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Human Molecular Genetics Advance Access originally published online on September 22, 2004
Human Molecular Genetics 2004 13(22):2793-2801; doi:10.1093/hmg/ddh303
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Human Molecular Genetics, Vol. 13, No. 22 © Oxford University Press 2004; all rights reserved

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

André B.P. van Kuilenburg1,*, Rutger Meinsma1, Eva Beke1, Birgit Assmann2, Antonia Ribes3, Isabel Lorente4, Rebekka Busch5, Ertan Mayatepek2, Nico G.G.M. Abeling1, Arno van Cruchten1, Alida E.M. Stroomer1, Henk van Lenthe1, Lida Zoetekouw1, Willem Kulik1, Georg F. Hoffmann6, Thomas Voit7, Ron A. Wevers8, Frank Rutsch5 and Albert H. van Gennip9

1Department of Clinical Chemistry, Academic Medical Center, Emma Children's Hospital, The Netherlands, 2Department of General Pediatrics, University Children's Hospital, Düsseldorf, Germany, 3Institut de Bioquímica Clínica, CDB, Hospital Clínic, Barcelona, Spain, 4Hospital Parc Taulí, Sabadell, Barcelona, Spain, 5Klinik für Kinder- und Jugendmedizin, Klinikum Dortmund gGmbH, Dortmund, Germany, 6Department of Pediatrics, University Hospital Heidelberg, Heidelberg, Germany, 7Department of Pediatrics, University Hospital Essen, Essen, Germany, 8Institute of Neurology, University Medical Center Nijmegen, Nijmegen, The Netherlands and 9Departments of Clinical Genetics and Clinical Chemistry, Academic Hospital Maastricht, Maastricht, The Netherlands

Received July 9, 2004; Accepted September 14, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ß-Ureidopropionase deficiency is an inborn error of the pyrimidine degradation pathway, affecting the cleavage of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid. In this study, we report the elucidation of the genetic basis underlying a ß-ureidopropionase deficiency in four patients presenting with neurological abnormalities and strongly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in plasma, cerebrospinal fluid and urine. No ß-ureidopropionase activity could be detected in a liver biopsy obtained from one of the patients, which reflected the complete absence of the ß-ureidopropionase protein. Analysis of the ß-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 ß-ureidopropionase enzyme without residual activity. Our results demonstrate that the N-carbamyl-ß-amino aciduria in these patients is due to a deficiency of ß-ureidopropionase, which is caused by mutations in the UPB1 gene. Furthermore, an altered homeostasis of ß-aminoisobutyric acid and/or increased oxidative stress might contribute to some of the clinical abnormalities encountered in patients with a ß-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 ß-ureidopropionase deficiency might not be as rare as is generally considered.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pyrimidine nucleotides are essential for a vast number of biological processes such as the synthesis of RNA, DNA, phospholipids and glycogen and the sialylation and glycosylation of proteins (1). There is, however, an increased awareness that pyrimidines play an important role in the regulation of the central nervous system and that metabolic changes affecting the levels of pyrimidines may lead to abnormal neurological activity (2). In humans, the pyrimidine bases uracil and thymine are degraded via a three-step pathway (Fig. 1). Dihydropyrimidine dehydrogenase (EC 1.3.1.2) is the initial and rate-limiting enzyme, catalyzing the reduction of thymine and uracil to 5,6-dihydrothymine and 5,6-dihydrouracil, respectively. The second step consists of a hydrolytic ring opening of the dihydropyrimidines, which is catalyzed by dihydropyrimidinase (EC 3.5.2.2). Finally, the resulting N-carbamyl-ß-aminoisobutyric acid and N-carbamyl-ß-alanine are converted, in the third step to, respectively, ß-aminoisobutyric acid and ß-alanine, ammonia and CO2 by ß-ureidopropionase (EC 3.5.1.6).



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  		Figure 1. Catabolic pathway of the pyrimidines uracil and thymine.

