Igf1sl1^C57BL/6J
QTL Variant Detail

Summary

• QTL variant: Igf1sl1^C57BL/6J
• Name: IGF-1 serum levels 1; C57BL/6J
• MGI ID: MGI:3046881
• QTL: Igf1sl1 Location: Chr6:52138693-116132228 bp Genetic Position: Chr6, cM position of peak correlated region/allele: 53.75 cM
• QTL Note: genome coordinates based on the marker associated with the peak LOD score

Variant origin

• Strain of Specimen: C57BL/6J

Variant description

• Allele Type: QTL

Expression

In Structures Affected by this Mutation:

1 anatomical structure(s)

Notes

Candidate Genes

J:99471

Igf1sl1 is a QTL previously identified in a congenic line named B6.C3-(D6Mit93-D6Mit150). Authors refer to this line as 6T. The congenic carries a C3H/HeJ-derived interval between D6Mit93 (26.3 cM) to D6Mit150 (51 cM) on a C57BL/6J genetic background. The congenic exhibits 30%-50% decreased IGF-1 expression in bone, liver, and fat tissue, and a 20% decrease in serum IGF-1 levels compared to background strain C57BL/6J. IGFBP-2 expression also appears to be decreased in bone. The congenic also exhibits decreased rate of bone formation and significantly increased percent body fat compared to C57BL/6J.

B6.C3-(D6Mit93-D6Mit150) did not respond well to parathyroid hormone (PTH) treatment as bone mineral density did not increased significantly after 4 week of daily injections with PTH.

Alox5 (53.2 cM) is a potential candidate gene mapping near Igf1sl1.

J:241559

Previously QTL, Bmd8, for the phenotype of total femoral volumetric bone mineral density (BMD) and Igf1sl1, for serum IGF-1 were mapped to a mid-dsital region of Chromosome 6 in a cross between C3H/HeJ (C3H) and C57BL/6J (B6) inbred mouse strains [J:78754 and J:66640, respectively].

In the current study a congenic strain, B6.C3H-(D6Mit93-D6Mit216)/J, (B6.C3H-6T), was developed for gaining insight into the biology underlying these QTL by generating mice that were homozygous for B6 alleles for the entire genome, except for the introgressed segment which was homozygous for C3H alleles.

Female B6.C3H-6T congenic mice have lower femoral volumetric bone mineral density (vBMD), smaller periosteal circumference, slightly shorter femurs and lower serum IGF-1 than either B6 or C3H mice. The congenic mice also exhibit a decrease in trabecular bone volume fraction of the distal femur that is coincident with an increase in marrow adipocytes and an impairment in osteoblast differentiation.

During the development of the congenic strain it was observed that no recombination events occurred between D6Mit124 and D6Mit150. Further experimentation determined that there was a 25 cM paracentric inversion on mid-distal Chr 6 in the C3H/HeJ strain and that foundation stocks were homozygous for the inversion. Both the D6Mit124 and the D6Mit150 markers were within the inverted region but D6Mit93 was not. The upper inversion breakpoint was further resolved to be distal to rs13478767 (55.2 Mb) but proximal to D6Mit124 (71.3 Mb) and the lower inversion breakpoint was distal to D6Mit150 (116.1 Mb) but proximal to D6Mit254 (125.3 Mb)

To identify candidate genes related to the QTL of interest the question of whether the inversion per se, or alleles within the QTL, or both, were responsible for the development

of a unique skeletal and metabolic phenotype that included a profound change in stromal cell allocation from preosteoblasts into adipocytes was explored. Disruption in the organization and order of genes within the genome, without disturbing the structure of a gene unit, can cause a variety of diseases.

Liver was collected from three 8-week-old B6 and three B6.C3H-6T female mice. Images were quantified using GeneChipTM Operating Software(GCOS) v1.2. Probe level data were imported into the R software environment and expression values were summarized using the Robust

MultiChip Average (RMA) function in the R/affy package. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE5959. The pattern of differential gene expression on Chr 6 in the liver of B6.C3H-6T and B6 mice was examined by microarray. At approximately 72 Mb and 116 Mb a clear pattern of gene expression emerged. At the approximate breakpoints of the inversion the majority of genes are clearly down regulated in liver of B6.C3H-6T congenic mice. [Fig 2.]

