Experiment
Non-alcoholic fatty liver disease (NAFLD) is often associated with obesity and type 2 diabetes. The authors mapped 1H nuclear magnetic resonance (NMR) metabolomic and disease-related phenotypes in a mouse F2 cross derived from strains showing resistance (BALB/c) and increased susceptibility (129S6) to these diseases.
BALB/c and 129S6 mice were used to produce F1 mice which were sib-mated to derive a cohort of 351 male F2 mice fed carbohydrate (CHD) chow. At 5 weeks, one group of F2 mice (n = 181) was transferred to a 40% high fat diet (HFD). A separate group (n = 148) remained on CHD.
To map the genetic regulation of NAFLD-related phenotypes, liver histopathology grades were used to test for linkage to SNP markers in CHD- and HFD-fed F2 mice. Genotyping was carried out with 1538 mouse SNP markers using the Illumina Golden Gate assay protocol (GRCm38). The Rqtl package was used for linkage analysis using a genetic map constructed in the cross.
Two signifcant linkages to macrovesicular lesions were detected in CHD-fed mice:
Mvlq1 (macrovesicular liver lesion QTL 1) maps to Chromosome 11 with a peak LOD score of 3.4 at rs3690511.
Mvlq2 (macrovesicular liver lesion QTL 2) maps to Chromosome 18 with a peak LOD score of 5.3 at rs13483492 (54 cM). 129S6 genotypes at Mvlq2 are associated with increased macrovesicular scores.
Four liver-related QTL were identified in HFD-fed mice:
Mvlq3 (macrovesicular liver lesion QTL 3) maps to Chromosome 4 with a peak LOD score of 3.8 at rs13477976 (38.6 cM).
Mvlq4 (macrovesicular liver lesion QTL 4) maps to Chromosome 18 with a peak LOD score of 5.3 at rs13483370 (20.3cM). 129S6 alleles contributed to increased phenotype severity at Mvlq4.
Mvlq5 (microvesicular liver lesion QTL 5) maps to Chromosome 18 with a peak LOD score of 9.3 at rs13483370 (20.3cM). 129S6 alleles contributed to increased phenotype severity at Mvlq5.
Liviq1 (liver inflammation QTL 1) maps to Chromosome 18 with a peak LOD score of 4.2 at rs13483370 (20.3cM). 129S6 alleles contributed to increased phenotype severity at Liviq1.
F2 mice carrying BALB/c homozygous genotypes at Liviq1 showed normal liver histology, whereas mice carrying 129S6 homozygous genotypes at the locus showed micro- and macrovesicular steatosis or microvesicular steatosis associated with infammation. In these examples, mice carrying the homozygous 129S6 allele at Liviq1, which exhibited evidence of liver histopathology, showed elevated adiposity index (59x103 and 61x103) and liver triglycerides content (118.0 mg/g and 144.4 mg/g) when compared to mice homozygous for BALB/c at the locus (adiposity index: 19x103; liver triglycerides: 10.9 mg/g).
The authors verifed that 129S6 mice fed HFD showed signifcantly elevated concentration of liver triglycerides when compared to 129S6 mice fed CHD and BALB/c mice fed HFD, whereas F2 mice showed intermediate values and broad distribution of this phenotype. Evidence of marginal linkage to liver triglycerides was detected at two QTL:
Ltgq7 (liver triglyceride QTL 7) maps to Chromosome 4 with a peak LOD score of 3.15 at rs3702881 (33.4 cM). The 129S6 allele is associated with elevated concentration of triglycerides at Ltgq7.
Ltgq8 (liver triglyceride QTL 8) maps to Chromosome 18 with a peak LOD score of 2.56 at rs13483370 (20.4 cM). The 129S6 allele is associated with elevated concentration of triglycerides at Ltgq8.
Four QTL were linked to body weight and adiposity index in CHD-fed F2 mice:
Bw41 (body weight QTL 41) maps to Chromosome 6 with a peak LOD score of 4.0 at rs13478819.