 
The pyrimidine degradation pathway plays an important role in the synthesis of ß-alanine and ß-aminoisobutyric acid (3). ß-Alanine is a structural analog of {gamma}-aminobutyric acid and glycine, which are the major inhibitory neurotransmitters in the central nervous system. Recently, a G protein-coupled receptor has been identified in dorsal root ganglia, which proved to be specific for ß-alanine (4). Furthermore, ß-aminoisobutyric acid has been shown to be a partial agonist of the glycine receptor (5). Thus, the altered homeostasis of these ß-amino acids, as observed in patients with a dihydropyrimidine dehydrogenase deficiency, might underlie some of the clinical abnormalities encountered in these patients (3).

To date, approximately 50 patients have been described with a dihydropyrimidine dehydrogenase defect (MIM 274270) and nine patients with a dihydropyrimidinase deficiency (MIM 222748). The clinical phenotype of these patients was highly variable, but centered around neurological problems (6–8). Until now, only two patients have been reported with a metabolic profile in urine characteristic of a ß-ureidopropionase deficiency (MIM 606673) (9–11). The cloning of the cDNA coding for human ß-ureidopropionase and the sequence of the entire human ß-ureidopropionase gene (UPB1) has now allowed the detection of the defect at the molecular level (12,13).

In this study, we report the clinical, biochemical and genetic findings of four patients with a complete ß-ureidopropionase deficiency. Furthermore, we show that an altered homeostasis of ß-aminoisobutyric acid and/or increased oxidative stress might be involved in the pathology of the disease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Clinical evaluation
The first patient was a 17-month-old girl who presented with muscular hypotonia, dystonic movements, scoliosis and severe developmental delay (9).

The second patient was a girl and the first-born child of consanguineous Turkish parents. The pregnancy was uneventful although the mother indicated that at the end of the pregnancy there was insufficient amniotic fluid. The child was born after 39 weeks by caesarean section, weighing 2470 g and 47 cm in length. At 6 months the child experienced an episode with high fever, and the presence of meningitis was later discounted. Subsequently, at 8 months, she suffered from afebrile seizures and showed a motor retardation of 2–3 months. An MRI scan of the brain showed delayed myelinization and the EEG showed the presence of hypsarrythmia. In addition, audiological tests were inconclusive. Subsequent therapy with vitamin B6 induced an allergic reaction. The therapy with Sultiam and Vigabatrin, however, proved to be effective and the convulsions stopped after 4 weeks. After 1 year of therapy with Vigabatrin, the dose could be gradually reduced and the EEG showed no epileptic activity. At 3 years of age, the patient showed psychomotor retardation (she learned to walk at 2 years of age), severe mental retardation and a speech disorder, although visual contact appeared to be normal.

The third patient was a boy from non-consanguineous parents. At the age of 4 months, he was found cyanosed with his head covered by his blanket. At admission to the hospital, he was febrile, alert and without respiratory abnormalities, but with a heart rate of about 240 beats per minute. Electrolytes and glucose were normal but compensated metabolic acidosis and elevated lactate in blood and cerebrospinal fluid (CSF) was present. Two hours later, he went into a status epilepticus that was particularly hard to overcome, requiring generalized anesthesia. No infectious agent could be detected in blood, CSF, urine, stool and gastric secretions. The child vomited once and had moderate diarrhea. Neither cranial CT scan nor ultrasound revealed cerebral edema. In due course, the levels of lactate in CSF normalized.

The fourth patient was a girl, born to healthy African non-consanguineous parents. The family history indicated that a brother of the patient died 24 h after birth and that another brother died at the age of 9 months. According to the parents, the latter experienced a number of convulsive crises. The patient was hospitalized as a result of an upper respiratory tract infection when she was 1 month and 10 days old. In addition to a loss of consciousness, which lasted for 30 min, she was diagnosed as having severe muscular hypertonia. The patient responded to the administration of Diazepam. After the incident, physical and neurological (EEG, cerebral echography and cerebral MRI) examination of the patient did not indicate any abnormalities. The patient had normal psychomotor and neurological development at 12 months. She started to walk independently at the age of 10 months and started to talk at 12 months. However, the parents indicated that whenever she had a cold, she would easily collapse.

