. The 1.5 LOD support interval mapped from 12.0 to 20.6 Mb influencing collagen area in female mice.
QTL Lfibq3 (liver fibrosis QTL 3) mapped to Chromosome 3 in the single QTL scan, LRS=15.0 between SNPs rs13477485 and rs13477494. The 1.5 LOD support interval mapped from 146.1 to 157.0 Mb influencing collagen area in female mice.
QTL Lfibq4 (liver fibrosis QTL 4) mapped to Chromosome 5 in both single and composite interval mapping. LRS=18.1 between markers mCV23582150 and rs6392739 with a 1.5 LOD support interval mapping between 3.1 and 20.1 Mb in the male and the combined mapping populations influencng collagen area.
QTL Lfibq5 (liver fibrosis QTL 5) mapped to Chromosome 5 in both single and composite interval mapping. LRS=23.1 between SNPs rs3678577 and rs6167407 with a 1.5 LOD support interval between 85.1 and 97.9 Mb in the female and the combined mapping populations influencing collagen area.
QTL Lfibq6 (liver fibrosis QTL 6) mapped to Chromosome 7 in the single QTL scan, LRS=12.5 between markers gnf07.050.858 and rs6166250. The 1.5 LOD support interval mapped between 55.3 and 74.2 Mb influencing collagen area in female mice.
QTL Lifbq7 (liver fibrosis QTL 7) mapped to Chromosome 7 in the single QTL scan between SNPs rs13479559 and rs3659292. The 1.5 LOD support interval mapped between 141.7 and 149.0 Mb influencing collagen area in male mice.
QTL Lifbq8 (liver fibrosis QTL 8) mapped to Chromosome 9 in the single QTL scan, LRS=14.4 between markers D9Mit227 and rs13480169. The 1.5 LOD support interval mapped between 41.1 and 46.6 Mb influencing collagen area in female mice.
QTL Lifbq9 (liver fibrosis QTL 9) mapped to Chromosome 13 in the single QTL scan, LRS=16.7 between SNPs rs6411274 and rs3682400. The 1.5 LOD support interval mapped between 45.5 and 51.7 MB influencing collagen area in the male and the combined mapping populations.
QTL Lifbq10 (liver fibrosis QTL 10) mapped to Chromosome 15 in the single QTL scan, LRS=13.9 between SNPs rs3710055 and rs13482702. The 1.5 LOD support interval mapped between 82.3 and 95.8 Mb influencing collagen area in male mice.
QTL Lifbq11 (liver fibrosis QTL 11) mapped to Chromosome 19 in the single QTL scan, LRS=13.3 between SNPs rs13483649 and rs8257607. The 1.5 LOD support interval mapped between 36.2 and 52.2 Mb influencing collagen area in male mice.
QTL Lifbq12 (liver fibrosis QTL 12) mapped to Chromosome X in the single QTL scan, LRS=17.1 between markers gnfX.023.534 and gnfX.026.801. The 1.5 LOD support interval mapped between 25.2 and 41.9 Mb influencing collagen area in male mice.
The alleles of the fibrosis susceptible strain, DBA/2J increased the trait value with an additive effect for QTL Lfibq1 through Lfibq12 with the exception of QTL Lifbq8, where the C57BL/6J allele exerted the most influence on the collagen area in female mice.
QTL Lifbq13 (liver fibrosis QTL 13) mapped to Chromosome 4 in both the single and composite interval mapping. LRS=17.4 between SNPs rs6254381 and rs13477745 with a 1.5 LOD support interval mapped between 55.1 and 73.9 Mb influencing hydroxyproline levels (Hyp) in the female and the combined mapping populations.
QTL Lifbq14 (liver fibrosis QTL 14) mapped to Chromosome 7 in both the single and composite interval mapping. LRS=16.3 between SNPs rs3703247 and rs8255275 with a 1.5 LOD support interval mapped between 52.8 and 56.7 Mb influencing hydroxyproline levels (Hyp) in female and the combined mapping populations.
QTL Lifbq15 (liver fibrosis QTL 15) mapped to Chromosome 5 in both the single and composite interval mapping, LRS=14.4 between SNPs rs13478413 and rs6228198 with a 1.5 LOD support interval mapped between 82.8 and 103.9 Mb influencing hydroxyproline levels (Hyp) in female and the combined mapping populations
QTL Lifbq16 (liver fibrosis QTL 16) mapped to Chromosome 12 in both the single and composite interval mapping, LRS=25.0 between SNPs rs3716547 and rs13481511 with a 1.5 LOD support interval mapped between 60.5 and 73.3 Mb influencing hydroxyproline levels (Hyp) in female and the combined mapping populations.
QTL Lifbq17 (liver fibrosis QTL 17) mapped to Chromosome X in both the single QTL scan, LRS=12.9 at marker CEL-X_154048891 with a 1.5 LOD support interval that mapped between 145.8 and 162.6 Mb influencing hydroxyproline levels (Hyp) in female mice.
The alleles of the fibrosis susceptible strain, DBA/2J increased the trait value with an additive effect for QTL Lfibq13 through Lfibq17 with the exception of QTL Lifbq16, where the C57BL/6J allele exerted the most influence on Hyp.
