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Using gene expression databases for classical trait QTL candidate gene discovery in the BXD recombinant inbred genetic reference population: mouse forebrain weight.

Lu L, Wei L, Peirce JL, Wang X, Zhou J, Homayouni R, Williams RW, Airey DC - BMC Genomics (2008)

Bottom Line: Successful strategies for QTL gene identification benefit from combined experimental and bioinformatic approaches.The intersection of gene lists that were within +/- 10 Mb of the strongest associated location, that had forebrain mRNA abundance correlated with forebrain weight among the BXD, and that had forebrain mRNA abundance differing between C57BL/6J and DBA/2J, produced two candidates, Tnni1 (troponin 1) and Asb3 (ankyrin repeat and SOCS box-containing protein 3).Delivery of well supported candidate genes following a single quantitative trait locus mapping experiment is difficult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jiangsu Key Laboratory of Neuroregeneration, Nantong University, PR China. lulu@utmem.edu

ABSTRACT

Background: Successful strategies for QTL gene identification benefit from combined experimental and bioinformatic approaches. Unique design aspects of the BXD recombinant inbred line mapping panel allow use of archived gene microarray expression data to filter likely from unlikely candidates. This prompted us to propose a simple five-filter protocol for candidate nomination. To filter more likely from less likely candidates, we required candidate genes near to the QTL to have mRNA abundance that correlated with the phenotype among the BXD lines as well as differed between the parental lines C57BL/6J and DBA/2J. We also required verification of mRNA abundance by an independent method, and finally we required either differences in protein levels or confirmed DNA sequence differences.

Results: QTL mapping of mouse forebrain weight in 34 BXD RI lines found significant association on chromosomes 1 and 11, with each C57BL/6J allele increasing weight by more than half a standard deviation. The intersection of gene lists that were within +/- 10 Mb of the strongest associated location, that had forebrain mRNA abundance correlated with forebrain weight among the BXD, and that had forebrain mRNA abundance differing between C57BL/6J and DBA/2J, produced two candidates, Tnni1 (troponin 1) and Asb3 (ankyrin repeat and SOCS box-containing protein 3). Quantitative RT-PCR confirmed the direction of an increased expression in C57BL/6J genotype over the DBA/2J genotype for both genes, a difference that translated to a 2-fold difference in Asb3 protein. Although Tnni1 protein differences could not be confirmed, a 273 bp indel polymorphism was discovered 1 Kb upstream of the transcription start site.

Conclusion: Delivery of well supported candidate genes following a single quantitative trait locus mapping experiment is difficult. However, by combining available gene expression data with QTL mapping, we illustrated a five-filter protocol that nominated Asb3 and Tnni1 as candidates affecting increased mouse forebrain weight. We recommend our approach when (1) investigators are working with phenotypic differences between C57BL/6J and DBA/2J, and (2) gene expression data are available on http://www.genenetwork.org that relate to the phenotype of interest. Under these circumstances, measurement of the phenotype in the BXD lines will likely also deliver excellent candidate genes.

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Western blot of Asb3 in C57BL/6J, DBA/2J, BXD25, and BXD40. C57BL/6J (B6) and BXD25 show elevated Asb3 protein abundance relative to DBA/2J (D2) and BXD40. The uniform intensity of the β-tubulin staining across all samples validates the consistency of the total protein loaded for each strain.
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Figure 6: Western blot of Asb3 in C57BL/6J, DBA/2J, BXD25, and BXD40. C57BL/6J (B6) and BXD25 show elevated Asb3 protein abundance relative to DBA/2J (D2) and BXD40. The uniform intensity of the β-tubulin staining across all samples validates the consistency of the total protein loaded for each strain.

Mentions: To verify gene expression differences with an independent method, reverse transcriptase PCR was performed on C57BL/6J and DBA/2J forebrain total RNA. Both Tnni1 and Asb3 showed greater transcript abundance in C57BL/6J relative to DBA/2J, with Tnni1 3.3-fold different, and Asb3 2.5-fold different. Two BXD strains with high (BXD25) and low (BXD40) microarray expression for Asb3 were also analyzed by RT-PCR, indicating a 8.4 fold increase in mRNA expression in BXD25 forebrain compared to BXD40 forebrain. Finally, using an antibody against Asb3 protein, Western blot analysis showed a 2.3 fold increase in BXD25 forebrain compared to BXD40 forebrain (Figure 6).


