title: High-throughput association testing on DNA pools to identify genetic variants that confer susceptibility to acute myeloid leukemia.
abstract: We have evaluated the use of allele-specific PCR (AS PCR) on DNA pools as a tool for screening inherited genetic variants that may be associated with risk of adult acute myeloid leukemia (AML). Two DNA pools were constructed, one of 444 AML cases, and another of 823 matched controls. The pools were validated using individual genotyping data for GSTP1 and LTalpha variants. Allele frequencies for variants in GSTP1 and LTalpha were estimated using quantitative AS PCR, and when compared to individual genotyping data, a high degree of concordance was seen. AS primer pairs were designed for nine candidate genetic variants in DNA repair and cell cycle/apoptotic regulatory genes, including Cyclin D1 [codon 870 splice site variant (A>G)]; BRCA1, P871L; ERCC2, K751Q; FAS -1377 (G>A); hMLH1 -93 (G>A) and V219I; p21, S31R; and the XRCC1 R194W and R399Q variants. For six of these assays, there was at least 95% concordance between AS PCR genotyping and an alternative approach carried out on individual samples. Furthermore, these six AS PCR assays all accurately estimated allele frequencies in the pools that had been calculated using individual genotyping data. A significant disease association was seen with AML for the -1377 variant in FAS (odds ratio 1.76, 95% confidence interval 1.26-2.44). These data suggest that quantitative AS PCR can be used as an efficient screening technique for disease associations of genetic variants in DNA pools made from case-control studies.
authors.affiliation: Leukaemia Research Fund Epidemiology and Genetics Unit, School of Medicine, University of Leeds, United Kingdom.
authors.forename: Sara
authors.initials: S
authors.lastname: Rollinson
authors.forename: James M
authors.initials: JM
authors.lastname: Allan
authors.forename: Graham R
authors.initials: GR
authors.lastname: Law
authors.forename: Philippa L
authors.initials: PL
authors.lastname: Roddam
authors.forename: Martyn T
authors.initials: MT
authors.lastname: Smith
authors.forename: Christine
authors.initials: C
authors.lastname: Skibola
authors.forename: Alexandra G
authors.initials: AG
authors.lastname: Smith
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Forrest
authors.forename: Kathryn
authors.initials: K
authors.lastname: Sibley
authors.forename: Russell
authors.initials: R
authors.lastname: Higuchi
authors.forename: Soren
authors.initials: S
authors.lastname: Germer
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Morgan
mesh_terms: D001483:Base Sequence; D016022:Case-Control Studies; D016001:Confidence Intervals; D005260:Female; D005787:Gene Frequency; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D016017:Odds Ratio; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*