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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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eQTL interval mapping of Tnni1. eQTL mapping of Tnni1 transcript abundance across BXD lines in two independent databases, based on (A) Affymetrix U74Av2 and (B) M430 microarray platforms. In both databases, significant association overlies the location of Tnni1, suggesting a potential cis-eQTL. This figure is directly from . The blue curve is the LRS trace; the red curve follows the right Y axis (effect size in standard deviations). The red horizontal line indicates genome wide significance. The triangle on the baseline is the position of Tnni1. The orange chatter along the X axis indicates the density of SNPs present in the BXD. The multicolored chatter along the top of the graph are hyperlinks to sites with additional genetic and sequence information (when a graph is viewed live at ).
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Figure 4: eQTL interval mapping of Tnni1. eQTL mapping of Tnni1 transcript abundance across BXD lines in two independent databases, based on (A) Affymetrix U74Av2 and (B) M430 microarray platforms. In both databases, significant association overlies the location of Tnni1, suggesting a potential cis-eQTL. This figure is directly from . The blue curve is the LRS trace; the red curve follows the right Y axis (effect size in standard deviations). The red horizontal line indicates genome wide significance. The triangle on the baseline is the position of Tnni1. The orange chatter along the X axis indicates the density of SNPs present in the BXD. The multicolored chatter along the top of the graph are hyperlinks to sites with additional genetic and sequence information (when a graph is viewed live at ).

Mentions: When either Tnni1 or Asb3 transcript abundance is mapped to locate QTLs controlling expression, both show strong evidence of control by cis-eQTLs (Figure 4, 5). Tnni1 transcript abundance maps to the chromosomal location of itself with an LRS of 25.2 (U74Av2 data set) and 30.6 (M430 data set). Asb3 transcript abundance maps to the chromosomal location of itself with an LRS of 36.1 (U74Av2 data set) and 51.5 (M430 data set).


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)

eQTL interval mapping of Tnni1. eQTL mapping of Tnni1 transcript abundance across BXD lines in two independent databases, based on (A) Affymetrix U74Av2 and (B) M430 microarray platforms. In both databases, significant association overlies the location of Tnni1, suggesting a potential cis-eQTL. This figure is directly from . The blue curve is the LRS trace; the red curve follows the right Y axis (effect size in standard deviations). The red horizontal line indicates genome wide significance. The triangle on the baseline is the position of Tnni1. The orange chatter along the X axis indicates the density of SNPs present in the BXD. The multicolored chatter along the top of the graph are hyperlinks to sites with additional genetic and sequence information (when a graph is viewed live at ).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: eQTL interval mapping of Tnni1. eQTL mapping of Tnni1 transcript abundance across BXD lines in two independent databases, based on (A) Affymetrix U74Av2 and (B) M430 microarray platforms. In both databases, significant association overlies the location of Tnni1, suggesting a potential cis-eQTL. This figure is directly from . The blue curve is the LRS trace; the red curve follows the right Y axis (effect size in standard deviations). The red horizontal line indicates genome wide significance. The triangle on the baseline is the position of Tnni1. The orange chatter along the X axis indicates the density of SNPs present in the BXD. The multicolored chatter along the top of the graph are hyperlinks to sites with additional genetic and sequence information (when a graph is viewed live at ).
Mentions: When either Tnni1 or Asb3 transcript abundance is mapped to locate QTLs controlling expression, both show strong evidence of control by cis-eQTLs (Figure 4, 5). Tnni1 transcript abundance maps to the chromosomal location of itself with an LRS of 25.2 (U74Av2 data set) and 30.6 (M430 data set). Asb3 transcript abundance maps to the chromosomal location of itself with an LRS of 36.1 (U74Av2 data set) and 51.5 (M430 data set).

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