Allelic imbalance and somatic mutations in folate pathway genes in childhood acute lymphoblastic leukaemia

Bosson, Geoffrey (2008) Allelic imbalance and somatic mutations in folate pathway genes in childhood acute lymphoblastic leukaemia. Doctoral thesis, Northumbria University.

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Abstract

ABSTRACT Acute lymphoblastic leukaemia (ALL) is the commonest of the childhood cancers, but thankfully responds well to chemotherapeutic agents, with 80% of children achieving long-term survival. However, for the remaining 20% who relapse, outcome is bleak. Increasing knowledge and understanding of pharmacogenetics indicates that constitutive or acquired resistance to the drugs used in the treatment of cancer contribute to relapse. Methotrexate (MTX) is one of the most important drugs used in the treatment of ALL and the work presented here contributes to the body of knowledge relevant to the understanding of resistance to this drug. The aim of this research is to determine if changes in genes involved in folate metabolism contribute to methotrexate resistance and subsequent relapse of childhood ALL. The reduced folate carrier (RFC) is required to transport methotrexate into the cell where it competitively inhibits dihydrofolate reductase (DHFR) and other key enzymes of folate and 1-carbon metabolism. Following the design of primers and optimisation of PCR amplification, the entire coding regions of both RFC and DHFR were screened for novel SNPs or mutations using denaturing high performance liquid chromatography (DHPLC) on genomic DNA from 40 normal and 40 relapse childhood ALL patients. Ethical approval for use of the samples was granted under WCRLEC no347 and reference 2002/111 for normal and relapse samples respectively. The relapse group was made up of 29 males and 11 females with an average age of 6.59 years (range 0.8-14.1) and was made up of 5 T-cell; 30 B-cell; 4 mixed lineage; and 1 null classification. The screening method was shown to be sufficiently sensitive to detect single base changes. Several of the exons in both genes have a high G-C content and required destabilisation agents and/or use of a new DNA polymerase (OptimaseTM) to achieve sufficient PCR amplification. Dimethyl sulphoxide (DMSO) and betaine were shown to be effective agents which did not interfere with subsequent DHPLC. The results show that somatic mutations in the coding sequences of RFC and DHFR are rare in relapsed ALL and that while it is recognised as a mechanism of methotrexate resistance in vitro, it is unlikely to contribute to relapse in children with ALL. However, for one patient who suffered multiple relapses, a novel acquired mutation was identified in the 5'-UTR of the RFC-1 gene (C-37T). The significance of the C-37T mutation on RFC transcription requires further study, but it may decrease RFC mRNA quantity or stability and thus protein levels. DHPLC analysis also detected common SNPs. In terms of frequency, there were no significant differences between relapse and normal samples for the genotypes; RFC G80A (38.3% G/G; 49.4% G/A; 12.3% A/A in 81 normal samples; 31.8%; 56.8%; 11.4% respectively in 44 relapse samples; x2 p = 0.656); RFC C696T (97.8% C/C; 2.2% C/T; 0% T/T in 45 normal samples; 92.5%; 5%; 2.5% respectively in 40 relapse samples; x2 p = 0.458). However, there was a significant difference in the 5'-UTR -19 base pair deletion in the DHFR gene between the normal and relapse groups (0% WT/WT; 100% WT/-19 or -19/-19 in 45 normals; 26.7% and 73.3% respectively in 15 relapse samples; Fisher's exact probability p = 0.017) and needs to be confirmed in a larger cohort. Estimated copy number and the probability of loss of heterozygosity (LOH) were generated from the Affymetrix 50K SNP microarray for the RFC, DHFR and other genes involved in folate metabolism using gDNA from 73 presentation and 20 relapse childhood ALL cases. Mann-Whitney non-parametric comparison of the presentation and relapse data showed that for estimated copy number, statistically significant differences were seen for MTHFR (p = 0.0072), MS (0.0025), FPGS (0.00048), TS (0.00046), CBS (0.0002) and RFC (0.0001). When the data at the nearest SNP location to the gene was presented as a scatterplot, the difference in each case was due to a bimodal distribu...

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