Inherited genetic variation in ATM and susceptibility to Non-Hodgkin lymphoma

Abstract

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The Ataxia Telangiectasia Mutated (ATM) gene is critical for the detection and repair of double stranded breaks. Mutations in this gene cause the autosomal recessive syndrome ataxia telangiectasia (AT), a feature of which is a high risk of cancer, particularly lymphoma. The hypothesis of this study is that inherited sequence variation in the ATM gene in the general population influences susceptibility to Non-Hodgkin Lymphoma (NHL). Objective: To determine whether inherited variation in ATM confers a risk of developing NHL. Design: In the SNP discovery phase of this project we are determining the extent of sequence variation in the promoter and all exons of ATM in 90 NHL patients, including both T and B cell lymphoma patients. We will then use single nucleotide polymorphisms (SNPs) and multi-SNP haplotypes in genetic association tests to determine if germline variants of this gene are associated with elevated risk of NHL in a case/control group of 800 cases and 800 controls. All consenting NHL cases aged 20-79 diagnosed between March 2000 and February 2004 and residing in the greater Vancouver or greater Victoria metropolitan areas of British Columbia, Canada constitute the case group. Controls were ascertained from the same geographic areas, and are frequency matched to cases by age and sex. Materials and Methods: Following PCR and cycle sequencing reactions, sequencing is carried out on ABI capillary sequencers. The bi-directional sequences are analyzed using PolyPhred (Nickerson et al., 1997) and visualized in Consed (Gordon et al., 1998). Results: The promoter region and exons of the 64-exon ATM gene comprise 66 PCR amplicons for sequencing. To date, 42 of these amplicons have been analyzed and 54 variants identified. Of these, 23 are not present in dbSNP (http://www.ncbi.nlm.nih.gov/SNP/), the largest public repository of SNP information. Using the variants identified in the SNP discovery phase, haplotypes will be estimated. SNPs will then be chosen for genotyping based on frequency and on inter-marker linkage disequilibrium. Variants that may have functional significance, for instance deletions or missense mutations, will be included in genotyping experiments. This combined sequencing and genotyping strategy will allow the assessment of both common (likely subtle) and rare (potentially stronger) deleterious ATM variants in susceptiblity to NHL. Conclusion: Preliminary sequencing of the ATM gene in the germline of 90 NHL patients has revealed 23 new variants, 9 of which result in non-synonymous amino acid changes. Two algorithms, Sorting Intolerant from Tolerant (SIFT) (Ng et al., 2001) and Polymorphism Phenotyping (PolyPhen) (Sunyaev et al., 2001), were employed to predict the impact of these amino acid substitutions on protein activity. Five of the changes were classified as “possibly or probably damaging” including one that results in a premature stop codon. Seven of 90 (7.7%) NHL patients were heterozygous at these loci.