Experiment
In the current study muscle mass is examined in AIL crosses of inbred mouse strains to better characterize the genetic factors underlying variation in skeletal mass.
Analysis of male and female mice from multiple advanced intercross lines (AILs) Aap:B6,D2-F8 and mice from Bliz:B6,D2-F9-F13 were combined to map QTL underlying muscle weight variation in the tibialis anterior (TA), the extensor digitorum longus (EDL), the gastrocnemius (Gastroc) and the soleus (Soleus) muscles. Across all 4 muscles, using a stringent criterion for significance, strong evidence was established for independent genetic factors on chromosomes 1,2,4,5,6,7,8,9,12,14,17 and 18.
Equal numbers of males (212) and females (213) were chosen from the F8 subpopulations. Mice were killed at 14 wks of age and one hindlimb was removed and stored.
Mice were drawn from each generation of F9 through F13 - 77, 91, 73, 65 and 101 respectively for a total of 205 males and 202 females for analysis from the F9 and F13 generations. These mice were killed at 29 weeks of age and the hindlimb was also removed and stored. All hindlimbs were genotyped in the same way.
The four separate muscles TA, EDL, gastro and soleus, were dissected and weighed to a precision of 0.01 mg. Discrepancies expected between the F8 and F9-F13 mice were expected as they were maintained at different facilities, fed different diets and their muscle weights were measured at different ages. These systematic differences were accounted for in the analysis.
The F8 AIL samples were genotyped using Illumina Mouse Medium Density Linkage Pane. Genotypes at 900 SNPs on the X and autosomal chromosomes were called. Of the 900 SNPs 95% corresponded to SNPs that are polymorphic between B6 and D2 strains. In the F9-F13 cohort SNPs were genotyped using the Mega Mouse Universal Genotyping Array that provided genotypes at 20,691 SNPs on the X and autosomal chromosomes. To analyze the combined data the genotypes of SNPs that were not directly genotyped in both F8 and F9-F13 cohorts were estimated. All SNP information and genomic positions were based on NCBI release 37 and build 128 of dbSNP database.
Genetic markers were used to estimate familial relationships. The QTLRel mixed model framework, which was originally developed for AIL mapping was used. QTLRel models the phenotype of individual 'i' as a linear combination of the genotype at any given SNP. In all analysis sex was included as a covariate (muscles in male mice were consistently larger than those in females). In the combined analysis a binary covariate was included that indicated whether the sample originated from the F8 or the F9-F13 cohort. The likelihood ratio statistic was calculated in QTLRel by fitting 2 models to the data, a model with additive and dominance effects included for the given SNP, and another model assuming that no markers have an effect on the phenotype (the null model). A significant LOD score was determined by estimating the distribution of maximum LOD scores under the null hypothesis.
Table 3: QTL identified using data combined from the two separate AIL crosses:
QTL Tammq1 (tibialis anterior muscle mass QTL 1) mapped to Chromosome 1 between 170.0-177.6 Mb with a LOD score of 5.36 at peak marker rs31218323 when using a model that allowed for sex-specific effects. The D2 allele was associated with increased muscle mass at Tammq1.
QTL Edlmmq1 (extensor digitorum longus muscle mass QTL 1) mapped to Chromosome 1 between 170.0-176.1 Mb with a LOD score of 9.75 at peak marker rs31218323 when using a model that allowed for sex-specific effects. The D2 allele was associated with increased muscle mass at Edlmmq1.
QTL Gmmq1 (gastrocnemius muscle mass QTL 1) mapped to Chromosome 1 between 173.2-174.3 with a LOD score of 7.96 at peak marker rs31218323 when using a model that allowed for sex-specific effects. The D2 allele was associated with increased muscle mass at Gmmq1.
QTL Edlmmq2 (extensor digitorum longus muscle mass QTL 2) mapped to Chromosome 2 between 113.2-134.2 Mb with a LOD score of 5.15 at peak marker rs4138562 when using a model that did not differentiate effects of SNPs in males and females. The B6 allele was associated with increased muscle mass at Edlmmq2.
