CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders.

PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

Plasma dopa decarboxylase activity in treatment-resistant recent-onset psychosis patients.

Treatment resistance (TR) in psychosis is a major clinical problem. A biomarker predicting TR against conventional antipsychotic drugs would be relevant, potentially reducing unnecessary delay to adequate treatment with clozapine. Dopa decarboxylase (DDC) activity in the striatum, measured with positron emission tomography, is elevated in responders, but not in treatment-resistant patients. Plasma DDC activity could be a surrogate marker for DDC brain activity, and thus a potential biomarker that could be used in daily clinical practice. Therefore, we determined plasma DDC activity in 40 male patients with recent-onset psychosis, of whom the majority had started treatment, whereby 21 turned out to be treatment responders and 19 treatment resistant during follow up. We observed no significant group differences. Furthermore, symptom severity was not associated with plasma DCC activity. We did observe a trend level difference in the distribution of plasma DDC activity across categories of medication, with subsequent post hoc analysis showing lower DDC activity in risperidone-using patients. This may suggest that risperidone could influence plasma DDC activity. Based on these results, plasma DDC activity does not appear to be a promising biomarker for TR in recent-onset psychosis patients who are already receiving antipsychotic treatment.

Genome sequencing reveals a novel genetic mechanism underlying dihydropyrimidine dehydrogenase deficiency: A novel missense variant c.1700G>A and a large intragenic inversion in DPYD spanning intron 8 to intron 12.

Dihydropyrimidine dehydrogenase (DPD) deficiency is associated with a variable clinical presentation. A family with three DPD-deficient patients presented with unusual clinical phenotypes including pregnancy-induced symptoms, transient visual impairment, severe developmental delay, cortical blindness, and delayed myelination in the brain. DPYD Sanger sequencing showed heterozygosity for the c.1905+1G>A mutation and a novel missense variant c.1700G>A (p.G567E). The recombinantly expressed p.G567E DPD variant showed increased temperature lability probably caused by structural rearrangements within the DPD protein. Genome sequencing of the affected son established compound heterozygosity for the c.1700G>A and an imperfect 115,731 bp inversion with breakpoints at chr1: 98,113,121 (intron 8) and chr1: 97,997,390 (intron 12) of the DPYD associated with a 4 bp deletion (chr1: 97,997,386_97,997,389del). Whole exome and mitochondrial DNA analyses for the mother and daughter did not reveal additional mutated genes of significance. Thus, an inversion in DPYD should be considered in patients with an inconclusive genotype or unusual clinical phenotype.

Capecitabine-based treatment of a patient with a novel DPYD genotype and complete dihydropyrimidine dehydrogenase deficiency.

Fluoropyrimidines are frequently used anti-cancer drugs. It is known that patients with reduced activity of dihydropyrimidine dehydrogenase (DPD), the key metabolic enzyme in fluoropyrimidine inactivation, are at increased risk of developing severe fluoropyrimidine-related toxicity. Upfront screening for DPD deficiency and dose reduction in patients with partial DPD deficiency is recommended and improves patient safety. For patients with complete DPD deficiency, fluoropyrimidine-treatment has generally been discouraged. During routine pretreatment screening, we identified a 59-year-old patient with a sigmoid adenocarcinoma who proved to have a complete DPD deficiency. Genetic analyses showed that this complete absence of DPD activity was likely to be caused by a novel DPYD genotype, consisting of a combination of amplification of exons 17 and 18 of DPYD and heterozygosity for DPYD*2A. Despite absence of DPD activity, the patient was treated with capecitabine-based chemotherapy, but capecitabine dose was drastically reduced to 150 mg once every 5 days (0.8% of original dose). Pharmacokinetic analyses showed that the area under the concentration-time curve (AUC) and half-life of 5-fluorouracil were respectively tenfold and fourfold higher than control values of patients receiving capecitabine 850 mg/m2 . When extrapolating from the dosing schedule of once every 5 days to twice daily, the AUC of 5-fluorouracil was comparable to controls. Treatment was tolerated well for eight cycles by the patient without occurrence of capecitabine-related toxicity. This case report demonstrates that a more comprehensive genotyping and phenotyping approach, combined with pharmacokinetically-guided dose administration, enables save fluoropyrimidine-treatment with adequate drug exposure in completely DPD deficient patients.

Dihydropyrimidinase deficiency in four East Asian patients due to novel and rare DPYS mutations affecting protein structural integrity and catalytic activity.

