Rutger Meinsma

Genotype and phenotype in patients with dihydropyrimidine dehydrogenase deficiency

Abstract Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterised by thymine-uraciluria in homozygous deficient patients and has been associated with a variable clinical phenotype. In order to understand the genetic and phenotypic basis for DPD deficiency, we have reviewed 17 families presenting 22 patients with complete deficiency of DPD. In this group of patients, 7 different mutations have been identified, including 2 deletions [295–298delTCAT, 1897delC], 1 splice-site mutation [IVS14+1G>A)] and 4 missense mutations (85T>C, 703C>T, 2658G>A, 2983G>T). Analysis of the prevalence of the various mutations among DPD patients has shown that the G→A point mutation in the invariant splice donor site is by far the most common (52%), whereas the other six mutations are less frequently observed. A large phenotypic variability has been observed, with convulsive disorders, motor retardation and mental retardation being the most abundant manifestations. A clear correlation between the genotype and phenotype has not been established. An altered β-alanine, uracil and thymine homeostasis might underlie the various clinical abnormalities encountered in patients with DPD deficiency.

Increased risk of grade IV neutropenia after administration of 5‐fluorouracil due to a dihydropyrimidine dehydrogenase deficiency: High prevalence of the IVS14+1g>a mutation

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate‐limiting enzyme in the catabolism of 5‐fluorouracil (5‐FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk of developing severe 5‐FU‐associated toxicity. We evaluated the importance of DPD deficiency, gender and the presence of the IVS14+1G>A mutation in the etiology of 5‐FU toxicity. In 61% of cases, decreased DPD activity could be detected in peripheral blood mononuclear cells. Furthermore, the number of females (65%) in the total group of patients appeared to be higher than the number of males (35%) (p = 0.03). Patients with partial DPD deficiency appeared to have a 3.4‐fold higher risk of developing grade IV neutropenia than patients with normal DPD activity. Analysis of the DPYD gene of patients suffering from grade IV neutropenia for the presence of the IVS14+1G>A mutation showed that 50% of the patients investigated were heterozygous or homozygous for the IVS14+1G>A mutation. Adopting a threshold level for DPD activity of 70% of that observed in the normal population, 14% of the population is prone to the development of severe 5‐FU‐associated toxicity. Below this threshold level, 90% of individuals heterozygous for a mutation in the DPYD gene can be identified. Considering the common use of 5‐FU in the treatment of cancer, the severe 5‐FU‐related toxicities in patients with low DPD activity and the apparently high prevalence of the IVS14+1G>A mutation, screening of patients at risk before administration of 5‐FU is warranted.

A point mutation in an invariant splice donor site leads to exon skipping in two unrelated Dutch patients with dihydropyrimidine dehydrogenase deficiency

SummaryDihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria and associated with a variable clinical phenotype. In order to identify the molecular defect underlying complete DPD deficiency in a Dutch patient previously shown to have a 165 base pair deletion in the mature DPD mRNA, we cloned the genomic region encompassing the skipped exon and its flanking intron sequences. Sequence analysis revealed that the patient was homozygous for a single G→A point mutation in the invariant GT dinucleotide splice donor site downstream of the skipped exon. The same mutation was identified in another, unrelated, Dutch patient. Because this mutation destroys a uniqueMaeII restriction site, rapid screening using restriction enzyme cleavage of the amplified genomic region encompassing this mutation is possible. Analysis of 50 controls revealed no individuals heterozygous for this mutation

Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and expression of missense mutations C29R, R886H and R235W

Abstract Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype. Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity. In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug. Sequence analysis in a patient with complete DPD deficiency, previously shown to be heterozygous for the ΔC1897 frameshift mutation, revealed the presence of a novel missense mutation, R235W. Expression of this novel mutation and previously identified missense mutations C29R and R886H in Escherichia coli showed that both C29R and R235W lead to a mutant DPD protein without significant residual enzymatic activity. The R886H mutation, however, resulted in about 25% residual enzymatic activity and is unlikely to be responsible for the DPD-deficient phenotype. We show that the E. coli expression system is a valuable tool for examining DPD enzymatic variants. In addition, two new patients who were both heterozygous for the C29R mutation and the common splice donor site mutation were identified. Only one of these patients showed convulsive disorders during childhood, whereas the other showed no clinical phenotype, further illustrating the lack of correlation between genotype and phenotype in DPD deficiency.

Identification of novel point mutations in the dihydropyrimidine dehydrogenase gene.

Dihydropyrimidine dehydrogenase deficiency (McKusick 274270) is an autosomal recessive disorder leading to thymine-uraciluria. Dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) catalyses the first and rate-limiting step in the catabolism of uracil, thymine and the analogue 5-fluorouracil. Patients do not exhibit a characteristic clinical phenotype, although in about half the cases with a complete or near-complete deficiency of the enzyme convulsive disorders are observed (Berger et al 1984; van Gennip et al 1989, 1994; Braakhekke et al 1987). In patients with a nearly complete enzyme defect, the initial diagnosis can be made on the presence of large amounts of both thymine and uracil in the patients body fluids, whereas the diagnosis can be confirmed by measurement of the enzyme activity in either peripheral mononuclear cells or fibroblasts (Van Kuilenburg et al 1996). The recent cloning of the dihydropyrimidine dehydrogenase cDNA now allows detection of the defect at the molecular level (Yokota et al 1994). We previously described a 165 base pair deletion in mRNA-derived cDNA, caused by exon skipping, in a patient with a complete deficiency of DPD (Meinsma et al 1995). Analysis of the flanking intron sequences revealed that exon skipping was due to a G → A point mutation in the invariant GT splice donor sequence in the intron downstream of the skipped exon (Vreken et al 1996). So far, no other mutations in the DPD gene have been described. We now report a new frameshift mutation (ΔC1897) and two missense mutations (T85C and G2658A) leading to amino acid substitutions C29R and R886H.