QTL Gmmq2 (gastrocnemius muscle mass QTL 2) mapped to Chromosome 2 between 112.8-127.8 with a LOD score of 6.51 at peak marker rs4138562 when using a model that did not differentiate effects of SNPs in males and females. The B6 allele was associated with increased muscle mass at Gmmq2.
QTL Edlmmq3 (extensor digitorum longus muscle mass QTL 3) mapped to Chromosome 4 between 95.4-103.3 Mb with a LOD score of 6.51 at peak marker rs28094289 when using a model that did not differentiate effects of SNPs in males and females. The B6 allele was associated with increased muscle mass at Edlmmq3.
QTL Smmq1 (soleus muscle mass QTL 1) mapped to Chromosome 5 between 112.0-120.1 Mb with a LOD score of 6.43 at peak marker rs45922215 when using a model that did not differentiate effects of SNPs in males and females. The B6 allele was associated with increased muscle mass at Smmq1.
QTL Tammq2 (tibialis anterior muscle mass QTL 2) mapped to Chromosome 5 between 3.3-16.0 Mb with a LOD score of 4.95 at peak marker rs45922215 when using a model that allowed for sex-specific effects. The B6 allele was associated with increased muscle mass at Tammq2.
QTL Edlmmq4 (extensor digitorum longus muscle mass QTL 4) mapped to Chromosome 6 between 3.98-16.0 Mb with a LOD score of 5.83 at peak marker rs30074418 when using a model that allowed for sex-specific effects.
QTL Gmmq3 (gastrocnemius muscle mass QTL 3) mapped to Chromosome 6 between 5.38-12.0 Mb with a LOD score of 6.94 at peak marker rs30074418 when using a model that allowed for sex-specific effects.
QTL Tammq3 (tibialis anterior muscle mass QTL 3) mapped to Chromosome 6 between 34.5-43.7 Mb with a LOD score of 9.93 at peak marker rs30074418 when using a model that allowed for sex-specific effects.
QTL Edlmmq5 (extensor digitorum longus muscle mass QTL 5) mapped to Chromosome 6 between 34.5-43.7 Mb with a LOD score of 6.29 at peak marker rs47723713 when using a model that allowed for sex-specific effects.
QTL Gmmq4 (gastrocnemius muscle mass QTL 4) mapped to Chromosome 6 between 34.1-43.7 Mb with a LOD score of 8.24 at peak marker rs47723713 when using a model that allowed for sex-specific effects.
QTL Tammq4 (tibialis anterior muscle mass QTL 4) mapped to Chromosome 6 between 4.6-17.7 Mb with a LOD score of 4.57 at peak marker rs47723713 when using a model that allowed for sex-specific effects.
The Chromosome 6 QTL, Edlmmq4, Gmmq3, Tammq3, Edlmmq5, Gmmq4 and Tmmq4 appeared to be male specific QTL with the B6 allele associated with increased muscle mass.
QTL Edlmmq6 (extensor digitorum longus muscle mass QTL 6) mapped to Chromosome 7 between 4.69-17.2 Mb with a LOD score of 5.68 at peak marker rs31137734 when using a model that allowed for sex-specific effects.
QTL Gmmq5 (gastrocnemius muscle mass QTL 5) mapped to Chromosome 7 between 4.69-17.7 Mb with a LOD score of 8.74 at peak marker rs31137734 when using a model that allowed for sex-specific effects.
QTL Tammq5 (tibialis anterior muscle mass QTL 5) mapped to Chromosome 7 between 121.2-130.3 Mb with a LOD score of 5.12 at peak marker rs31137734 when using a model that allowed for sex-specific effects.
The Chromosome 7 QTL Edlmmq6, Gmmq5 and Tammq5 also appeared to be a male specific QTL with the D2 allele associated with increased muscle mass.