Bw42 (body weight QTL 42) maps to Chromosome 11 with a peak LOD score of 4.0 at rs3690511.
Adip28 (adiposity 28) maps to Chromosome 6 with a peak LOD score of 4.6 at rs13478819.
Adip29 (adiposity 29) maps to Chromosome 11 with a peak LOD score of 5.0 at rs3690511.
These QTL explain a high proportion of the phenotypic variances of body weight (10.4 - 10.7%) and adiposity index (8.8 - 12.3%). BALB/c alleles were associated with increased body weight and adiposity index at Bw41 and Adip28, and decreased body weight and adiposity index at Bw42 and Adip29, indicating balancing effects of 129S6 and BALB/c alleles at these loci to maintain these phenotypes within similar ranges as observed in the parental strains.
Four QTL were linked to body weight and adiposity index in HFD-fed F2 mice:
Bw43 (body weight QTL 43) maps to Chromosome 3 with a peak LOD score of 4.7.
Bw44 (body weight QTL 44) maps to Chromosome 4 with a peak LOD score of 5.1.
Bw43 and Bw 44 together explain 13.1 - 13.7% of the phenotypic variance of body weight in the cross.
Bw45 (body weight QTL 45) maps to Chromosome 4 with a peak LOD score of 5.6 at rs3702881 (122.49 Mb; 33.4 cM). 129S6 alleles are associated with increased body weight at Bw45.
Adip30 (adiposity 30) maps to Chromosome 4 with a peak LOD score of 5.6 at rs3702881 (122.49 Mb; 33.4 cM). 129S6 alleles are associated with increased adiposity index at Adip30.
To identify molecular phenotypes specifcally contributing to NAFLD, the authors carried out H NMR metabolic profling of liver extracts in HFD-fed F2 mice. Spectral peaks were aligned and quantifed using methods previously used in the cross in order to map metabolomic QTL in the mouse genome. The authors mapped 49 QTL signifcantly linked to untargeted metabolic features (listed below), including a QTL on chromosome 18 (25 cM) linked to a metabolite at 7.34ppm, which maps in the close vicinity to the QTL for liver histopathology and infammation. 129S6 alleles at the locus contributed to increased concentration of the metabolite. Further statistically-aided structural analyses (statistical total correlation spectroscopy, STOCSY) using the 7.34ppm signal as the driver peak identifed other correlated peaks around 6.92 and 3.25ppm, a pattern characteristic of octopamine.
Forty-nine QTL linked to untargeted matabolic features:
Nmrs1 (NAFLD-associated magnetic resonance shift 1) maps to Chromosome 1 with a peak LOD score of 4.72 at rs13476036 (47.2 cM), with a minimum resonance of 4.159, a maximum resonance of 4.164, and a ppm of 4.1615. No potential candidate metabolites are given for Nmrs1.
Nmrs2 (NAFLD-associated magnetic resonance shift 2) maps to Chromosome 3 with a peak LOD score of 5.33 at rs13477108 (23.6 cM), with a minimum resonance of 0.85185, a maximum resonance of 0.8612, and a ppm of 0.856525. Cholesterol C25 C26 and Lipid CH3 are given as potential candidate metabolites for Nmrs2.
Nmrs3 (NAFLD-associated magnetic resonance shift 3) maps to Chromosome 3 with a peak LOD score of 5.87 at rs134771081 (23.6 cM), with a minimum resonance of 1.2781, a maximum resonance of 1.288, and a ppm of 1.28305. Isoleucine and Lipid CH2CH2CH2CO are given as potential candidate metabolites for Nmrs3.
Nmrs4 (NAFLD-associated magnetic resonance shift 4) maps to Chromosome 3 with a peak LOD score of 4.91 at rs134771082 (23.6 cM), with a minimum resonance of 2.0822, a maximum resonance of 2.0872, and a ppm of 2.0847. Glutamate and Glutamine are given as potential candidate metabolites for Nmrs4.