Pyrimidine bases and degradation products in body fluids
As part of a selective screening program for inborn errors, the pyrimidine bases, uracil and thymine, and their degradation products were analyzed in urine, plasma and CSF samples. The urine samples of the four patients showed normal to marginally elevated levels of uracil and thymine when compared with controls, whereas the concentration of the dihydropyrimidines was moderately elevated. In contrast, the concentrations of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid were strongly elevated, when compared with those observed in controls (Table 1). The observed N-carbamyl-ß-amino aciduria in these patients strongly suggests a deficiency of ß-ureidopropionase.


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  		Table 1. Mutation data and pyrimidine degradation metabolites of patients with a ß-ureidopropionase deficiency
 
Whereas a similar pattern of the pyrimidine bases and their degradation products was observed in urine and plasma, a completely different metabolic profile was observed in CSF. The concentration of the N-carbamyl-ß-amino acids in CSF, although elevated when compared with that observed in controls, was often even lower than that of the dihydropyrimidines (Table 1).

ß-Ureidopropionase in a liver biopsy
The activity of ß-ureidopropionase and dihydropyrimidine dehydrogenase was determined in a liver biopsy obtained from patient 3. Figure 2A shows that the activity of ß-ureidopropionase was readily detected in control liver (119 nmol/mg/h; controls 69±37, n=10) whereas no ß-ureidopropionase activity (<0.17 nmol/mg/h) could be detected in a liver biopsy of the patient (Fig. 2B). In contrast, normal activity of dihydropyrimidine dehydrogenase was present in the liver biopsy of the patient (8.6 nmol/mg/h) when compared with controls (6.4±3.9 nmol/mg/h, n=14).



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  		Figure 2. ß-Ureidopropionase activity and expression level in human liver. The HPLC elution profiles are shown from a control (A) and patient 3 (B), obtained after incubation of the liver homogenates with radiolabeled N-carbamyl-ß-alanine (N-C-ß-alanine). (C) Immunoblot analysis of ß-ureidopropionase in human liver. Equal amounts of protein were separated by 7.5% SDS–PAGE and analyzed on immunoblot with ß-ureidopropionase-specific antibodies.

 
Immunblot analysis of the liver homogenate, using antibodies directed against rat ß-ureidopropionase, showed that the lack of ß-ureidopropionase activity was due to the absence of the ß-ureidopropionase protein itself (Fig. 2C).

Mutation analysis of UPB1
Analysis of the genomic sequences of exons 1–11 of UPB1, including their flanking intronic sequences, showed that patient 1 was a compound heterozygote for two splice acceptor site mutations: the first mutation is IVS1-2A>G in intron 1 and the second mutation is IVS8-1G>A in intron 8 (Table 1) (13). Analysis of the parents revealed that the father was heterozygous for the intron 1 mutation and the mother was heterozygous for the intron 8 mutation. Patient 2 proved to be homozygous for the IVS1-2A>G mutation, and analysis of the UPB1 gene of the parents showed that both parents were heterozygous for this mutation. Patient 3 proved to be homozygous for the IVS8-1G>A mutation, with both parents being heterozygous for this mutation.

Analysis of the genomic sequences of UPB1 from patient 4 showed that the patient was homozygous for a missense mutation 254C>A in exon 2 of UPB1, leading to the amino acid substitution A85E (Fig. 3A). Alignment of various eukaryotic ß-ureidopropionase protein sequences revealed that the alanine at position 85 is replaced by a threonine in ß-ureidopropionase from mouse, rat, Drosophila melanogaster, Caenorhabditis elegans and Dictyostelium discoideum.



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  		Figure 3. Genomic organization and mutation analyses of the UPB1 gene. (A) UPB1 gene consisting of 11 exons with an open reading frame of 1152 bp (depicted in gray). The different mutations identified in the three patients with a complete ß-ureidopropionase deficiency are indicated. (B) ß-Ureidopropionase activity and immunoblot analysis of wild-type and mutant ß-ureidopropionase expressed in E. coli as well as that of human liver. The results represent the mean±SD of three independent experiments, with each experiment being performed in duplicate. For immunoblot analysis, equal amounts of protein were separated by 7.5% SDS–PAGE and analyzed on immunoblot with ß-ureidopropionase-specific antibodies (right-hand side panel).