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyzes the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 31 genetically confirmed patients with a DHP deficiency have been reported and the clinical, biochemical and genetic spectrum of DHP deficient patients is, therefore, still largely unknown. Here, we show that 4 newly identified DHP deficient patients presented with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in urine and a highly variable clinical presentation, ranging from asymptomatic to infantile spasm and reduced white matter and brain atrophy. Analysis of the DHP gene (DPYS) showed the presence of 8 variants including 4 novel/rare missense variants and one novel deletion. Functional analysis of recombinantly expressed DHP mutants carrying the p.M250I, p.H295R, p.Q334R, p.T418I and the p.R490H variant showed residual DHP activities of 2.0%, 9.8%, 9.7%, 64% and 0.3%, respectively. The crystal structure of human DHP indicated that all point mutations were likely to cause rearrangements of loops shaping the active site, primarily affecting substrate binding and stability of the enzyme. The observation that the identified mutations were more prevalent in East Asians and the Japanese population indicates that DHP deficiency may be more common than anticipated in these ethnic groups.

Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). Genetic variations in DPD have emerged as predictive risk factors for severe fluoropyrimidine toxicity. Here, we report novel and rare genetic variants underlying DPD deficiency in 9 cancer patients presenting with severe fluoropyrimidine-associated toxicity. All patients possessed a strongly reduced DPD activity, ranging from 9 to 53% of controls. Analysis of the DPD gene (DPYD) showed the presence of 21 variable sites including 4 novel and 4 very rare aberrations: 3 missense mutations, 2 splice-site mutations, 1 intronic mutation, a deletion of 21 nucleotides and a genomic amplification of exons 9-12. Two novel/rare variants (c.2843T>C, c.321+1G>A) were present in multiple, unrelated patients. Functional analysis of recombinantly-expressed DPD mutants carrying the p.I948T and p.G284V mutation showed residual DPD activities of 30% and 0.5%, respectively. Analysis of a DPD homology model indicated that the p.I948T and p.G284V mutations may affect electron transfer and the binding of FAD, respectively. cDNA analysis showed that the c.321+1G>A mutation in DPYD leads to skipping of exon 4 immediately upstream of the mutated splice-donor site in the process of DPD pre-mRNA splicing. A lethal toxicity in two DPD patients suggests that fluoropyrimidines combined with other therapies such as radiotherapy might be particularly toxic for DPD deficient patients. Our study advocates a more comprehensive genotyping approach combined with phenotyping strategies for upfront screening for DPD deficiency to ensure the safe administration of fluoropyrimidines.

Phenotypic and clinical implications of variants in the dihydropyrimidine dehydrogenase gene.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil, thymine and the antineoplastic agent 5-fluorouracil. Genetic variations in the gene encoding DPD (DPYD) have emerged as predictive risk alleles for 5FU-associated toxicity. Here we report an in-depth analysis of genetic variants in DPYD and their consequences for DPD activity and pyrimidine metabolites in 100 Dutch healthy volunteers. 34 SNPs were detected in DPYD and 15 SNPs were associated with altered plasma concentrations of pyrimidine metabolites. DPD activity was significantly associated with the plasma concentrations of uracil, the presence of a specific DPYD mutation (c.1905+1G>A) and the combined presence of three risk variants in DPYD (c.1905+1G>A, c.1129-5923C>G, c.2846A>T), but not with an altered uracil/dihydrouracil (U/UH2) ratio. Various haplotypes were associated with different DPD activities (haplotype D3, a decreased DPD activity; haplotype F2, an increased DPD activity). Functional analysis of eight recombinant mutant DPD enzymes showed a reduced DPD activity, ranging from 35% to 84% of the wild-type enzyme. Analysis of a DPD homology model indicated that the structural effect of the novel p.G401R mutation is most likely minor. The clinical relevance of the p.D949V mutation was demonstrated in a cancer patient heterozygous for the c.2846A>T mutation and a novel nonsense mutation c.1681C>T (p.R561X), experiencing severe grade IV toxicity. Our studies showed that the endogenous levels of uracil and the U/UH2 ratio are poor predictors of an impaired DPD activity. Loading studies with uracil to identify patients with a DPD deficiency warrants further investigation.

Novel Genetic Mutations in the First Swedish Patient with Purine Nucleoside Phosphorylase Deficiency and Clinical Outcome After Hematopoietic Stem Cell Transplantation with HLA-Matched Unrelated Donor.

Purine nucleoside phosphorylase (PNP) is an enzyme active in the purine salvage pathway. PNP deficiency caused by autosomal recessive mutations in the PNP gene leads to severe combined immunodeficiency (SCID) and in two thirds of cases also to neurological effects such as developmental delay, ataxia, and motor impairment.PNP deficiency has a poor outcome, and the only curative treatment is allogenic hematopoietic stem cell transplantation (HSCT). We present the first Swedish patient with PNP deficiency with novel mutations in the PNP gene and the immunological results of the HSCT and evaluate the impact of HSCT on the neurological symptoms. The patient presented early in life with neurological symptoms and suffered later from repeated serious respiratory tract infections. Biochemical tests showed severe reduction in PNP activity (1% residual activity). Genetic testing revealed two new mutations in the PNP gene: c.729C>G (p.Asn243Lys) and c.746A>C (p.Tyr249Cys). HSCT was performed with an unrelated donor, resulting in prompt and sustained engraftment and complete donor chimerism. There was no further aggravation of the patient's neurological symptoms at 21 months post HSCT, and appropriate developmental milestones were achieved. HSCT is curative for the immunological defect caused by PNP deficiency, and our case strengthens earlier reports that HSCT is effective as a treatment even for neurological symptoms in PNP deficiency.