Nmrs5 (NAFLD-associated magnetic resonance shift 5) maps to Chromosome 3 with a peak LOD score of 4.53 at rs134771083 (23.6 cM), with a minimum resonance of 2.4227, a maximum resonance of 2.4331, and a ppm of 2.4279. Carnitine and Alpha-ketoglutarate are given as potential candidate metabolites for Nmrs5.
Nmrs6 (NAFLD-associated magnetic resonance shift 6) maps to Chromosome 3 with a peak LOD score of 3.98 at rs3719360 (23.6 cM), with a minimum resonance of 3.9071, a maximum resonance of 3.922, and a ppm of 3.91455. Creatine is given as a potential candidate metabolite for Nmrs6.
Nmrs7 (NAFLD-associated magnetic resonance shift 7) maps to Chromosome 3 with a peak LOD score of 3.91 at rs37193601 (23.6 cM), with a minimum resonance of 4.197, a maximum resonance of 4.2058, and a ppm of 4.2014. D(-)-3-Phosphoglycerate, o-Phosphoserine, and p-Hydroxyphenyllacate are given as potential candidate metabolites for Nmrs7.
Nmrs8 (NAFLD-associated magnetic resonance shift 8) maps to Chromosome 3 with a peak LOD score of 6.23 at rs37193602 (23.6 cM), with a minimum resonance of 6.1747, a maximum resonance of 6.1836, and a ppm of 6.17915. No potential candidate metabolites are given for Nmrs8.
Nmrs9 (NAFLD-associated magnetic resonance shift 9) maps to Chromosome 3 with a peak LOD score of 4.74 at rs37193603 (23.6 cM), with a minimum resonance of 7.9413, a maximum resonance of 7.9512, and a ppm of 7.94625. No potential candidate metabolites are given for Nmrs9.
Nmrs10 (NAFLD-associated magnetic resonance shift 10) maps to Chromosome 3 with a peak LOD score of 6.18 at rs134771084 (23.6 cM), with a minimum resonance of 8.5513, a maximum resonance of 8.5601, and a ppm of 8.5557. No potential candidate metabolites are given for Nmrs10.
Nmrs11 (NAFLD-associated magnetic resonance shift 11) maps to Chromosome 3 with a peak LOD score of 3.92 at c3.loc25 (25 cM), with a minimum resonance of 0.72975, a maximum resonance of 0.7391, and a ppm of 0.734425. No potential candidate metabolites are given for Nmrs11.
Nmrs12 (NAFLD-associated magnetic resonance shift 12) maps to Chromosome 3 with a peak LOD score of 3.96 at c3.loc251 (25 cM), with a minimum resonance of 2.4337, a maximum resonance of 2.4441, and a ppm of 2.4389. Carnitine and Alpha-ketoglutarate are given as potential candidate metabolites for Nmrs12.
Nmrs13 (NAFLD-associated magnetic resonance shift 13) maps to Chromosome 3 with a peak LOD score of 3.83 at c3.loc252 (25 cM), with a minimum resonance of 4.0017, a maximum resonance of 4.0303, and a ppm of 4.016. No potential candidate metabolites are given for Nmrs13.
Nmrs14 (NAFLD-associated magnetic resonance shift 14) maps to Chromosome 3 with a peak LOD score of 4.05 at c3.loc253 (25 cM), with a minimum resonance of 8.273, a maximum resonance of 8.279, and a ppm of 8.276. No potential candidate metabolites are given for Nmrs14.
Nmrs15 (NAFLD-associated magnetic resonance shift 15) maps to Chromosome 3 with a peak LOD score of 4.43 at c3.loc254 (25 cM), with a minimum resonance of 8.5326, a maximum resonance of 8.5486, and a ppm of 8.5406. No potential candidate metabolites are given for Nmrs15.