 
An analysis for the presence of the IVS1-2A>G, the IVS8-1G>A and the 254C>A mutation in 95 controls did not identify individuals either heterozygous or homozygous for the IVS1-2A>G or 254C>A mutation. Thus, the allele frequency of the IVS1-2A>G and 254C>A mutation in the normal population is <0.5%. However, one individual was identified who proved to be heterozygous for the IVS8-1G>A mutation, resulting in an allele frequency of 0.5%.

Expression analysis of the A85E mutation
To investigate the effect of the A85E mutation on the activity of ß-ureidopropionase, the mutation was introduced into the pSE420-UP vector by site-directed mutagenesis and expressed in E. coli. No endogenous ß-ureidopropionase activity (<1 nmol/mg/h) could be detected in the E. coli strain used for the expression of the constructs. Introduction of the wild type ß-ureidopropionase construct increased the ß-ureidopropionase activity more than 75x above the background level. The activity of ß-ureidopropionase from human liver was comparable to that of the wild-type enzyme expressed in E. coli. Expression of the ß-ureidopropionase construct containing the A85E mutation yielded no detectable activity of ß-ureidopropionase (Fig. 3B).

To exclude the possibility that the lack of enzyme activity was the result of an inability to produce the mutant protein in E. coli, the expression level was analyzed by immunoblotting. Figure 3B shows that the mutant protein carrying the A85E mutation was expressed. Furthermore, no ß-ureidopropionase protein could be detected in mock-transfected cells. Thus, the lack of ß-ureidopropionase activity in E. coli transfected with the pSE420-UP-A85E construct is not due to rapid degradation of the mutant ß-ureidopropionase protein in the E. coli lysates.

ß-Alanine and ß-aminoisobutyric acid levels
To investigate whether the impaired degradation of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid would result in altered levels of ß-alanine and ß-aminoisobutyric acid, respectively, we measured the levels of these ß-amino acids in body fluids of controls and patients with a ß-ureidopropionase deficiency (Table 2). In general, normal levels of ß-alanine were observed in the urine, plasma and CSF of patients with ß-ureidopropionase deficiency. The high concentration of ß-alanine in urine of patient 2, obtained during treatment with Vigabatrin, reflects the inhibition of {gamma}-aminobutyric acid transaminase by Vigabatrin (14). In addition, high levels of ß-alanine were present in the CSF of patient 3. In contrast, the concentration of ß-aminoisobutyric acid in the plasma was approximately 5–30x lower compared with that observed in controls and undetectable in the CSF.


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  		Table 2. Levels of ß-aminoacids and 8-hydroxydeoxyguanosine in body fluids
 
Oxidative stress related parameters
To investigate whether patients with a ß-ureidopropionase deficiency suffer from increased oxidative stress, the levels of 8-hydroxydeoxyguanosine were measured in urine (Table 2). Normal concentrations of 8-hydroxydeoxyguanosine were present in the urine from patients 1 and 4 when compared with age-matched controls. However, an increased concentration of 8-hydroxydeoxyguanosine was present in the urine from patients 2 and 3, suggesting that patients 2 and 3 are suffering from increased oxidative stress. This phenomenon would be in line with the presence of 5-hydroxymethyluracil in the plasma (0.44 µM) and CSF (0.32 µM) of patient 2, as this metabolite was not detected in the plasma (<0.1 µM, n=97) and CSF (<0.1 µM, n=29) from age-matched controls.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this paper, we describe the elucidation of the genetic cause underlying ß-ureidopropionase deficiency, an inborn error of the pyrimidine degradation pathway. The UPB1 gene has been mapped to chromosome 22q11.2 and consists of 11 exons spanning ~20 kB of genomic DNA (Fig. 3A). The cDNA coding for human ß-ureidopropionase contains an open reading frame of 1152 nucleotides, corresponding to a protein of 384 amino acids with a calculated molecular weight of 43 158 Da (12). Mammalian ß-ureidopropionase appears to have been only relatively conserved throughout evolution, as a comparison of the deduced amino acid sequences of human ß-ureidopropionase with that of rat showed a homology of 84% (12,15). Nevertheless, the analysis of a variety of eukaryotic ß-ureidopropionases showed that they are functionally related, despite a high degree of structural diversity (16).

