DDD/Sgn mice have significantly higher plasma lipid concentrations than C57BL/6J mice. In the present study, we performed quantitative trait loci (QTL) mapping for plasma total-cholesterol (CHO) and triglyceride (TG) concentrations in reciprocal F2 male intercross populations between the two strains. By single-QTL scans, we identified four significant QTL on chromosomes (Chrs) 1, 5, 17, and 19 for CHO and two significant QTL on Chrs 1 and 12 for TG. By including cross direction as an interactive covariate, we identified separate significant QTL on Chr 17 for CHO but none for TG. When the large phenotypic effect of QTL on Chr 1 was controlled by composite interval mapping, we identified three additional significant QTL on Chrs 3, 4, and 9 for CHO but none for TG. QTL on Chr 19 was a novel QTL for CHO and the allelic effect of this QTL significantly differed between males and females. Whole-exome sequence analysis in DDD/Sgn mice suggested that
Plasma lipid concentrations are representative quantitative traits; that is, they are controlled by multiple genes under the influence of nonheritable environmental effects. Among plasma lipids, cholesterol (CHO) and triglyceride (TG) have clinical implications in atherosclerosis and coronary artery disease; therefore, it is crucially important to identify genes influencing variations in plasma CHO and TG concentrations [
We previously performed QTL mapping studies to identify plasma lipid concentrations using mouse intercrosses [
The inbred mouse strains DDD and B6 were maintained at the National Institute of Agrobiological Sciences (NIAS, Tsukuba, Japan). Reciprocal crosses between DDD and B6 strains produced DB (♀DDD × ♂B6) F1 and BD (♀B6 × ♂DDD) F1 mice, both of which were intercrossed to produce DB F2 (
All mice were weaned at 4 weeks of age and 4-5 mice were housed together in a cage during the experiments. All mice were maintained in a specific pathogen-free facility with a regular light cycle and controlled temperature and humidity. Food (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and water were freely available throughout the experimental period. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of NIAS.
Plasma lipid concentrations were determined at the age of 11 to 14 weeks in DDD, B6, and F1 mice and at the age of 11 to 12 weeks (71–80 days after birth) in F2 mice.
Mice were euthanized with an overdose of ether immediately after weighing in the morning. Blood was collected from the heart of an individual mouse using heparin as an anticoagulant. Plasma was separated by centrifugation at 2,000 ×g for 15 min at 4°C and was stored at −80°C until use. Plasma CHO and TG concentrations were determined by enzymatic methods using clinical chemical kits (Wako Pure Chemical Industries, Osaka, Japan).
Microsatellite sequence length polymorphisms were identified by electrophoresis after PCR amplification of genomic DNA. PCR amplification was carried out by use of a TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio Inc., Shiga, Japan) under the following conditions: 1 cycle at 94°C for 3 min; 35 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 45 s; 1 cycle at 72°C for 7 min. PCR products were separated on 10% polyacrylamide gel (Nacalai Tesque Inc., Kyoto, Japan) and were visualized by ethidium bromide (Nacalai Tesque) staining. A total of 117 microsatellite loci were genotyped. Their chromosomal positions were retrieved from MGI.
Normality of the trait data was assessed by Shapiro-Wilk
QTL mapping was performed using R/qtl version 1.38-4 [
Genomic DNA was extracted from the tail of DDD mice using a genomic DNA purification kit (Wizard Genomic DNA Purification Kit, Promega KK, Tokyo, Japan) and was submitted to Filgen, Inc. (Nagoya, Aichi, Japan) for exome capture and sequencing. Briefly, genomic DNA was subjected to the agarose gel and OD ratio tests to confirm the purity and concentration prior to Bioruptor (Diagenode, Inc., Denville, NJ, USA) fragmentation. Fragmented genomic DNAs were tested for size distribution and concentration using an Agilent Bioanalyzer 2100 and Nanodrop (Agilent Technologies, Wilmington, DE, USA). Illumina libraries were made from qualified fragmented genomic DNA using SPRIworks HT reagent kit (Beckman Coulter, Inc., Indianapolis, IN, USA), and the resulting libraries were subjected to exome enrichment using SureSelect XT Mouse All Exon Kit (Agilent Technologies) following the manufacturer’s instructions. Enriched libraries were tested for enrichment by qPCR and for size distribution and concentration by an Agilent Bioanalyzer 2100. The samples were then sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA, USA), which generated paired-end reads of 90 or 100 nucleotides. Data was analyzed for data quality using FASTQC (Babraham Institute, Cambridge, UK). Sequence reads were mapped to the mouse reference genome (GRCm38, mm10). Read mapping and variant analyses were performed using CLC Genomics Workbench 7.0.4 and 8.5.1 (Filgen).
