Transcript week7
How do you go from QTL to gene? Fine mapping • Progeny testing • Collaborative cross • Heterogeneous stocks • Other outbred populations From www.complextrait.org/Powerpoint/ ctc2004/karl_broman.ppt x How do you go from QTL to gene? Fine mapping • Limited resolution? • Incomplete annotation of genes in region • Regulatory sequence within region (separate from genes) How do you prove a gene is responsible for the effect of a QTL? “The nature and identification of quantitative trait loci: a community’s view” by Members of the Complex Trait Consortium. Nature Reviews Genetics (2003) 4: 911-916. Standards will vary between taxa? How do you prove a gene is responsible for the effect of a QTL? Most conclusive: allele replacement by knock-in Or knock-outs, rescued by alternate alleles? How do you prove a gene is responsible for the effect of a QTL? Circumstantial evidence • Polymorphisms in coding or regulatory regions • Gene function • Expression differences • Homology • Knock-out studies • Mutational analysis • In vitro functional studies • Transgenesis with bacterial artificial chromosomes (BAC) • Quantitative complementation test Complementation test heterozygous for QTL segment heterozygous for gene X knock out Complementation test If gene X is the gene responsible for the effect of the QTL, the genotypes at the QTG will be If gene X is not the gene responsible for the effect of the QTL, the genotypes at the QTG will be Assumptions! white / KO dark / KO The phenotypic difference between these two genotypes white / white > will be greater than dark / white white / white dark / white the phenotypic difference between these two genotypes. white / white dark / white = The phenotypic will be the phenotypic equal to difference between difference between these two these two genotypes genotypes Example of gene identification (?): Klein et al. 2004; Science 303: 229-232 • • • • • • Mapped QTL affecting BMD in mice to a 31 MB region. Analysed gene expression by microarray. Only one gene within region was differentially expressed. Confirmed with RT PCR. Gene was in a pathway known to inhibit bone formation. Expression of bone formation markers was altered in cell cultures from the two genotypes. • Overexpression of the gene in cell culture affected markers of bone formation. • Phenotype of knock-out mouse was consistent with difference in expression between QTL genotypes (high expression -> low BMD). • Pharmacological inhibitors of the gene’s product affected BMD in a manner consistent with the QTL genotypes/ knock-outs (improved BMD in rodent models of osteoporosis).