Until recently, only patients with an acquired (secondary) ß-ureidopropionase deficiency have been reported with severe forms of propionic acidemia, due to the inhibition of this enzyme by propionic acid (17). The identification of two splice acceptor mutations and a missense mutation in UPB1 demonstrated that the observed N-carbamyl-ß-amino aciduria in the four patients was indeed due to a primary deficiency of ß-ureidopropionase. In addition, homozygosity for the IVS8-1G>A mutation resulted in a complete absence of enzyme activity, and no residual ß-ureidopropionase protein could be detected in a liver biopsy of this patient. Previously, we have shown that no residual ß-ureidopropionase activity could be detected in a liver biopsy of patient 1, who was a compound heterozygote for the IVS1-2A>G and IVS8-1G>A mutation (18,19).

In urine, the concentration of N-carbamyl-ß-alanine was approximately 1.5x higher than that of N-carbamyl-ß-aminoisobutyric acid. This might reflect the fact that the catalytic efficiency of dihydropyrimidinase with dihydrouracil is also 1.5x higher than that of dihydrothymine (20,21). In addition, it has been shown that N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid are weak inhibitors of dihydropyrimidinase (21), which might explain the moderately increased levels of dihydrouracil and dihydrothymine in the patients.

In humans, ß-ureidopropionase is expressed only in the liver and kidney (22). Hence, the presence of N-carbamyl-ß-amino acids in the CSF is most likely attributable to the transport of these metabolites from the blood to the brain. Furthermore, the fact that the concentration of the N-carbamyl-ß-amino acids in plasma is ~10–20-fold higher than that of CSF indicates that the polar nature of these metabolites may prevent proper transport through the blood–brain barrier.

To date, the mechanism underlying the clinical symptoms observed in our patients with a ß-ureidopropionase deficiency is not known. It has been suggested that N-carbamyl-ß-alanine, one of the accumulating substrates, might function as an endogenous neurotoxin (23), as high concentrations of N-carbamyl-ß-alanine inhibited the mitochondrial energy metabolism that was accompanied by the induction of oxidative stress. The observation, however, that the concentrations of N-carbamyl-ß-alanine were only marginally elevated in the CSF of patients argues against a direct role for N-carbamyl-ß-alanine in the neuropathology.

A conceivable possibility would be that the altered homeostasis of ß-aminoisobutyric acid underlies some of the clinical abnormalities encountered in the four patients with a ß-ureidopropionase deficiency. ß-Aminoisobutyric acid is a structural analog of {gamma}-aminobutyric acid and glycine, which are the major inhibitory neurotransmitters in the central nervous system. In addition, it has been shown that ß-aminoisobutyric acid is a partial agonist of the glycine receptor (5). Thus, the decreased concentration of ß-aminoisobutyric acid in the CSF of patients with a ß-ureidopropionase deficiency might, therefore, have a profound effect on the degree of activation of the glycine receptor and the efficacy of {gamma}-aminobutyric acid.

A conspicuous finding was the presence of 5-hydroxymethyluracil in plasma of one of the patients. Typically, 5-hydroxymethyluracil is formed during the oxidative damage of DNA by reactive oxygen species (24). Furthermore, the elevated levels of 8-hydroxydeoxyguanosine in two patients indicate that they are suffering from increased oxidative stress. Thus, an altered homeostasis of ß-aminoisobutyric acid and/or increased oxidative stress may be pathological factors underlying the clinical abnormalities encountered in our patients with a ß-ureidopropionase deficiency.

During a neonatal screening in Japan, an asymptomatic child with a putative ß-ureidopropionase deficiency was identified (11), although it is not known whether the child has since developed clinical symptoms. Nevertheless, the wide spectrum of clinical abnormalities, encountered in patients with a dihydropyrimidine dehydrogenase or dihydropyrimidinase deficiency, may also occur for deficiencies of the last enzyme of the pyrimidine degradation pathway. Although the limited number of patients prevents an analysis of genotype–phenotype correlations, it is of interest that the splice-site mutations were present in three patients, all of whom developed a severe disabling encephalopathy, whereas the fourth patient, homozygous for a missense mutation, presented with transient neurological abnormalities only.