Nmrs16 (NAFLD-associated magnetic resonance shift 16) maps to Chromosome 3 with a peak LOD score of 5.09 at c3.loc30 (30 cM), with a minimum resonance of 0.8656, a maximum resonance of 0.87495, and a ppm of 0.870275. No potential candidate metabolites are given for Nmrs16.
Nmrs17 (NAFLD-associated magnetic resonance shift 17) maps to Chromosome 3 with a peak LOD score of 3.94 at c3.loc301 (30 cM), with a minimum resonance of 8.7234, a maximum resonance of 8.7295, and a ppm of 8.72645. Nicotinurate is given as a potential candidate metabolite for Nmrs17.
Nmrs18 (NAFLD-associated magnetic resonance shift 18) maps to Chromosome 4 with a peak LOD score of 3.91 at CEL.4_95976899 (23.9 cM), with a minimum resonance of 2.6669, a maximum resonance of 2.6718, and a ppm of 2.66935. Acetylcarnitine is given as a potential candidate metabolite for Nmrs18.
Nmrs19 (NAFLD-associated magnetic resonance shift 19) maps to Chromosome 4 with a peak LOD score of 4.05 at rs13477899 (27.3 cM), with a minimum resonance of 2.6509, a maximum resonance of 2.6575, and a ppm of 2.6542. Acetylcarnitine is given as a potential candidate metabolite for Nmrs19.
Nmrs20 (NAFLD-associated magnetic resonance shift 20) maps to Chromosome 4 with a peak LOD score of 3.93 at rs134778991 (27.3 cM), with a minimum resonance of 2.9259, a maximum resonance of 2.9353, and a ppm of 2.9306. No potential candidate metabolites are given for Nmrs20.
Nmrs21 (NAFLD-associated magnetic resonance shift 21) maps to Chromosome 5 with a peak LOD score of 4.43 at rs3697291 (6.5 cM), with a minimum resonance of 9.593, a maximum resonance of 9.5991, and a ppm of 9.59605. No potential candidate metabolites are given for Nmrs21.
Nmrs22 (NAFLD-associated magnetic resonance shift 22) maps to Chromosome 6 with a peak LOD score of 3.90 at rs6182329 (5.1 cM), with a minimum resonance of 7.085, a maximum resonance of 7.0927, and a ppm of 7.08885. No potential candidate metabolites are given for Nmrs22.
Nmrs23 (NAFLD-associated magnetic resonance shift 23) maps to Chromosome 8 with a peak LOD score of 4.27 at D8Mit291 (59 cM), with a minimum resonance of 2.5871, a maximum resonance of 2.597, and a ppm of 2.59205. No potential candidate metabolites are given for Nmrs23.
Nmrs24 (NAFLD-associated magnetic resonance shift 24) maps to Chromosome 8 with a peak LOD score of 3.83 at c8.loc60 (60 cM), with a minimum resonance of 2.5118, a maximum resonance of 2.5365, and a ppm of 2.52415. No potential candidate metabolites are given for Nmrs24.
Nmrs25 (NAFLD-associated magnetic resonance shift 25) maps to Chromosome 8 with a peak LOD score of 4.28 at c8.loc65 (65 cM), with a minimum resonance of 1.0361, a maximum resonance of 1.057, and a ppm of 1.04655. Branched chain amino acids are given as potential candidate metabolites for Nmrs25.
Nmrs26 (NAFLD-associated magnetic resonance shift 26) maps to Chromosome 9 with a peak LOD score of 3.82 at c9.loc40 (40 cM), with a minimum resonance of 1.0361, a maximum resonance of 1.057, and a ppm of 1.04655. Branched chain amino acids are given as potential candidate metabolites for Nmrs26.