Plasma lipid concentrations are represented as the mean ± SEM (mg/dL). Statistical differences between two groups were analyzed using Student’s or Welch’s
Table
Plasma lipid concentrations in DDD, B6, DB F1, and BD F1 mice.
Plasma lipids | Sex | Mean ± SEM plasma lipids (mg/dL) |
|
Mean ± SEM plasma lipids (mg/dL) ( |
| ||
---|---|---|---|---|---|---|---|
DDD | B6 | DB F1 | BD F1 | ||||
CHO | Males | 173 ± 4 ( |
102 ± 5 ( |
<0.0001 | 137 ± 4 ( |
128 ± 5 ( |
NS |
Females | 162 ± 3 ( |
92 ± 3 ( |
<0.0001 | 119 ± 3 ( |
110 ± 3 ( |
<0.05 | |
TG | Males | 154 ± 11 ( |
109 ± 15 ( |
<0.03 | 200 ± 15 ( |
112 ± 16 ( |
<0.0005 |
Females | 194 ± 10 ( |
43 ± 12 ( |
<0.0001 | 108 ± 9 ( |
65 ± 10 ( |
<0.005 |
NS, not significant.
Histograms showing the distributions of plasma CHO and TG concentrations in 300 F2 males (data from 150 BD F2 and 150 DB F2 mice are combined) are presented in Figures
Genome-wide scan for plasma CHO concentrations in F2 male mice. (a) A histogram showing the distribution of plasma CHO concentrations. (b) Genome-wide LOD score plot of single-QTL scans for plasma CHO concentrations (solid lines: cross direction as an additive covariate; broken lines: cross direction and body weight as additive covariates). The
Genome-wide scan for plasma TG concentrations in F2 male mice. (a) A histogram showing the distribution of plasma TG concentrations. (b) Genome-wide LOD score plot of single-QTL scans for plasma TG concentrations (solid lines: cross direction as an additive covariate; broken lines: cross direction and body weight as additive covariates). The
Genome-wide LOD score plots obtained via single-QTL scans for plasma CHO and TG concentrations in F2 males are shown in Figures
Significant and suggestive QTL identified by genome-wide scans of F2 males.
Trait | QTL |
Chr | Peak cM | 95% CI |
LOD |
Nearest marker | High strain |
Overlapping QTL | |
---|---|---|---|---|---|---|---|---|---|
Name | Reference | ||||||||
CHO |
|
1 | 80.5 | 77.5–85.5 |
|
|
DDD, Add |
|
[ |
3 | 23.8 | 10.8–56.8 | 2.3 |
|
B6 | ||||
|
5 | 59.8 | 17.8–75.8 | 2.9 |
|
DDD |
|
[ | |
9 | 37.0 | 12.0–59.6 | 2.2 |
|
DDD | ||||
|
17 | 35.1 | 17.1–51.1 |
|
|
B6, Add |
|
[ | |
|
19 | 8.0 | 3.0–19.0 |
|
|
DDD, Rec |
|
[ | |
|
|||||||||
TG |
|
1 | 84.5 | 77.5–93.5 |
|
|
DDD, Dom |
|
[ |
5 | 50.8 | 17.8–66.8 | 2.6 |
|
DDD | ||||
|
12 | 47.0 | 13.0–62.0 | 2.9 |
|
B6 |
|
[ | |
14 | 60.3 | 15.3–66.1 | 2.3 |
|
DDD | ||||
15 | 53.9 | 40.8–53.9 | 2.4 |
|
DDD |
QTL, quantitative trait loci; CI, confidence interval; LOD, logarithm of the odds.
Cross direction was included as an additive covariate in all analyses.
Multiple-regression analysis for plasma lipid concentrations.
Plasma lipid | Chromosome (cM) |
df |
Variance, % |
|
---|---|---|---|---|
CHO | Chr1@80.5 | 2 | 34.1 | 113.0 |
Chr3@23.8 | 2 | 2.0 | 6.6 | |
Chr5@59.8 | 2 | 3.3 | 11.0 | |
Chr9@37.0 | 2 | 4.0 | 13.3 | |
Chr17@35.1 | 2 | 4.7 | 15.5 | |
Chr19@8.0 | 2 | 3.1 | 10.1 | |
Total | 12 | 56.7 | ||
|
||||
TG | Chr1@84.5 | 2 | 15.1 | 30.1 |
Chr5@50.8 | 2 | 2.0 | 4.0 | |
Chr12@47.0 | 2 | 2.7 | 5.4 | |
Chr14@60.3 | 2 | 2.5 | 5.1 | |
Chr15@53.9 | 2 | 1.6 | 3.2 | |
Total | 10 | 27.5 |
Cross direction was also included as a covariate.
