Reference
The Diversity Outbred heterogeneous stock (J:DO) is a developing mouse population derived from progenitor lines of the Collaborative Cross (CC). The CC is a panel of recombinant inbred (RI) mouse strains that combines the genomes of eight genetically diverse founder strains - A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ - to capture nearly 90% of the known variation present in laboratory mice (Churchill et al. 2004). Animals from 160 incipient CC lines at early stages of inbreeding were used to establish the DO population, which is maintained by a randomized outbreeding strategy that avoids brother-sister matings. The DO and CC populations thus capture the same set of natural allelic variants derived from a common set of eight founder strains, with DO mice being outbred and the CC population being inbred.
CTC (2004), Churchill, G. A., et al. The Collaborative Cross, a community resource for the genetic analysis of complex traits. Nat Genet. 36, 1133-7.
Experiment
The intestinal microbiota has profound effects on host physiology and health. The microbial communities that inhabit the distal gut of humans and other mammals exhibit large inter-individual variation. While host genetics is a known factor that influences gut microbiota composition, the mechanisms underlying this variation remain largely unknown. Bile acids (BAs) are hormones that are produced by the host and chemically modified by gut bacteria. BAs serve as environmental cues and nutrients to microbes, but they can also have antibacterial effects. We hypothesized that host genetic variation in BA metabolism and homeostasis influence gut microbiota composition.
To address this, the authors characterized the fecal microbiota composition and plasma and cecal BA profiles from 400 Diversity Outbred (J:DO) mice maintained on a high-fat high-sucrose diet for ~22 weeks.
The authors performed LC-MS/MS analyses of plasma and cecal contents to assess relative variation in the levels of 27 BAs. Both plasma and cecal bile acids were measured to provide a comprehensive picture of systemic BA homeostasis. Additionally, they examined gut microbiota composition (n = 399) using 16S rRNA gene amplicon sequencing of DNA extracted from fecal samples collected at the end of the experiment. For subsequent analysis, they identified a core measurable microbiota (CMM), which they defined as taxon found in at least 20% of the mice. In total, the CMM was comprised of 86 ESVs and 42 agglomerated taxa. The CMM traits represent a small fraction of the total microbes detected, but account for 94.5% of the rarefied sequence reads, and therefore constitute a significant portion of the identifiable microbiota.
To identify associations between regions of the mouse genome and the clinical and molecular traits discussed above, the authors performed QTL analysis using the R/qtl2 package. They used sex, days on the diet, and experimental wave as covariates. Genotyping was performed using the Mouse Universal Genotyping Array (GigaMUGA; 143,259 markers) at Neogen (Lincoln, NE). Genotypes were converted to founder strain-haplotype reconstructions using a hidden Markov model (HMM) implemented in the R/qtl2 package. They interpolated the GigaMUGA markers onto an evenly spaced grid with 0.02-cM spacing and added markers to fill in regions with sparse physical representation, which resulted in 69,005 pseudomarkers.
The authors identified 13 significant QTL (LOD >= 7.66; P <= 0.05) and 50 suggestive QTL (LOD >= 6.80; P <= 0.2) for bacterial, bile acid, and body weight traits (all genome coordinates relative to GRCm38/mm10):
Bal1 (bile acid level 1, plasma TUDCA) maps to Chr 8: 93.0702 - 95.1271 Mb with a peak LOD score of 13.4383 at 94.9356 Mb.
Lwq13 (liver weight QTL 13) maps to Chr 9: 62.2738 - 67.8503 Mb with a peak LOD score of 9.3300 at 66.7099 Mb.
Micab19 (microbial abundance 19, Proteobacteria) maps to Chr 18: 23.2653 - 23.7547 Mb with a peak LOD score of 8.6701 at 23.7341 Mb.
Micab20 (microbial abundance 20, 16S ESV) maps to Chr 2: 164.2552 - 165.1435 Mb with a peak LOD score of 8.2516 at 164.3141 Mb.
Micab21 (microbial abundance 21, 16S ESV) maps to Chr 12: 31.2622 - 33.3823 Mb with a peak LOD score of 8.1564 at 33.2267 Mb.
Micab22 (microbial abundance 22, Turicibacterales) maps to Chr 8: 4.3166 - 6.9358 Mb with a peak LOD score of 8.0438 at 4.3166 Mb.
Micab23 (microbial abundance 23, Turicibacteraceae) maps to Chr 8: 4.3166 - 6.9358 Mb with a peak LOD score of 8.0438 at 4.3166 Mb.
Micab24 (microbial abundance 24, Turicibacter) maps to Chr 8: 4.3166 - 6.9358 Mb with a peak LOD score of 8.0438 at 4.3166 Mb.