Nmrs27 (NAFLD-associated magnetic resonance shift 27) maps to Chromosome 10 with a peak LOD score of 3.91 at D10Mit123 (0 cM), with a minimum resonance of 1.0697, a maximum resonance of 1.0746, and a ppm of 1.07215. Alpha-ketobutyrate is given as a potential candidate metabolite for Nmrs27.
Nmrs28 (NAFLD-associated magnetic resonance shift 28) maps to Chromosome 10 with a peak LOD score of 4.50 at rs6378338 (5.4 cM), with a minimum resonance of 3.218, a maximum resonance of 3.2268, and a ppm of 3.2224. Acetylcholine, Homoserine, and Carnitine are given as potential candidate metabolites for Nmrs28.
Nmrs29 (NAFLD-associated magnetic resonance shift 29) maps to Chromosome 12 with a peak LOD score of 4.00 at rs3724198 (7.9 cM), with a minimum resonance of 4.3636, a maximum resonance of 4.3702, and a ppm of 4.3669. Uridine is given as a potential candidate metabolite for Nmrs29.
Nmrs30 (NAFLD-associated magnetic resonance shift 30) maps to Chromosome 12 with a peak LOD score of 4.10 at rs37241981 (7.9 cM), with a minimum resonance of 8.1184, a maximum resonance of 8.125, and a ppm of 8.1217. N-Formylglycine is given as a potential candidate metabolite for Nmrs30.
Nmrs31 (NAFLD-associated magnetic resonance shift 31) maps to Chromosome 12 with a peak LOD score of 4.18 at rs37241982 (7.9 cM), with a minimum resonance of 8.207, a maximum resonance of 8.2125, and a ppm of 8.20975. Adenine is given as a potential candidate metabolite for Nmrs31.
Nmrs32 (NAFLD-associated magnetic resonance shift 32) maps to Chromosome 12 with a peak LOD score of 4.10 at rs37241983 (7.9 cM), with a minimum resonance of 8.5755, a maximum resonance of 8.5854, and a ppm of 8.58045. Purine is given as a potential candidate metabolite for Nmrs32.
Nmrs33 (NAFLD-associated magnetic resonance shift 33) maps to Chromosome 12 with a peak LOD score of 3.89 at c12.loc25 (25 cM), with a minimum resonance of 3.2757, a maximum resonance of 3.2839, and a ppm of 3.2798. Betaine, Taurine, and TMAO are given as potential candidate metabolites for Nmrs33.
Nmrs34 (NAFLD-associated magnetic resonance shift 34) maps to Chromosome 13 with a peak LOD score of 6.13 at rs6307428 (0.6 cM), with a minimum resonance of 2.1977, a maximum resonance of 2.2027, and a ppm of 2.2002. Adipate is given as a potential candidate metabolite for Nmrs34.
Nmrs35 (NAFLD-associated magnetic resonance shift 35) maps to Chromosome 13 with a peak LOD score of 3.86 at rs13481825 (25.7 cM), with a minimum resonance of 8.1949, a maximum resonance of 8.2037, and a ppm of 8.1993. Adenine is given as a potential candidate metabolite for Nmrs35.
Nmrs36 (NAFLD-associated magnetic resonance shift 36) maps to Chromosome 17 with a peak LOD score of 4.46 at rs3682923 (19.7 cM), with a minimum resonance of 2.5976, a maximum resonance of 2.6036, and a ppm of 2.6006. No potential candidate metabolites are given for Nmrs36.
Nmrs37 (NAFLD-associated magnetic resonance shift 37) maps to Chromosome 17 with a peak LOD score of 3.98 at rs13482999 (20.8 cM), with a minimum resonance of 7.5871, a maximum resonance of 7.5965, and a ppm of 7.5918. N-formyktryptophan and Nicotinurate are given as potential candidate metabolites for Nmrs37.
Nmrs38 (NAFLD-associated magnetic resonance shift 38) maps to Chromosome 17 with a peak LOD score of 4.80 at rs134829991 (20.8 cM), with a minimum resonance of 7.597, a maximum resonance of 7.6031, and a ppm of 7.60005. N-formyktryptophan and Nicotinurate are given as potential candidate metabolites for Nmrs38.
