Experiment
Angiogenesis is the process by which new blood vessels are formed from existing vessels. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases. Using the murine corneal micro pocket assay more than ten-fold differences have been observed in angiogenic responsiveness among various mouse strains. The degree of difference was observed in either bFGF or VEGF induced corneal neovascularization. In this study F2 crosses between C57BL/6J, SJL/6J and 129 substrains new QTL affecting angiogenic responsiveness were identified.
In the first cross, 77 (C57BL/6J x 129P1/ReJ)F2 animals were analyzed. The angiogenic response of F2 mice was determined using the corneal neovascularization assay using a dose of 10 ng bFGF. Markers with uncorrected p values of <0.05 were detected for association between vessel area and genotype but none of these was significant at the genome-wide level.
The next cross examined was between C57BL/6J and 129P3/J mice, also assessed at 10 ng bFGF. Simple association tests for each of the markers demonstrated uncorrected p values <0.05 for regions on chr 7, proximal chr 12 (D12Mit105 at 6 cM, p<0.001; D12Mit136 at 13 cM, p<0.001; and D12Mit46 at 16 cM) and at Chr 14 between D14Mit228 at 46, cM, p<0.05 and D14Mit198 at 54 cM, p<0.05 [Fig 2/Table 2].
In interval mapping, the linked region on Chr 12 exhibited the strongest effect, with an estimated effect on vessel area of ~0.6mm2.
The analysis also revealed linkage centered around marker D14Mit265 that appeared to reduce vessel area in 129 mice. The region exceed the genome-wide permutation threshold for p<0.05. This locus was named Angfq7, angiogenesis due to FGF2 QTL 7.
To test the role of parental sex a reciprocal cross using C57BL/6J and 129/P3J mice was performed. In male mice only the F1 value was dependent on the genetic background of the parent, suggesting that one of the sex chromosomes had a locus that affected angiogenesis. This was confirmed using 129S1/SvImJ-Chr YC57BL/6J/NaJ mice which has the C57BL/6J- Y chromosome on a 129S1/SvImJ background. When the angiogenic response of the 129S1/SvImJ-Chr YC57BL/6J/NaJ mice was compared with that of the 129S1/SvImJ parent using a 20 ng bFGF pellet, a 22% increase in angiogenic response was observed. Results were consistent with the difference observed between F2 animals with 129 vs B6 Y chromosomes [Fig 3b]. This locus was labeled Angfq8, angiogenesis due to FGF2 QTL 8, and was confirmed in several 129 substrains.
The final crosses were between SJL/J and C57BL/6J mice. Three distinct F2 crosses (72, 66 and 186 animals) resulted in 325 animals available for analysis. The angiogenic response of the resulting animals was determined using the corneal neovascularization assay using a dose of 80g bFGF. In the complete data set association between animal genotype and vessel area was observed at markers on chromosomes 1, 3, 5, 7, 1, and 17. Interval mapping on the full dataset revealed a single region of genome-wide highly significant linkage near D7Mit20 on Chromosome 7 that accounted for approximately 10% of trait variance.
Several chromosome 7 congenics were also generated from C57BL/6J x SJL/6J mice [Fig 5]. Animals heterozygous for D7Mit229, D7Mit211, D7Mit233, D7Mit62 and D7Mit301, but homozygous for the B6 allele at D7Mit332 were selected, and backcrossed for ten generations before intercrossing. These strains exhibited a decrease in bFGF-induced angiogenesis when compared to C57BL/6J controls, with the B6 allele exhibiting partial dominance. The Chromosome 7 locus was named Angfq5, angiogenesis due to FGF2 QTL 5. Based on shared haplotypes among closely related 129 strains [Fig 5] an analysis reduced the Angfq5 QTL to 4 regions of appropriate haplotype containing 1, 15, 7 and 2 annotated genes distributed across a total of 5.5.Mbp. Of the annotated genes only 2 exhibited SNPs predicted to alter amino-acid sequence, Herc2 and Oca2.
To directly test whether a pink-eyed dilution mutation could suppress angiogenesis the Ocap-J mutation was used. A comparison of the bFGF-induced vessel areas in C3H/HeJ and C3H/HeJ-Ocap-J animals, strains that differ only in a deletion at the pink-eyed dilution locus, showed that a pink-eyed dilution mutation suppressed angiogenesis to an extent that was sufficient to explain Angfq5.
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