Fine-mapping a locus for glucose tolerance using heterogeneous stock rats

Abstract

Heterogeneous stock (HS) animals provide the ability to map quantitative trait loci at high resolution [<5 Megabase (Mb)] in a relatively short time period. In the current study, we hypothesized that the HS rat colony would be useful for fine-mapping a region on rat chromosome 1 that has previously been implicated in glucose regulation. We administered a glucose tolerance test to 515 HS rats and genotyped these animals with 69 microsatellite markers, spaced an average distance of <1 Mb apart, on a 67 Mb region of rat chromosome 1. Using regression modeling of inferred haplotypes based on a hidden Markov model reconstruction and mixed model analysis in which we accounted for the complex family structure of the HS, we identified one sharp peak within this region. Using positional bootstrapping, we determined the most likely location of this locus is from 205.04 to 207.48 Mb. This work demonstrates the utility of HS rats for fine-mapping complex traits and emphasizes the importance of taking into account family structure when using highly recombinant populations.

Fig. 1.

Glucose response curves after a glucose challenge in inbred founders (A) and heterogeneous stock (HS) rats (B). Founder curves represent the mean of 6–14 animals within each inbred strain. Each line in the HS graphs represent an individual animal (200 representative HS rats are shown in graph).

Fig. 2.

Mapping of Glucose_AUC within a 67 Mb region previously implicated for glucose tolerance on rat chromosome 1. The x-axis shows position in Mb, and the y-axis gives the -log₁₀P value of association. Dashed lines represent region-wide 5% significance thresholds for additive (red) and full (additive + dominance; green) genetic models. A: results based on a mixed-model analysis incorporating family structure in both locus model and threshold procedure. B: results based on an analysis excluding family structure from the locus model with thresholds calculated so that family structure is accounted for (upper dashed lines) and ignored (lower dashed lines). AUC, area under the curve.

Fig. 3.

Positional bootstrapping of the significant peak from Fig. 2A. Histogram shows the number of times (y-axis) each marker interval (x-axis, locations in Mb) represented the strongest association in the subregion (205.04–207.48 Mb) when the analysis was applied to each of 1,000 bootstrapped sets of rats. Shading indicates the central quantile regions: e.g., for at least 50% of bootstraps the highest association was within 205.04–207.48 Mb.

Fig. 4.

Effect plot for marker D1Rat112 on phenotype Glucose_AUC in HS rats. SSLP genotypes are shown on the x-axis (note that 4 alleles were found in the HS for this marker). Glucose_AUC is shown on the y-axis, with means ± SE for animals within each genotype group plotted on the graph. Founder allele sizes are listed in the graph. Note that the 133 allele (ACI and WN) appears to protect against high glucose, while HS animals with either the 161 (WKY) the 172 (F344) allele exhibit significantly higher glucose levels during the glucose tolerance test than those with the other alleles, except when heterozygous with the protective 133 allele. The BN allele at this locus, 157, was not represented within the HS colony.

Fig. 5.

Quantitative trait loci (QTLs) identified in multiple rat F2 crosses within a 67 Mb region on rat chromosome 1. Vertical bars to the right of chromosome 1 (shown from 200 Mb to the end of the chromosome) represent 95% confidence interval of the QTL. The black arrow indicates the location of the 2.44 Mb locus we have mapped using HS rats. Information for each QTL can be found by number in the accompanying table. Phenotypes in the table are abbreviated: PPGluc, postprandial glucose; FGluc, fasting glucose; BW, body weight; Chol, cholesterol; Trig, triglycerides; Ins, postprandial insulin.