Clinical,biochemical and molecular analysis of 13 Japanese patients with β-ureidopropionase deficiency demonstrates high prevalence of the c.977G > A (p.R326Q) mutation

β-ureidopropionase (βUP) deficiency is an autosomal recessive disease characterized by N-carbamyl-β-amino aciduria. To date, only 16 genetically confirmed patients with βUP deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 13 Japanese βUP deficient patients. In this group of patients, three novel missense mutations (p.G31S, p.E271K, and p.I286T) and a recently described mutation (p.R326Q) were identified. The p.R326Q mutation was detected in all 13 patients with eight patients being homozygous for this mutation. Screening for the p.R326Q mutation in 110 Japanese individuals showed an allele frequency of 0.9 %. Transient expression of mutant βUP enzymes in HEK293 cells showed that the p.E271K and p.R326Q mutations cause profound decreases in activity (≤ 1.3 %). Conversely, βUP enzymes containing the p.G31S and p.I286T mutations possess residual activities of 50 and 70 %, respectively, suggesting we cannot exclude the presence of additional mutations in the non-coding region of the UPB1 gene. Analysis of a human βUP homology model revealed that the effects of the mutations (p.G31S, p.E271K, and p.R326Q) on enzyme activity are most likely linked to improper oligomer assembly. Highly variable phenotypes ranging from neurological involvement (including convulsions and autism) to asymptomatic, were observed in diagnosed patients. High prevalence of p.R326Q in the normal Japanese population indicates that βUP deficiency is not as rare as generally considered and screening for βUP deficiency should be included in diagnosis of patients with unexplained neurological abnormalities.     

Lethal outcome of a patient with a complete dihydropyrimidine dehydrogenase (DPD) deficiency after administration of 5-fluorouracil: frequency of the common IVS14+1G>A mutation causing DPD deficiency.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk from developing a severe 5FU-associated toxicity. In this study, we demonstrated that a lethal toxicity after a treatment with 5FU was attributable to a complete deficiency of DPD. Analysis of the DPD gene for the presence of mutations showed that the patient was homozygous for a G-->A mutation in the invariant GT splice donor site flanking exon 14 (IVS14+1G>A). As a consequence, no significant residual activity of DPD was detected in peripheral blood mononuclear cells. To determine the frequency of the IVS14+1G>A mutation in the Dutch population, we developed a novel PCR-based method allowing the rapid analysis of the IVS14+1G>A mutation by RFLP. Screening for the presence of this mutation in 1357 Caucasians showed an allele frequency of 0.91%. In our view, the apparently high prevalence of the IVS14+1G>A mutation in the normal population, with 1.8% heterozygotes, warrants genetic screening for the presence of this mutation in cancer patients before the administration of 5FU.     

High prevalence of the IVS14 + 1G>A mutation in the dihydropyrimidine dehydrogenase gene of patients with severe 5-fluorouracil-associated toxicity

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU) and a DPD deficiency is increasingly being recognized as an important pharmacogenetic factor in the aetiology of severe 5FU-associated toxicity. In this study, we evaluated the DPD activity and the prevalence of the common splice site mutation IVS14 + 1G>A in tumour patients suffering from severe grade 3-4 toxicity after the administration of 5FU. DPD activity was measured with a radiochemical assay and screening for the presence of the IVS14 + 1G>A mutation was performed by restriction fragment length polymorphism. A decreased DPD activity could be detected in peripheral blood mononuclear cells in 60% of the cases. Furthermore, a high prevalence of the IVS14 + 1G>A mutation was noted as 28% of all patients were heterozygous or homozygous for this mutation. In patients with a low DPD activity, 42% were heterozygous and one patient (3%) was homozygous for the IVS14 + 1G>A mutation. In contrast, the IVS14 + 1G>A mutation could be detected in only one out of 24 (4%) patients with a normal DPD activity. Our study demonstrates that a DPD deficiency is the major determinant of 5FU-associated toxicity. The apparently high prevalence of the IVS14 + 1G>A mutation warrants genetic screening for this mutation in cancer patients before the administration of 5FU.     

Clinical, biochemical and genetic findings in two siblings with a dihydropyrimidinase deficiency

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and it catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine to N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid, respectively. To date, only nine individuals have been reported suffering from a complete DHP deficiency. We report two siblings presenting with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. One of the siblings had a severe delay in speech development and white matter abnormalities, whereas the other one was free of symptoms. Analysis of the DHP gene (DPYS) showed that both patients were compound heterozygous for the missense mutation 1078T>C (W360R) in exon 6 and a novel missense mutation 1235G>T (R412M) in exon 7. Heterologous expression of the mutant enzymes in Escherichia coli showed that both missense mutations resulted in a mutant DHP enzyme without residual activity. Analysis of the crystal structure of eukaryotic DHP from the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum suggests that the W360R and R412M mutations lead to structural instability of the enzyme which could potentially impair the assembly of the tetramer.     

