Fine mapping of genes determining extrafusal fiber properties in murine soleus muscle

Peer reviewedPostprin

Intragenic deletions and a deep intronic mutation affecting pre-mRNA splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5-fluorouracil toxicity

Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme acting in the catabolism of the widely used antineoplastic agent 5-fluorouracil (5FU). DPD deficiency is known to cause a potentially lethal toxicity following administration of 5FU. Here, we report novel genetic mechanisms underlying DPD deficiency in patients presenting with grade III/IV 5FU-associated toxicity. In one patient a genomic DPYD deletion of exons 21–23 was observed. In five patients a deep intronic mutation c.1129–5923C>G was identified creating a cryptic splice donor site. As a consequence, a 44 bp fragment corresponding to nucleotides c.1129–5967 to c.1129–5924 of intron 10 was inserted in the mature DPD mRNA. The deleterious c.1129–5923C>G mutation proved to be in cis with three intronic polymorphisms (c.483 + 18G>A, c.959–51T>G, c.680 + 139G>A) and the synonymous mutation c.1236G>A of a previously identified haplotype. Retrospective analysis of 203 cancer patients showed that the c.1129–5923C>G mutation was significantly enriched in patients with severe 5FU-associated toxicity (9.1%) compared to patients without toxicity (2.2%). In addition, a high prevalence was observed for the c.1129–5923C>G mutation in the normal Dutch (2.6%) and German (3.3%) population. Our study demonstrates that a genomic deletion affecting DPYD and a deep intronic mutation affecting pre-mRNA splicing can cause severe 5FU-associated toxicity. We conclude that screening for DPD deficiency should include a search for genomic rearrangements and aberrant splicing

Cell cycle perturbation and acquired 5-fluorouracil chemoresistance

Acquired chemoresistance is one of the obstacles for success of 5-fluorouracil (5-FU)-based cancer chemotherapy. Some molecular mechanisms of acquired 5-FU resistance are still unknown. We have recently demonstrated down-regulation of a group of cell cycle related genes in acquired 5-FU resistant human cancer cell lines. In this study, the bivariate distribution of propidium iodide versus BrdU in acquired 5-FU resistant colon (H630(R10)) and breast (T47D(FU2.5)) cancer cell lines was compared with their parental cell lines using flow cytometric analysis. The resistant cell lines showed significantly lower labelling index (T47DFU2.5) and cell cycle delay in G1 and G1/S boundary and prolonged DNA synthesis time (H630R10). Both resistant cell lines demonstrated significantly prolonged potential doubling time (T-pot). The protein expression levels of some G1 and S phase transition-related genes were also analysed by Western blot. CDK2 protein and Thr-160 phosphorylated CDK2 were remarkably reduced in the resistant cell lines. Cyclin D3 and cyclin A were also decreased in the resistant cells. Total pRB expression was unaltered but hypophosphorylation of pRB (Ser780, Ser795 and Ser807/811) was detected in the resistant cancer cells. Our data suggest that there may be a slow down in cell cycle traverse preventing incorporation of 5-FU metabolites into DNA and also providing cancer cells with sufficient time to correct the mis-incorporated nucleotides. The cell cycle perturbation may be involved in acquired 5-FU resistance