Dihydropyrimidinase deficiency: Phenotype, genotype and structural consequences in 17 patients.

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 11 individuals have been reported suffering from a complete DHP deficiency. Here, we report on the clinical, biochemical and molecular findings of 17 newly identified DHP deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological and gastrointestinal abnormalities and markedly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. Analysis of DPYS, encoding DHP, showed nine missense mutations, two nonsense mutations, two deletions and one splice-site mutation. Seventy-one percent of the mutations were located at exons 5-8, representing 41% of the coding sequence. Heterologous expression of 11 mutant enzymes in Escherichia coli showed that all but two missense mutations yielded mutant DHP proteins without significant activity. Only DHP enzymes containing the mutations p.R302Q and p.T343A possessed a residual activity of 3.9% and 49%, respectively. The crystal structure of human DHP indicated that the point mutations p.R490C, p.R302Q and p.V364M affect the oligomerization of the enzyme. In contrast, p.M70T, p.D81G, p.L337P and p.T343A affect regions near the di-zinc centre and the substrate binding site. The p.S379R and p.L7V mutations were likely to cause structural destabilization and protein misfolding. Four mutations were identified in multiple unrelated DHP patients, indicating that DHP deficiency may be more common than anticipated.

Pyrimidine Degradation Defects and Severe 5‐Fluorouracil Toxicity

5‐Fluorouracil (5FU) remains one of the most frequently prescribed chemotherapeutic drugs for the treatment of cancer. Recently, the pivotal role of the catabolic pathway of 5FU in the determination of toxicity towards 5FU has been highlighted. Patients with a (partial) dihydropyrimidine dehydrogenase deficiency proved to be at risk of developing severe toxicity after the administration of 5FU. A partial dihydropyrimidinase deficiency proved to be a novel pharmacogenetic disorder associated with severe 5FU toxicity.

In vitro inhibition of cytidine triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia

Cytidine triphosphate (CTP) synthetase is a key enzyme for the synthesis of cytosine (deoxy)ribonucleotides, catalysing the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in several malignancies. In this preclinical study, the enzyme activity and mRNA expression of the enzyme and (deoxy)ribonucleotide concentrations were analysed in leukaemic cells of 57 children suffering from acute lymphocytic leukaemia (ALL). In addition, in vitro experiments were performed with the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC). A significantly higher activity of CTP synthetase (6·5 ± 3·9 nmol CTP/mg/h) was detected in ALL cells than in lymphocytes of healthy controls (1·8 ± 0·9 nmol CTP/mg/h, P < 0·001) that was independent of white blood cell (WBC) count, blast percentage, age, gender or type of ALL. The enzyme activity was not correlated with the CTP synthetase mRNA expression. The activity of CTP synthetase in ALL cells compared with non‐malignant CD34+ bone marrow controls (5·6 ± 2·4 nmol CTP/mg/h) was not statistically different. In vitro treatment of ALL cells with CPEC induced a dose‐dependent decrease of the CTP concentration. The lowest concentration of CPEC (0·63 µm) induced a depletion of CTP of 41 ± 20% and a depletion of dCTP of 27 ± 21%. The degree of CTP depletion of ALL cells after treatment with CPEC was positively correlated with the activity of CTP synthetase. The inhibition of CTP synthetase in situ was confirmed by flux studies using radiolabelled uridine. From these results, it can be expected that CPEC has a cytostatic effect on lymphoblasts of children with ALL.

Identification of a four-base deletion (delTCAT296-299) in the dihydropyrimidine dehydrogenase gene with variable clinical expression

Abstract Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting step in the breakdown of thymine, uracil, and the widely used antineoplastic drug, 5-fluorouracil. Sequence analysis of the dihydropyrimidine dehydrogenase cDNA in a Dutch consanguineous family identified a novel four-base deletion (delTCAT296–299) leading to premature termination of translation. The deletion is located in a TCAT tandem-repeat sequence and most likely results from unequal crossing-over or slipped mispairing. In this family we identified three homozygous individuals for this mutation. Two of these showed convulsive disorders but one was clinically normal. This observation suggests that, at least in this family, there is no clear correlation between the dihydropyrimidine dehydrogenase genotype and phenotype.

Identification of three novel mutations in the dihydropyrimidine dehydrogenase gene associated with altered pre-mRNA splicing or protein function

Abstract Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil and thymine, as well as of the widely used chemotherapeutic drug 5-fluorouracil (5FU). Analysis of the DPD gene (DPYD) in two patients presenting with complete DPD deficiency and the parents of an affected child showed the presence of three novel mutations, including one splice site mutation IVS11+1G→T and the missense mutations 731A→C (E244V) and 1651G→A (A551T). The G→T mutation in the invariant GT splice donor site flanking exon 11 (IVS11+1G→T) created a cryptic splice site within exon 11. As a consequence, a 141-bp fragment encoding the aminoacid residues 400–446 of the primary sequence of the DPD protein was missing in the mature DPD mRNA. Analysis of the crystal structure of pig DPD suggested that the E244V mutation might interfere with the electron flow between NADPH and the pyrimidine binding site of DPD. The A551T point mutation might prevent binding of the prosthetic group FMN and affect folding of the DPD protein. The identification of these novel mutations in DPYD will allow the identification of patients with an increased risk of developing severe 5FU-associated toxicity.