To determine if the low BMD and low serum IGF-1 phenotypes of the B6.C3H-6T congenic mouse

were a function of allelic differences from C3H, or due entirely to the Chr 6 inversion, a new congenic strain, B6.BXH6-(D6Mit102-rs3727110)/J, (B.H-6) was generated by introgressing an app 30 Mb region from BXH6 RI line onto a B6 background. Mice were typed at each generation and backcrossing continued for 8 generations. N8F1 mice were intercrossed producing N8F2 progeny. Mice homozygous for the C3H-like alleles were intercrossed for 2 more generations, yielding N8F4 and creating B.H-6.

Areal BMD and body composition was assessed; all bones for density and architecture analysis were collected from 16 week old female mice; femur lengths were measured for density; total vBMD values were calculated; femurs were scanned to evaluate trabecular bone volume fraction and microarchitecture in the secondary spongiosa of the distal femur; statistical evaluation of bone and body composition was conducted.

At 16 weeks of age, female B.H-6 congenic mice weighed more than both B6 and the B6.C3H-6T congenic mice. B.H-6 mice also had higher absolute fat mass and a higher percentage of total body fat that either B6 or B6.C3H-6T mice. In addition the strain had a lower lean mass. Unlike the B6.C3H-6T mice, the B.H-6 female mice had a similar total femoral vBMD compared to B6 mice. Most striking, the B.H-6 mice had an increase in trabecular bone volume compared to either B6 or B6.C3H-6T.

Results show that the B6.C3H-6T and B.H-6 congenic strains, despite having the same C3H/HeJ alleles on distal Chr 6 and identical B6 backgrounds, have a drastically different anthropomorphic and skeletal phneotype. The B6.C3H-6T mouse is a small lean mouse with very low BMD but marrow adipogenesis, where B.H-6 is fatter with higher BMD. These data support the thesis that the chromosomal inversion, not the allelic differences between C3H and B6, is responsible for the metabolic and skeletal phenotypes of B6.C3H-6T.

The most likely candidate gene to be disrupted by this inversion because of its genomic location is Pparg, a nuclear receptor that is essential for adipogenesis, and also a negative regulator of osteoblastogenesis when activated by specific ligands.

Mapping and Phenotype information for this QTL, its variants and associated markers

J:66640

Phenotypically extreme female animals from a (C57BL/6J x C3H/HeJ)F2 intercross were genotyped at 114 polymorphic loci at a resolution of 14 cM to identify QTLs associated with serum IGF-1 levels and bone mineral density (BMD). Parental strain C3H/HeJ exhibits increased serum IGF-1 and increased BMD compared to C57BL/6J. Since osteoblasts from C3H/HeJ animals secrete more IGF-1 compared to osteoblasts from C57BL/6J animals a correlation between IGF-1 levels and BMD was investigated. 682 F2 female animals were used in the final analysis.

Significant linkage mapped to 51 cM on mouse Chromosome 6 near D6Mit150 (LOD=9). This locus in named Igf1sl1 (IGF-1 serum level 1). C3H/HeJ-derived alleles confer decreased serum IGF-1 at this locus. A correlation betweenserum IGF-1 levels and BMD was observed for Igf1sl1.

Igf1sl2 mapped to 51 cM on mouse Chromosome 10 near D10Mit95 (LOD=4.5). C3H/HeJ-derived alleles confer increased serum IGF-1 at this locus. Correlation between serum IGF-1 levels and BMD was not observed for Igf1sl2. The Igf1 (48 cM) gene maps very close to this locus, just 3 cM proximal to Igf1sl2.

Igf1sl3 mapped to 32 cM on mouse Chromosome 15 near D15Mit209 (LOD=4.4). C3H/HeJ-derived alleles confer increased serum IGF-1 at this locus. Correlationbetween serum IGF-1 levels and BMD was not observed for Igf1sl1.