There is a significant correlation between body weight and plasma lipid concentrations. That is, based on Spearman’s rank correlation coefficient, the correlation between body weight and CHO concentration was 0.4855 (Spearman’s
We next searched for possible QTL that interact with cross direction (BD versus DB) by including cross direction as an interactive covariate. For CHO (but not TG), we identified significant QTL that interacted with cross direction on Chr 17@60.7 cM with LOD score 2.6 (threshold LOD score for significant QTL × covariate interaction was 2.4) (Figure
Genome-wide scan for QTL × cross direction interaction for plasma CHO concentrations. (a) Genome-wide LOD score plot. The
Because of the prominent phenotypic effect exerted by the Chr 1 QTL on both traits, we performed composite interval mapping by including the nearest marker for
Significant and suggestive QTL when
Trait | QTL |
Chr | Peak cM | 95% CI |
LOD |
Nearest marker | High strain |
Overlapping QTL | |
---|---|---|---|---|---|---|---|---|---|
Name | Reference | ||||||||
CHO |
|
3 | 19.8 | 10.8–35.8 |
|
|
B6, Add |
|
[ |
|
4 | 23.1 | 9.1–37.1 |
|
|
B6, Add |
|
[ | |
|
5 | 59.8 | 17.8–71.8 |
|
|
DDD, Rec |
|
[ | |
|
8 | 39.0 | 16.5–51.5 | 2.8 |
|
B6 | |||
|
9 | 37.0 | 12.0–59.0 |
|
|
DDD, Add |
|
[ | |
|
17 | 37.1 | 15.1–50.1 |
|
|
B6, Rec |
|
[ | |
|
19 | 5.0 | 3.0–19.0 |
|
|
DDD |
QTL, quantitative trait loci; CI, confidence interval; LOD, logarithm of the odds.
Cross direction was also included as an additive covariate.
Genome-wide LOD score plot for CHO concentrations by composite interval mapping. The
Finally, we combined the data from this study on males with the previously analyzed data on B6 × DDD.Cg-
Significant and suggestive QTL for CHO identified by genome-wide scans of combined F2 mice (
QTL |
Chr | Peak cM | 95% CI |
LOD |
Nearest marker | High strain |
---|---|---|---|---|---|---|
|
1 | 79.2 | 77.5–82.5 |
|
|
DDD |
|
3 | 41.8 | 12.8–62.8 | 2.5 |
|
B6 |
|
5 | 47.8 | 17.8–72.0 | 2.1 |
|
DDD |
|
8 | 21.2 | 14.5–53.5 |
|
|
B6 |
|
11 | 61.4 | 34.4–75.4 |
|
|
DDD |
12 | 60.6 | 23.0–62.2 | 2.5 |
|
B6 | |
|
17 | 19.7 | 13.1–49.1 | 3.3 |
|
B6 |
QTL, quantitative trait loci; CI, confidence interval; LOD, logarithm of the odds.
Sex was included as an additive covariate.
Genome-wide scan for QTL × sex interaction for plasma CHO concentrations. (a) Genome-wide LOD score plot. The
We submitted the term “abnormal circulating cholesterol level” as a query to the MGI database (Mammalian Phenotype Browser), resulting in the retrieval of 1197 genotypes with 1905 annotations (MGI search was done on April 28, 2017). We consulted the MGI database (Genes and Markers Query Form) to determine the chromosomal localization of candidate genes. We performed whole-exome sequence analysis to identify nonsynonymous single-nucleotide variation (nsSNV), frameshift, and nonsense mutations as well as insertion-deletion (indel) in the coding regions of candidate genes in DDD mice. Of note, we found an Asp94Gly amino-acid substitution in
This study identified a multifactorial basis for plasma lipid concentrations in F2 male mice generated by crosses between B6 and DDD inbred mice. We previously performed QTL mapping for body weight in the same F2 intercross population and identified four significant QTL on Chrs 1, 2, 5, and 17 [
There was a clear lineage effect on plasma lipid concentrations between DB F1 and BD F1 males; that is, DB F1 mice had higher plasma lipids than BD F1 mice. Based on the studies of Y-consomic mice, the lineage effect was not due to influence by Chr Y [
Most of the QTL identified in this study have coincidental QTL that had been reported by others [
Based on their chromosomal localizations, two QTL have been reported as coincidental QTL with
A relevant QTL mapped close to
Because of its chromosomal localization,
Although numerous QTL for plasma lipid concentrations have been reported, there are additional QTL that remain to be identified. QTL analyses using a new strain combination will identify additional QTL. Identification of coincidental QTL will further substantiate the candidate genes underlying previously reported QTL.
The authors declare that there are no conflicts of interest.
This study was supported in part by the NIAS (National Institute of Agrobiological Sciences) Strategic Research Fund.