Abstract 5113: Functional analysis of genomic variants identified through whole exome sequencing for susceptibility to lymphocytic leukemia
Authors: Saliba Jason , Trevino Lisa R. , Meng Qingchang , Zabriskie Ryan , Powell Bradford , Hicks Stephanie , Kimmel Marek , Cheung Hannah , Muzny Donna M. , Reid Jeffrey G. , Wheeler David , Gibbs Richard A. , Plon Sharon E. 
Published: August 2012

Abstract
The underlying genetic basis of many childhood cancers remains largely unknown, which places a significant focus on the discovery and understanding of cancer susceptibility genes. Our research is focused on the identification of novel high risk childhood cancer susceptibility genes through next generation sequencing methods and functional assays. One kindred contained four individuals diagnosed during childhood with lymphocytic leukemia or lymphoma transmitted in an autosomal dominant pattern of inheritance. Whole exome sequencing was performed on the constitutional DNA of three of the patients diagnosed with childhood cancer and one non-transmitting parent as an internal control. This analysis resulted in over 1000 unique single nucleotide variant (SNV) sites shared among the three affected family members. Of these SNVs, greater than 95% were found in control databases and excluded. Also, any variant found in the non-transmitting parent was eliminated. Systematic bioinformatics analysis resulted in a short list of seven missense SNVs predicted to be functionally important. Within this list is the missense mutation of a conserved residue (L254P) of Human Cytosolic 5′ Nucleotidase (NT5C1A). NT5C1A has a role in nucleoside metabolism as this protein primarily catalyzes the production of adenosine through the dephosphorylation of AMP. Adenosine metabolism is essential for lymphocyte maturation and viability, making NT5C1A an attractive leukemia susceptibility candidate to pursue further through functional genomic assays. Prior studies have shown that overexpression of wild type NT5C1A in HEK293 cells improves cell survival after treatment with purine and pyrimidine analogs also used in leukemia treatment. We created multiple HEK293 cell lines that stably overexpress either the NT5C1A wild type or L254P mutant protein. These cells were tested for quantitative changes in NT5C1A mediated cytotoxicity to the nucleoside analogs, Cladribine and Gemcitabine. We have replicated the prior findings in cells overexpressing wild type NT5C1A and demonstrated that resistance is dependent on the amount of exogenous NT5C1A protein expressed. Importantly, overexpression of the L254P protein is deficient in mediating resistance to these analogs, which is consistent with this novel variant interfering with NT5C1A activity. This finding has led us to hypothesize that the L254P mutation leads to a reduction in protein function, most likely through reduced substrate binding, which may increase AMP levels in early lymphocytes and predispose to tumorigenesis. Future directions include experiments to determine the L254P protein's substrate binding affinity and activity and the introduction of the mutation into murine leukemia models as well as analysis of L254P as a pharmacogenetic variant.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5113. doi:1538-7445.AM2012-5113