Abstract
The delayed infection hypothesis, first proposed by Mel Greaves in 1988, postulates that paediatric leukaemia is the result of a triggering infectious agent acting upon an unconditioned immune system. According to this hypothesis, childhood leukaemia develops in two sequential steps involving (i) the generation, in utero, of a pre-leukaemic clone; and (ii) the post-natal conversion, by an infectious agent, of this pre-leukaemic clone into a leukaemic transformant. The balance between the two major T cell subsets, Th1 and Th2, is critical for determining the type and extent of the human immune response. Children shielded early in life from common childhood infections have unconditioned immune systems showing Th1/Th2 anomalies that may pre-dispose them to developing leukaemia. Since the delayed infection hypothesis specifically proposes that the immune response to infection critically determines leukaemia development, we are studying genes encoding cytokines that affect this balance. We are looking for patterns of single nucleotide polymorphisms (SNPs) in paediatric acute lymphoblastic leukaemia (ALL) and comparing these with the patterns in non-ALL controls.
Our aims in this study are to collect and catalogue archived paediatric ALL and non-ALL control DNA samples from centres around Europe; to expand the collected stocks by whole genome amplification (WGA); and to determine the combination of immune response gene-related SNPs in paediatric ALL and non-ALL patients while piloting a high-throughput protocol for determining patterns of SNPs in multiple genes. Using the Taqman and SNPlex assays, we aim to evaluate relevant genes including the ones encoding interleukin (IL)-12, IL-10, IL-2, IL-4, interferon-γ and IFNγ receptor, all known to regulate Th1/Th2 balance.
300 paediatric ALL and 200 non-ALL control DNA samples were obtained from sample banks in the Royal Marsden Hospital in London and the Kinderspital in Munich. 90% of samples were successfully amplified from nanogram to microgram quantities using the GenomiPhi® WGA kit. Up to 1000-fold amplification was routinely achieved using this method, with a mean of 600-fold amplification. DNA fragments from samples were amplifiable by multiplex PCR using primers to five genomic regions (corresponding to AF4, PLZF, RAG1 and TBXAS1). In addition, sequencing of 700-bp amplicons from the IL-10, IL-18 and ERCC2 genes showed identity of pre- and post-GenomiPhi templates for the fragments sequenced, confirming that the products of WGA by this method are representative of the original templates. We will present data on our SNPlex analysis of 66 t(12;21)- and 84 hyperdiploidy-related paediatric ALL samples, together with 135 untyped samples, compared to 200 control samples, for 6 Th1/Th2-related genes. We will also present similar comparative data on 70 SNPs in 50 genes connected with DNA repair, xenobiotic metabolism, oxidative stress, neuropathies and bone development, as well as a control set of genes unrelated to leukaemia.
Kay Kendall Leukaemia Fund
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