Micab25 (microbial abundance 25, 16S ESV) maps to Chr 2: 106.4954 - 115.5507 Mb with a peak LOD score of 7.9848 at 115.1504 Mb.
Micab26 (microbial abundance 26, 16S ESV) maps to Chr 6: 135.7233 - 137.7530 Mb with a peak LOD score of 7.8509 at 136.5912 Mb.
Micab27 (microbial abundance 27, 16S ESV) maps to Chr 1: 18.5001 - 21.1273 Mb with a peak LOD score of 7.8391 at 19.3712 Mb.
Micab28 (microbial abundance 28, 16S ESV) maps to Chr 17: 62.8397 - 62.9952 Mb with a peak LOD score of 7.7337 at 62.8645 Mb.
Micab29 (microbial abundance 29, 16S ESV) maps to Chr 14: 24.5798 - 25.4152 Mb with a peak LOD score of 7.6780 at 24.9530 Mb.
For cecal deoxycholic acid (DCA) (Bal2-5), the WSB/EiJ founder haplotype was associated with higher levels of this BA, while the NOD/ShiLtJ founder haplotype was associated with lower levels. The opposite pattern was observed for plasma taurodeoxycholic acid, where the NOD/ShiLtJ and WSB/EiJ haplotype were associated with higher and lower levels, respectively. The suggestive QTL identified for cecal DCA are:
Bal2 (bile acid level 2, cecal DCA) maps to Chr 18: 41.7507 - 44.2413 Mb with a peak LOD score of 6.9832 at 42.2771 Mb.
Bal3 (bile acid level 3, cecal DCA) maps to Chr 12: 105.0624 - 106.2201 Mb with a peak LOD score of 6.4398 at 105.4887 Mb.
Bal4 (bile acid level 4, cecal DCA) maps to Chr 13: 25.7665 - 118.2735 Mb with a peak LOD score of 6.3664 at 108.8442 Mb.
Bal5 (bile acid level 5, cecal DCA) maps to Chr 1: 121.3282 - 127.6879 Mb with a peak LOD score of 6.2822 at 125.7685 Mb.
The authors also identified overlapping suggestive QTL on Chr 11 at ~71 Mbp for cecal levels of the secondary BAs lithocholic acid (LCA) (Bal6) and isolithocholic acid (ILCA) (Bal7), the isomer of LCA produced by bacterial epimerization:
Bal6 (bile acid level 6, cecal LCA) maps to Chr 11: 58.3553 - 77.9319 Mb with a peak LOD score of 6.4792 at 71.7827 Mb.
Bal7 (bile acid level 7, cecal ILCA) maps to Chr 11: 71.4398 - 78.3584 Mb with a peak LOD score of 7.3233 at 72.9745 Mb.
Higher levels of these cecal BAs are associated with the 129S1/SvImJ founder haplotype and lower levels are associated with the A/J founder haplotype. The authors identified the positional candidate gene Slc13a5, which is a sodium-dependent transporter that mediates cellular uptake of citrate, an important precursor in the biosynthesis of fatty acids and cholesterol. Recent evidence indicates that Slc13a5 influences host metabolism and energy homeostasis. Slc13a5 is a transcriptional target of pregnane X receptor (PXR), which also regulates the expression of genes involved in the biosynthesis, transport, and metabolism of BAs.
The authors focused their co-mapping analysis on Chr 8 at ~ 5.5 Mbp, where significant Turicibacter sp. QTL Micab24 overlaps with suggestive plasma cholic acid (CA) QTL Bal8:
Bal8 (bile acid level 8, plasma CA) maps to Chr 8: 4.3166 - 6.5812 Mb with a peak LOD score of 6.5561 at 4.3166 Mb.
These traits were particularly interesting because both have been shown to be influenced by host genetics by previous studies. Turicibacter has been identified as highly heritable in both mouse and human genetic studies, and multiple reports have found differences in CA levels as a function of host genotype. Furthermore, CA levels are influenced by both host genetics and microbial metabolism since it is synthesized by host liver enzymes from cholesterol and subsequently modified by gut microbes in the intestine. Notably, these co-mapping traits also share the same allele effects pattern, where the A/J and WSB/EiJ haplotypes have strong positive and negative associations, respectively. The overlapping QTL mapped to a locus containing the gene for the ileal bile acid transporter, Slc10a2. Mediation analysis and subsequent follow-up validation experiments suggest that differences in Slc10a2 gene expression associated with the different strains influences levels of both traits and revealed novel interactions between Turicibacter and BAs.
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