The three enzymes of the pyrimidine degradation pathway are not only responsible for the catabolism of the naturally occurring bases uracil and thymine, but also of the widely used chemotherapeutic agent 5-fluorouracil. In this light, deficiencies of dihydropyrimidine dehydrogenase and dihydropyrimidinase have been associated with severe toxicity after the administration of 5-fluorouracil (25,26). Reasoning along these lines, it is conceivable that patients with a complete deficiency of ß-ureidopropionase might also be at risk of developing 5-fluorouracil toxicity.

It should be realized that the vast majority of patients with defects of the pyrimidine degradation pathway have been detected by gas chromatography–mass spectrometry (6). In patients with a deficiency of ß-ureidopropionase, the accumulating secondary metabolites, dihydrouracil and dihydrothymine, can be detected by gas chromatography–mass spectrometry. Until recently, the detection of the N-carbamyl-ß-amino acids was seriously hampered by the fact that it required laborious and time-consuming procedures, most of which were not routinely performed as part of a screening protocol for inborn errors of metabolism. With the introduction of HPLC–tandem mass spectrometry in 2000 (27), a specific and rapid screening test for ß-ureidopropionase deficiency is now currently available. In the 4 year period since the introduction of this technique, we have identified three new patients with this defect. Furthermore, the analysis of 95 controls for the presence of the two splice site mutations and the missense mutation identified one carrier for the IVS8-1G>A mutation. Thus, a ß-ureidopropionase deficiency might not be as rare as is generally considered.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Analysis of pyrimidines in body fluids
The concentrations of uracil, thymine, dihydrouracil, dihydrothymine, N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine, plasma and CSF were determined using reversed-phase HPLC combined with electrospray tandem mass spectrometry (27,28).

Analysis of ß-amino acids and oxidative-stress related parameters
The concentrations of ß-alanine and ß-aminoisobutyric acid in urine, plasma and CSF were determined with a dual-column reversed-phase HPLC procedure, combined with fluorescence detection of the orthophthaldialdehyde derivatives (3).

The concentration of 5-hydroxymethyluracil was determined using reversed-phase HPLC combined with electrospray tandem mass spectrometry (29). 8-Hydroxydeoxyguanosine in urine was measured using reversed-phase HPLC with electrochemical detection (30).

Determination of the ß-ureidopropionase and dihydropyrimidine dehydrogenase activity
The activity of ß-ureidopropionase in human liver was determined in a standard assay mixture containing 200 mM MOPS (pH 7.4), 1 mM dithiothreitol and 500 µM [14C]-N-carbamyl-ß-alanine. Radiolabeled N-carbamyl-ß-alanine was prepared by hydrolysis of [6-14C]5,6-dihydrouracil (2035 MBq/mmol; Moravek Biochemicals, Brea, CA, USA) under alkaline conditions (31). Separation of radiolabeled N-carbamyl-ß-alanine from radiolabeled ß-alanine was performed isocratically (50 mM NaH2PO4, pH 4.5, at a flow rate of 1 ml/min) by reversed-phase HPLC on an Aqua C18 column (250x4.6 mm 5 µm particle size, Phenomenex, Torrance, CA, USA) and a guard column (Securityguard C18, 4x3.0 mm ID, Phenomenex) with on-line detection of the radioactivity.

The dihydropyrimidine dehydrogenase activity in human liver was determined in a reaction mixture containing 35 mM potassium phosphate (pH 7.4), 2.5 mM MgCl2, 1 mM dithiothreitol, 2.5 mM NADPH and 40 µM [414C]-thymine (2072 MBq/mmol; Moravek Biochemicals) (32). Separation of radiolabeled thymine and the radiolabeled reaction products dihydrothymine, N-carbamyl-ß-aminoisobutyric acid and ß-aminoisobutyric acid was performed isocratically (50 mM NaH2PO4, pH 4.5, and 7.5% methanol, at a flow rate of 1 ml/min) by reversed-phase HPLC on an Aqua C18 column (250x4.6 mm, 5 µm particle size, Phenomenex) and a guard column (Securityguard C18, 4x3.0 mm ID, Phenomenex) with on-line detection of the radioactivity. Protein concentrations were determined with a copper-reduction method using bicinchoninic acid, essentially as described by Smith et al. (33).