Nmrs39 (NAFLD-associated magnetic resonance shift 39) maps to Chromosome 17 with a peak LOD score of 4.05 at c17.loc25 (25 cM), with a minimum resonance of 0.6907, a maximum resonance of 0.6962, and a ppm of 0.69345. Cholesterol C18 is given as a potential candidate metabolite for Nmrs39.
Nmrs40 (NAFLD-associated magnetic resonance shift 40) maps to Chromosome 17 with a peak LOD score of 4.28 at c17.loc251 (25 cM), with a minimum resonance of 2.5871, a maximum resonance of 2.597, and a ppm of 2.59205. No potential candidate metabolites are given for Nmrs40.
Nmrs41 (NAFLD-associated magnetic resonance shift 41) maps to Chromosome 17 with a peak LOD score of 3.93 at c17.loc252 (25 cM), with a minimum resonance of 4.2091, a maximum resonance of 4.214, and a ppm of 4.21155. No potential candidate metabolites are given for Nmrs41.
Nmrs42 (NAFLD-associated magnetic resonance shift 42) maps to Chromosome 17 with a peak LOD score of 4.97 at c17.loc253 (25 cM), with a minimum resonance of 4.2635, a maximum resonance of 4.2701, and a ppm of 4.2668. No potential candidate metabolites are given for Nmrs42.
Nmrs43 (NAFLD-associated magnetic resonance shift 43) maps to Chromosome 17 with a peak LOD score of 3.88 at c17.loc254 (25 cM), with a minimum resonance of 8.2796, a maximum resonance of 8.2922, and a ppm of 8.2859. Nicotinate is given as a potential candidate metabolite for Nmrs43.
Nmrs44 (NAFLD-associated magnetic resonance shift 44) maps to Chromosome 17 with a peak LOD score of 4.20 at c17.loc255 (25 cM), with a minimum resonance of 8.5046, a maximum resonance of 8.5216, and a ppm of 8.5131. No potential candidate metabolites are given for Nmrs44.
Nmrs45 (NAFLD-associated magnetic resonance shift 45) maps to Chromosome 17 with a peak LOD score of 4.12 at c17.loc30 (30 cM), with a minimum resonance of 0.8656, a maximum resonance of 0.87495, and a ppm of 0.870275. Lipid CH3CH2CH2C= is given as a potential candidate metabolite for Nmrs45.
Nmrs46 (NAFLD-associated magnetic resonance shift 46) maps to Chromosome 17 with a peak LOD score of 3.98 at c17.loc301 (30 cM), with a minimum resonance of 4.4857, a maximum resonance of 4.4945, and a ppm of 4.4901. 1-Methylnicotinamide, Homocarnosine, and Anserine are given as potential candidate metabolites for Nmrs46.
Nmrs47 (NAFLD-associated magnetic resonance shift 47) maps to Chromosome 17 with a peak LOD score of 4.05 at c17.loc302 (30 cM), with a minimum resonance of 6.0477, a maximum resonance of 6.0543, and a ppm of 6.051. No potential candidate metabolites are given for Nmrs47.
Nmrs48 (NAFLD-associated magnetic resonance shift 48) maps to Chromosome 17 with a peak LOD score of 3.91 at rs3703241 (30.3 cM), with a minimum resonance of 6.1659, a maximum resonance of 6.172, and a ppm of 6.16895. No potential candidate metabolites are given for Nmrs48.
Nmrs49 (NAFLD-associated magnetic resonance shift 49) maps to Chromosome 18 with a peak LOD score of 4.31 at c18.loc25 (25 cM), with a minimum resonance of 7.3385, a maximum resonance of 7.3452, and a ppm of 7.34185. Octopamine is given as a potential candidate metabolite for Nmrs49.
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