Beta-ureidopropionase deficiency presenting with congenital anomalies of the urogenital and colorectal systems

Beta-ureidopropionase deficiency (McKusick 606673) is an autosomal recessive condition caused by mutations in the UPB1 gene. To date, five patients have been reported, including one putative case detected through newborn screening. Clinical presentation includes neurological and developmental problems. Here, we report another case of beta-ureidopropionase deficiency who presented with congenital anomalies of the urogenital and colorectal systems and with normal neurodevelopmental milestones. Analysis of a urine sample, because of the suspicion of renal stones on ultrasound, showed strongly elevated levels of the characteristic metabolites, N-carbamyl-beta-amino acids. Subsequent analysis of UPB1 identified a novel mutation 209 G>C (R70P) in exon 2 and a previously reported splice receptor mutation IVS1-2A>G. Expression studies of the R70P mutant enzyme showed that the mutant enzyme did not possess any residual activity. Long-term follow-up is required to determine the clinical significance of the beta-ureidopropionase deficiency in our patient.     

Cyclopentenyl cytosine-induced activation of deoxycytidine kinase increases gemcitabine anabolism and cytotoxicity in neuroblastoma

The effect of the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC) on the metabolism and cytotoxicity of 2′,2′-difluorodeoxycytidine (dFdC, gemcitabine) and the expression and activity of deoxycytidine kinase (dCK) was studied in human neuroblastoma cell lines. The cytotoxicity of dFdC and CPEC was studied in a panel of MYCN-amplified and MYCN-single-copy neuroblastoma cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-assays. dFdC-metabolism was studied in SK-N-BE(2)c cells using [³H]-radiolabeled dFdC. dCK activity and expression were measured using enzyme assays, immunoblot and quantitative PCR, respectively. Both MYCN-amplified and MYCN-single-copy neuroblastoma cell lines were highly sensitive to dFdC, with concentration of the drug resulting in 50% effect when compared to untreated controls (ED₅₀) values in the nanomolar range after a 3-h exposure to dFdC. There was no correlation of the observed ED₅₀ with the dCK activity. Treatment with dFdC induced cell death in MYCN-amplified cells whereas MYCN-single-copy-cell lines underwent neuronal differentiation. Pre-incubation with CPEC significantly increased dFdC-cytotoxicity from 1.3 to 5.3-fold in 13 out of 15 cell lines. [³H]dFdC-anabolism increased 6–44 fold in SK-N-BE(2)c cells after incubation with CPEC and was paralleled by a significant increase in expression and activity of dCK. In conclusion, the combination of dFdC and CPEC is highly toxic to neuroblastoma in vitro.     

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

Summary Dihydropyrimidine 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 GA 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     

beta-Ureidopropionase deficiency: an inborn error of pyrimidine degradation associated with neurological abnormalities

beta-Ureidopropionase deficiency is an inborn error of the pyrimidine degradation pathway, affecting the cleavage of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid. In this study, we report the elucidation of the genetic basis underlying a beta-ureidopropionase deficiency in four patients presenting with neurological abnormalities and strongly elevated levels of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid in plasma, cerebrospinal fluid and urine. No beta-ureidopropionase activity could be detected in a liver biopsy obtained from one of the patients, which reflected the complete absence of the beta-ureidopropionase protein. Analysis of the beta-ureidopropionase gene (UPB1) of these patients revealed the presence of two splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). Heterologous expression of the mutant enzyme in Escherichia coli showed that the A85E mutation resulted in a mutant beta-ureidopropionase enzyme without residual activity. Our results demonstrate that the N-carbamyl-beta-amino aciduria in these patients is due to a deficiency of beta-ureidopropionase, which is caused by mutations in the UPB1 gene. Furthermore, an altered homeostasis of beta-aminoisobutyric acid and/or increased oxidative stress might contribute to some of the clinical abnormalities encountered in patients with a beta-ureidopropionase deficiency. An analysis of the presence of the two splice site mutations and the missense mutation in 95 controls identified one individual who proved to be heterozygous for the IVS8-1G>A mutation. Thus, a beta-ureidopropionase deficiency might not be as rare as is generally considered.