Using gene expression databases for classical trait QTL candidate gene discovery in the BXD recombinant inbred genetic reference population: mouse forebrain weight.

Lu L, Wei L, Peirce JL, Wang X, Zhou J, Homayouni R, Williams RW, Airey DC - BMC Genomics (2008)

Western blot of Asb3 in C57BL/6J, DBA/2J, BXD25, and BXD40. C57BL/6J (B6) and BXD25 show elevated Asb3 protein abundance relative to DBA/2J (D2) and BXD40. The uniform intensity of the β-tubulin staining across all samples validates the consistency of the total protein loaded for each strain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570693&req=5

Figure 6: Western blot of Asb3 in C57BL/6J, DBA/2J, BXD25, and BXD40. C57BL/6J (B6) and BXD25 show elevated Asb3 protein abundance relative to DBA/2J (D2) and BXD40. The uniform intensity of the β-tubulin staining across all samples validates the consistency of the total protein loaded for each strain.
Mentions: To verify gene expression differences with an independent method, reverse transcriptase PCR was performed on C57BL/6J and DBA/2J forebrain total RNA. Both Tnni1 and Asb3 showed greater transcript abundance in C57BL/6J relative to DBA/2J, with Tnni1 3.3-fold different, and Asb3 2.5-fold different. Two BXD strains with high (BXD25) and low (BXD40) microarray expression for Asb3 were also analyzed by RT-PCR, indicating a 8.4 fold increase in mRNA expression in BXD25 forebrain compared to BXD40 forebrain. Finally, using an antibody against Asb3 protein, Western blot analysis showed a 2.3 fold increase in BXD25 forebrain compared to BXD40 forebrain (Figure 6).

Bottom Line: Successful strategies for QTL gene identification benefit from combined experimental and bioinformatic approaches.The intersection of gene lists that were within +/- 10 Mb of the strongest associated location, that had forebrain mRNA abundance correlated with forebrain weight among the BXD, and that had forebrain mRNA abundance differing between C57BL/6J and DBA/2J, produced two candidates, Tnni1 (troponin 1) and Asb3 (ankyrin repeat and SOCS box-containing protein 3).Delivery of well supported candidate genes following a single quantitative trait locus mapping experiment is difficult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jiangsu Key Laboratory of Neuroregeneration, Nantong University, PR China. lulu@utmem.edu

ABSTRACT

Background: Successful strategies for QTL gene identification benefit from combined experimental and bioinformatic approaches. Unique design aspects of the BXD recombinant inbred line mapping panel allow use of archived gene microarray expression data to filter likely from unlikely candidates. This prompted us to propose a simple five-filter protocol for candidate nomination. To filter more likely from less likely candidates, we required candidate genes near to the QTL to have mRNA abundance that correlated with the phenotype among the BXD lines as well as differed between the parental lines C57BL/6J and DBA/2J. We also required verification of mRNA abundance by an independent method, and finally we required either differences in protein levels or confirmed DNA sequence differences.

Results: QTL mapping of mouse forebrain weight in 34 BXD RI lines found significant association on chromosomes 1 and 11, with each C57BL/6J allele increasing weight by more than half a standard deviation. The intersection of gene lists that were within +/- 10 Mb of the strongest associated location, that had forebrain mRNA abundance correlated with forebrain weight among the BXD, and that had forebrain mRNA abundance differing between C57BL/6J and DBA/2J, produced two candidates, Tnni1 (troponin 1) and Asb3 (ankyrin repeat and SOCS box-containing protein 3). Quantitative RT-PCR confirmed the direction of an increased expression in C57BL/6J genotype over the DBA/2J genotype for both genes, a difference that translated to a 2-fold difference in Asb3 protein. Although Tnni1 protein differences could not be confirmed, a 273 bp indel polymorphism was discovered 1 Kb upstream of the transcription start site.

Conclusion: Delivery of well supported candidate genes following a single quantitative trait locus mapping experiment is difficult. However, by combining available gene expression data with QTL mapping, we illustrated a five-filter protocol that nominated Asb3 and Tnni1 as candidates affecting increased mouse forebrain weight. We recommend our approach when (1) investigators are working with phenotypic differences between C57BL/6J and DBA/2J, and (2) gene expression data are available on http://www.genenetwork.org that relate to the phenotype of interest. Under these circumstances, measurement of the phenotype in the BXD lines will likely also deliver excellent candidate genes.

Show MeSH
Related in: MedlinePlus