PCR amplification of coding exons of UPB1
Genomic DNA was isolated from peripheral blood mononuclear cells using the Wizard Genomic DNA Purification kit (Promega Benelux b.v., Leiden, The Netherlands). Exons 1–11 of UPB1 and their flanking sequences were amplified using the primer sets described in Table 3. Amplification was carried out in 25 µl reaction mixtures containing 10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.5 µM of each primer, 0.2 mM dNTPs and 0.6 U of Taq polymerase (Promega Benelux). After initial denaturation for 2 min at 96°C, amplification was carried out for 30 cycles (10 s 96°C, 30 s 55°C, 1 min 72°C) with a final extension step of 9 min at 72°C. PCR products were separated on 1% agarose gels, visualized with ethidium bromide and purified using a Qiaquick Gel Extraction kit (Qiagen, Hilden, Germany) and used for direct sequencing.


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  		Table 3. PCR primers used for mutation analysis of the UPB1 gene
 
Expression of ß-ureidopropionase in E. coli
The expression plasmid containing the wild-type human ß-ureidopropionase cDNA (pSE420-UP) was constructed by subcloning the NcoI–NaeI insert of a plasmid, containing the complete coding region of the cDNA encoding human ß-ureidopropionase, into the pSE420 vector (12). The 254C>A mutation (A85E) was introduced into the human ß-ureidopropionase sequence using the megaprimer technique (34) and subsequently subcloned in a pSE420 vector (pSE420-UP-A85E). The resulting clones were sequenced completely to verify the presence of the mutation and to exclude the presence of random mutations introduced by PCR artifacts. Expression plasmids were introduced into the E. coli strain BL21. A 25 ml LB broth culture, supplemented with 50 µg/ml ampicillin, was inoculated with 250 µl of an overnight preculture grown in LB broth. Cells were grown for 2 h at 37°C and induction was performed by the addition of isopropyl-ß-D-thiogalactoside to a final concentration of 1 mM. Cells were sedimented after 5 h, washed with an isolation buffer (35 mM potassium phosphate pH 7.4 and CompleteTM (Roche Diagnostics, Almere, The Netherlands) and resuspended in 2.5 ml of isolation buffer. The cell suspension was frozen for 16 h at –20°C, thawed on ice and lysed by sonication. The crude lysate was centrifuged at 18 000g at 4°C for 15 min and the resulting supernatant was stored at –80°C.

Western blot analysis
Cell extracts (10.5 µg) were fractionated on 7.5% (w/v) SDS–PAGE and transferred to a nitrocellulose filter. Blocking of the membrane was performed for 16 h at 4°C with TBS (10 mM Tris and 150 mM NaCl, pH 8.0) containing 5% (w/v) non-fat dry milk. Subsequently, the membrane was incubated for 1 h with a 1 : 1000 dilution of rabbit anti-rat ß-ureidopropionase polyclonal antibody in TBS, supplemented with 0.05% (v/v) Tween 20. The membranes were washed three times (5 min each) with TBS containing 0.05% (v/v) Tween 20 and incubated for 45 min with TBS containing 0.05% (v/v) Tween 20, 5% (w/v) non-fat dry milk and a 1 : 5000 dilution of a goat anti-rabbit secondary antibody conjugated to Alkaline Phosphatase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After rinsing the membrane three times (5 min each) with TBS containing 0.05% Tween 20 and once with TBS, detection was performed with Western Blue® (Promega Benelux).

Sequence analysis
Sequence analysis of genomic fragments amplified by PCR and expression plasmids was carried out on an Applied Biosystems model 3100 automated DNA sequencer using the dye-terminator method (Perkin Elmer Corp., Foster City, CA, USA).


   ACKNOWLEDGEMENTS
 
We thank Dr Koichi Matsuda and Professor Dr Nanaya Tamaki for the ß-ureidopropionase antibody and Dr Ries Duran and Fiona Ward for their critical reading of the manuscript. This paper is dedicated to the memory of Dr Peter Vreken (1962–2001).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Academic Medical Center, Laboratory Genetic Metabolic Diseases F0-224, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel: +31 205665958; Fax: +31 206962596; Email: a.b.vankuilenburg{at}amc.uva.nl


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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