QTL Lifbq18 (liver fibrosis QTL 18) mapped to Chromosome 2 in both the single and composite interval mapping, LRS=13.3 at marker rs3719468 with a 1.5 LOD support interval that mapped between 74.9 and 76.8 Mb influencing fibrosis stage score in both the male and the combined map populations.
QTL Lifbq19 (liver fibrosis QTL 19) mapped to Chromosome 2 in both the single and composite interval mapping, LRS=15.2 between SNPs rs6305540 and rs6402916 with a 1.5 LOD support interval mapped between 174.5 and 181.5 Mb influencing fibrosis stage scores in male, female and combined mapping populations.
QTL Lifbq20 (liver fibrosis QTL 20) mapped to Chromosome 4 in the single QTL scan, LRS=13.7 between SNPs rs4224919 and rs3669806 with a 1.5 LOD support interval mapped between 139.1 and 152.7 Mb influencing fibrosis stage scores in female mice.
QTL Lifbq21 (liver fibrosis QTL 21) mapped to Chromosome 7 in both the single and composite interval mapping, LRS=20.3 between SNPs rs3703247 and rs8255275 with a 1.5 LOD support interval mapped between 48.2 and 53.7 Mb influencing fibrosis stage scores in the male and the combined mapping populations.
QTL Lifbq22 (liver fibrosis QTL 22) mapped to Chromosome 8 in the single QTL scan, LRS=12.8 between SNPs rs3699325 and rs13479628 with a 1.5 LOD support interval mapped between 9.13 and 18.42 Mb influencing fibrosis stage scores in male mice.
QTL Lifbq23 (liver fibrosis QTL 23) mapped to Chromosome 11 in both the single and composite interval mapping, LRS=12.1 between SNPs rs13481150 and rs3688955 with a 1.5 LOD support interval mapped between 79.8 and 93.5 Mb influencing fibrosis stage scores in the male and the combined mapping populations.
QTL Lifbq24 (liver fibrosis QTL 24) mapped to Chromosome 13 in both the single and composite interval mapping, LRS=16.7 between SNPs rs3688207 and rs429721 with a 1.5 LOD support interval mapped between 44.2 and 52.7 Mb influencing fibrosis stage scores in the male and the combined mapping populations.
QTL Lifbq25 (liver fibrosis QTL 25) mapped to Chromosome 15 in the single QTL scan, LRS=12.9 between SNPs rs13482436 and rs4139555 with a 1.5 LOD support interval mapped between 9.4 and 15.3 Mb influencing fibrosis stage scores in female mice.
QTL Lifbq26 (liver fibrosis QTL 26) mapped to Chromosome 15 in the single QTL scan, LRS=16.7 between SNPs rs13482547 and rs3692040 with a 1.5 LOD support interval mapped between 44.2 and 51.7 Mb influencing fibrosis stage scores in female mice.
QTL Lifbq27 (liver fibrosis QTL 27) mapped to Chromosome 15 in the single QTL scan, LRS=13.8 between SNPs rs13482723 and rs3664692 with a 1.5 LOD support interval mapped between 92.8 and 95.9 Mb influencing fibrosis stage scores in male mice.
QTL Lifbq28 (liver fibrosis QTL 28) mapped to Chromosome 17 in the single QTL scan, LRS=22.0 between SNPs rs13483077 and rs13483081 with a 1.5 LOD support interval mapped between 64.9 and 71.1 Mb influencing fibrosis stage scores in female mice.
The alleles of the fibrosis susceptible strain, DBA/2J increased the trait value with an additive effect for QTL Lifbq18 through Lifpq28. Fibrosis stage scores were differentiated in stages 1-4, perivenular to pronounced fibrosis respectively.
(Fig S1 in File S1).
For microarray analysis of hepatic expression profiles total RNA was isolated from snap frozen individual liver samples from 30 BXD strains, the parental strains and B6XD2 F1 hybrids. The aim was to infer causal mechanisms for phenotype variation. Observed effects on gene expression that resulted from variants in an identified genomic region increased the confidence that a locus harbored causal candidates underlying the phenotype. Candidate gene search was restricted to the genes located in significant and phenotype overlapping QTL regions.
Nine QTL regions on Chromosomes 2, 4, 5, 7, 12, 13, 15 and 17 were further dissected using expression QTL mapping. The nine regions either conferred significant linkage or were associated with more than one phenotype. Eighty-five potential candidate genes were identified when comparing fibrosis-associated expression patterns that were also locally regulated within the QTL intervals. (Table 2, Fig.S2, File1). In further selection steps key regulatory candidates of fibrosis were inferred from among the 85 candidate genes that fulfilled a least one selection criterion (Table S3, File S2). Among the 85 candidate genes, 22 had nsSNPs in coding regions that segregated between the two parental strains. Only eleven genes fulfilled all 3 criteria: Afm, Fan1, Hsd17b14, Napsa, Nomo, Nin, Susd1, Klk1b5, Klk1b21, Klk1b22 and Klk1b26.
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