title: Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA.
abstract: The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19-142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a matching tumor type, and which represent candidate PAI regions warranting further investigation.
authors.affiliation: Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: de Smith
authors.affiliation: Division of Neuroepidemiology, Department of Neurological Surgery, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Walsh
authors.affiliation: Division of Neuroepidemiology, Department of Neurological Surgery, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Helen M
authors.initials: HM
authors.lastname: Hansen
authors.affiliation: Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Alyson A
authors.initials: AA
authors.lastname: Endicott
authors.affiliation: Division of Neuroepidemiology, Department of Neurological Surgery, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: John K
authors.initials: JK
authors.lastname: Wiencke
authors.affiliation: School of Public Health, University of California, Berkeley, California, United States of America.
authors.forename: Catherine
authors.initials: C
authors.lastname: Metayer
authors.affiliation: Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California, United States of America.|Division of Neuroepidemiology, Department of Neurological Surgery, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Wiemels
mesh_terms: D000483:Alleles; D022981:Allelic Imbalance; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D005787:Gene Frequency; D055106:Genome-Wide Association Study; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000473:pathology; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics
chemical_list: D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D014157:Transcription Factors