A fourth QTL, Igf1sl4, was identified via pairwise interaction analysis. Ifg1sl4 maps to 1.1 cM on mouse Chromosome 11 near D11Mit71 and is epistatic to Igf1sl1 on mouse Chromosome 6. Homozygosity for C3H/HeJ-derived alleles at Igf1sl4 negates the IGF-1 lowering effect of Igf1sl1. When Igf1sl4 is homozygous or heterozygous for C57BL/6J-derived alleles the effect of Igfsl1 can be observed. Correlation between serum IGF-1 levels and BMD wasobserved for Igf1sl4. Potential candidate genes mapping near Igf1sl4 are Igfbp1 (1.3 cM) and Igfbp3 (1.35 cM).

To test the theory that C3H/HeJ-derived alleles at Igfsl1 and C57BL/6J-derived alleles at Igfsl4 impact serum IGF-1 and BMD, a congenic line (B6.C3H-6) carrying C3H/HeJ-derived DNA on chromosome 6 on the genetic background of C57BL/76J was studied. Congenic animals exhibit a 16% decrease in serum IGF-1 and a 4% decrease in femoral BMD compared to C57BL/6J.

J:94512

A previously identified QTL named Igf1sl1 was confirmed and fine-mapped in the present study using congenic strain analysis. Igf1sl1 is associated with serum IGF-1 levels and bone mineral density (BMD), and maps to 51 cM on mouse Chromosome 6 in linkage to D6Mit150.

The congenic strain that was created carries a C3H/HeJ-derived interval that contains Igf1sl1 on a C57BL/6J genetic background. This congenic strain is called B6.C3-(D6Mit93-D6Mit150) and the C3H/HeJ-derived region spans approximately 26.3 cM to 51 cM. Although donor strain C3H/HeJ exhibits increased serum IGF-1 levels (25%-30% higher) compared to background strain C57BL/6J, the Igf1sl1 locus confers decreased serum IGF-1 levels when the alleles are from C3H/HeJ. Therefore, congenic strain B6.C3-(D6Mit93-D6Mit150) is expected to have lower serum IGF-1 levels compared to C57BL/6J and that is what is observed (serum IGF-1 is 15%-25% decreased). The congenic also exhibits decreased rate of bone formation and significantly reduced trabecularandcortical BMD of the L5 vertebrae compared to C57BL/6J.

DNA microarray analysis and Q-PCR (quantitative real-time PCR) were used to identify candidate genes within the Igf1sl1 interval. Pparg and Cav3 map within the Ifg1sl1 interval and are differentially expressed between congenic B6.C3-(D6Mit93-D6Mit150) and parental C57BL/6J.

J:112375

Igf1sl1 is previously identified QTL on mouse Chromosome 6 affecting levels of serum IGF-1. A congenic line named B6.C3-(D6Mit93-D6Mit150) carries a C3H/HeJ-derived interval encompassing Igf1sl1 between D6Mit93 (26.3 cM) to D6Mit150 (51 cM) on a C57BL/6J genetic background. Authors refer to this line as 6T, which is estimated to be approximately 99.8% C57BL/6J at the N9 generation. Congenic females display 11%-21% decreased serum IGF-1 at 6, 8, and 16 weeks of age, and congenic males display 13% decreased serum IGF-1 at 16weeks of age compared to C57BL/6J. Congenic osteoblasts also secreted 40% less IGF-1 compared to C57BL/6J osteoblasts. This study also examined the effects of serum IGF-1 on skeletal phenotypes.

Congenic animals display decreased body fat percentage at12 weeks of age compared to C57BL/6J. Female congenic animals at 16 weeks of age display decreased femoral length, periosteal circumference, and total body areal bone mineral density (BMD) compared to C57BL/6J females. Female congenic animalsalso display significantly decreased bone volume fraction and bone thickness in the femur and vertebral bodies between 8-16 weeks of age. It appears that C3H/HeJ-derived alleles at Igf1sl1 affect bone development and acquisition, especially during times of rapid growth around 8-12 weeks of age.

References

• Original: J:66640 Rosen CJ, et al., Mapping quantitative trait loci for serum insulin-like growth factor-1 levels in mice. Bone. 2000 Oct;27(4):521-8
• All: 5 reference(s)