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.
Highlights
Over the past decade, genome-wide association studies (GWAS) have identified common heritable genetic variants associated with most cancer types
For single nucleotide polymorphism (SNP) located in genomic loci frequently deleted in acute lymphoblastic leukemia (ALL), i.e. CDKN2A and IKZF1, we investigated whether allelic imbalance (AI) was caused by somatic copy number loss or by copy-neutral loss of heterozygosity (LOH)
For SNPs in CDKN2A and IKZF1, genes frequently deleted in ALL, we hypothesized a priori that the risk allele would be preferentially retained when somatic loss occurs at these loci in heterozygote cases
Summary
Genome-wide association studies (GWAS) have identified common heritable genetic variants associated with most cancer types. Tumor selection of cancer-associated single nucleotide polymorphism (SNP) risk alleles has been demonstrated in colorectal cancer [4,5,6] and squamous cell carcinoma [2, 7] This type of selection, known as preferential allelic imbalance (PAI), may occur when there is somatic loss or gain at a genomic locus containing a heritable cancer-associated SNP, whereby the risk allele is preferentially retained or gained relative to the protective allele. Several techniques have previously been used to investigate tumor PAI, including Sanger sequencing [6], microsatellite marker genotyping [2, 5], and SNP genotyping [4, 7] These methodologies do not allow absolute quantitation of allelic imbalance (AI), and provide no indication as to the clonality of the specific SNP allele loss or gain within the tumor
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