Experiment
To gain insight into the genetic control of body length, quantitative trait locus (QTL) analysis was performed in F2 female mice produced by crossing C57BL/6J females and DDD.Cg-Ay males. The congenic DDD.Cg-Ay strain was established by introgressing the Ay allele from the B6.Cg-Ay strain by backcrossing for 12 generations. DDD.Cg-Ay females were longer than B6.Cg-Ay females. QTL analysis was performed for QTL interacting with the Ay allele that might be identified for body length and for plasma insulin-like growth factor 1 (Igf1) levels because Igf1 is known to play essential roles in growth and development.
Both DDD.Cg-Ay and B6.Cg-Ay mice were referred to as 'Ay mice' and their control littermates DDD/SgnRbrc (DDD) and C57BL/6J (B6) were referred to as 'non-A-y' mice. At the age of 16 weeks the body weight of the mice, fasted for 4 hours, was measured. Mice were euthanized and whole blood was drawn from the heart. The Igf1 concentration was determined by ELISA. The anal nasal length of each mouse was measured to the nearest 0.01 mm just after the blood collection. Body length is defined as the anal nasal length. Genomic DNA isolation and genotyping of microsatellite markers were performed.
Of a total of 298 F2 female (148 were F2 non-Ay and 150 were F2 Ay) mice were analyzed. QTL analysis was performed using R/qtl. Data for F2 non-Ay and F2 Ay mice were combined and analyzed using the agouti locus genotype as a covariate. Threshold logarithm of odds (LOD) scores for suggestive (p < 0.63) and significant (p < 0.05) linkages were determined by performing 1,000 permutations for each trait. After single QTL scans, pair wise evaluations for potential interactions between loci were made.
Results: Table 3: QTL identified by single QTL scans with the agouti locus (Ay) as an additive covariate:
QTL Blndq1 (body length in DDD, QTL 1) mapped to Chromosome 15 peaking at 3.0 cM nearest marker D15Mit174 with a LOD score of 4.16. The 95% confidence interval spanned from 0.0 to 20.0 cM. The DDD allele was associated with increased body length at Blndq1.
QTL Blndq2 (body length in DDD, QTL 2) mapped to Chromosome 17 peaking at 14.0 cM nearest marker D17Mit176 with a LOD score of 3.94. The 95% confidence interval spanned from 0.0 to 37.0 cM. The B6 allele was associated with increased body length at Blndq2.
QTL Blndq1 and Blndq2 did not interact with the Ay allele, and the allele effect of these QTL was in the same direction in F2 non-Ay and F2 Ay mice. When the agouti locus genotype was included as a covariate in the analysis, no significant QTL covariate interactions were identified.
The 95% confidence intervals of Blndq1 and Blndq2 contained the genes Ghr and Igf2r, respectively, as potential candidate genes.
Two suggestive QTL were also identified for body length mapping to Chr 6 (35.0 cM, LOD=2.58) and Chr 11 (2.0 cM, LOD=2.58).
QTL Igfdq1 (Igf1 levels in DDD, QTL 1) mapped to Chromosome 10 peaking at 36.0 cM nearest marker D10Mit42 with a LOD score of 9.60. The 95% confidence interval spanned from 21.0 to 66.0 cM. The DDD allele was associated with higher Igf1 levels.
QTL Igfdq2 (Igf1 levels in DDD, QTL 2) mapped to Chromosome 12 peaking at 54.0 cM nearest marker D12Nds2 with a LOD score of 4.11. The 95% confidence interval spanned from 27.0 to 54.0 cM. The heterozygous phenotype was associated with higher Igf1 levels.
Four suggestive loci associated with Igf1 levels were mapped to Chr 8 (4.0 cM, LOD=3.07); Chr 13 (28.0 cM, LOD=2.78); Chr 14 (15.0 cM, LOD=2.96) and Chr 19 (27.0 cM, LOD-2.06).
When the QTL x agouti locus genotype was tested, one significant interacting QTL was identified on chromosome 19. QTL Igfdq3 (Igf1 levels in DDD, QTL 3) mapped to Chr 19 peaking at 61.0 cM with a LOD score of 3.62. The direction of allele effect of this QTL differed between F2 non-Ay and F2 Ay females. [Fig 4E].
The 95% CI for Igfdq3 contained the Kazal-type serine peptidase inhibitor domain 1 (Kazald1) also known as Igfbp-rp10, as a candidate gene.
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