QTL Edlmmq7 (extensor digitorum longus muscle mass QTL 7) mapped to Chromosome 8 between 126.2-130.3 Mb with a LOD score of 5.97 at peak marker rs33444220 when using a model that allowed for sex-specific effects.
QTL Gmmq6 (gastrocnemius muscle mass QTL 6) mapped to Chromosome 8 between 126.2-130.3 Mb with a LOD score of 6.76 at peak marker rs33444220 when using a model that allowed for sex-specific effects.
The Chromosome 8 QTL Edlmmq7 and Gmmq5 appeared to be female specific QTL with the B6 allele associated with decreased muscle mass in females.
QTL Tammq6 (tibialis anterior muscle mass QTL 6) mapped to Chromosome 9 with a LOD score of 5.32 (no peak marker given) when using a model that allowed for sex-specific effects.
QTL Edlmmq8 (extensor digitorum longus muscle mass QTL 8) mapped to Chromosome 9 between 34.7-49.6 Mb with a LOD score of 5.30 at peak marker rs6413270 when using a model that did not differentiate effects of SNPs in males and females.
QTL Tammq7 (tibialis anterior muscle mass QTL 7) mapped to Chromosome 9 between 106.3-111.5 Mb with a LOD score of 6.70 at peak marker rs13480407 when using a model that did not differentiate effects of SNPs in males and females.
QTL Edlmmq9 (extensor digitorum longus muscle mass QTL 9) mapped to Chromosome 9 between 106.2-111.8 Mb with a LOD score of 4.36 at peak marker rs13480407 when using a model that allowed for sex-specific effects.
QTL Gmmq7 (gastrocnemius muscle mass QTL 7) mapped to Chromosome 9 between 106.4-111.8 Mb with a LOD score of 5.01 at peak marker rs13480407 when using a model that did not differentiate effects of SNPs in males and females.
For the Chromosome 9 QTL Tammq6, Edlmmq8, Tammq7, Edlmmq9 and Gmmq7 the B6 allele was associated with increased muscle mass.
QTL Edlmmq10 (extensor digitorum longus muscle mass QTL 10) mapped to Chromosome 12 between 28.9-53.9 Mb with a LOD score of 4.85 at peak marker rs29138639 when using a model that did not differentiate effects of SNPs in males and females. The D2 allele was associated with increased muscle mass at Edlmmq10.
QTL Smmq2 (soleus muscle mass QTL 2) mapped to Chromosome 17 between 51.9-55.4 Mb with a LOD score of 4.44 at peak marker rs6272475 when using a model that did not differentiate effects of SNPs in males and females. Heterozygous mice at this locus exhibited the largest muscle weight.
Potential candidate genes for the above QTL are listed in Table 3.
Two QTL mapped with significance in the F8 AIL cross but did not achieve significance in the combine cross data:
QTL Gmmq8 (gastrocnemius muscle mass QTL 8) mapped to Chromosome 5 between 5.43-25.3 Mb with a LOD score of 6.57 at peak marker rs3714258 when using a model that allowed for sex-specific effects. The B6 allele was associated with increased muscle mass at this locus.
QTL Smmq3 (soleus muscle mass QTL 3) mapped to Chromosome 18 between 25.47-66.6 Mb with a LOD score of 4.25 at peak marker rs3676196. The B6 allele was associated with increased muscle mass at this locus. Gene Abca3 is a candidate gene for QTL Smmq3 carrying a nonsense mutation.
QTL Tammq8 (tibialis anterior muscle mass QTL 8) was identified in only the F9-F13 AIL mapping population. It was mapped to Chromosome 17 between 24.54-31.1 Mb with a LOD score of 4.97 at peak marker rs49469183. The B6 allele was associated with increased muscle mass at this locus. Gene Bin1 was identified as a candidate gene with a potentially damaging missense mutation.
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