Report of two never treated adult sisters with aromatic L-amino Acid decarboxylase deficiency: a portrait of the natural history of the disease or an expanding phenotype?

Two sisters were diagnosed in their adulthood with aromatic L-amino acid decarboxylase (AADC) deficiency (OMIM#608643). They experienced early myasthenia-like manifestations, myoclonic jerks, oculogyric crises, tremors, and developmental delay during childhood; clinical stabilization afterwards; and spontaneous improvement during adolescence and young adulthood. Two novel pathogenic mutations on DDC gene [p.Tyr37Thrfs*5 (c.105delC) and p.F237S (c.710 T>C)] were associated with undetectable enzyme activity in plasma and only a mild reduction of biogenic amines in cerebrospinal fluid (CSF). The increase of both 3-O-methyldopa and 5-hydroxytryptophan on CSF was the most relevant biochemical alteration denoting AADC defect in these subjects. Transdermal rotigotine remarkably improved their gross motor functions and the asthenic status they complained. The present cases broaden the phenotypic spectrum of AADC deficiency and suggest that (1) AADC defect is not a progressive neurological disease and behaves rather as a neurodevelopmental disorder that improves during the second decade of life; (2) treatment-naïve adults can still respond well to neurotransmitter therapy; and (3) the possibility of a mild presentation of AADC deficiency should be considered when examining young adults with asthenic and parkinsonian symptoms.

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

ß-ureidopropionase (ßUP) deficiency is an autosomal recessive disease characterized by N-carbamyl-ß-amino aciduria. To date, only 16 genetically confirmed patients with ßUP deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 13 Japanese ßUP 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 ßUP enzymes in HEK293 cells showed that the p.E271K and p.R326Q mutations cause profound decreases in activity (≤ 1.3 %). Conversely, ßUP 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 ßUP 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 ßUP deficiency is not as rare as generally considered and screening for ßUP deficiency should be included in diagnosis of patients with unexplained neurological abnormalities.

An algorithm to predict phenotypic severity in mucopolysaccharidosis type I in the first month of life.

INTRODUCTION: Mucopolysaccharidosis type I (MPS I) is a progressive multisystem lysosomal storage disease caused by deficiency of the enzyme α-L-iduronidase (IDUA). Patients present with a continuous spectrum of disease severity, and the most severely affected patients (Hurler phenotype; MPS I-H) develop progressive cognitive impairment. The treatment of choice for MPS I-H patients is haematopoietic stem cell transplantation, while patients with the more attenuated phenotypes benefit from enzyme replacement therapy. METHODS: Thirty patients were included in this study. Genotypes were collected from all patients and all patients were phenotypically categorized at an age of > 18 months based on the clinical course of the disease. In 18 patients, IDUA activity in fibroblast cultures was measured using an optimized IDUA assay. Clinical characteristics from the first month of life were collected from 23 patients. RESULTS: Homozygosity or compound heterozygosity for specific mutations which are associated with MPS I-H, discriminated a subset of patients with MPS I-H from patients with more attenuated phenotypes (specificity 100%, sensitivity 82%). Next, we found that enzymatic analysis of IDUA activity in fibroblasts allowed identification of patients affected by MPS I-H. Therefore, residual IDUA activity in fibroblasts was introduced as second step in the algorithm. Patients with an IDUA activity of < 0.32 nmol x mg(-1) × hr(-1) invariably were MPS I-H patients, while an IDUA activity of > 0.66 nmol × mg(-1) × hr(-1) was only observed in more attenuated patients. Patients with an intermediate IDUA activity could be further classified by the presence of differentiating clinical characteristics, resulting in a model with 100% sensitivity and specificity for this cohort of patients. CONCLUSION: Using genetic, biochemical and clinical characteristics, all potentially available in the newborn period, an algorithm was developed to predict the MPS I phenotype, allowing timely initiation of the optimal treatment strategy after introduction of NBS.

ß-ureidopropionase deficiency: phenotype, genotype and protein structural consequences in 16 patients.

ß-ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyzes the conversion of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, ammonia and CO(2). To date, only five genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 11 newly identified ß-ureidopropionase deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological abnormalities (intellectual disabilities, seizures, abnormal tonus regulation, microcephaly, and malformations on neuro-imaging) and markedly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine and plasma. Analysis of UPB1, encoding ß-ureidopropionase, showed 6 novel missense mutations and one novel splice-site mutation. Heterologous expression of the 6 mutant enzymes in Escherichia coli showed that all mutations yielded mutant ß-ureidopropionase proteins with significantly decreased activity. Analysis of a homology model of human ß-ureidopropionase generated using the crystal structure of the enzyme from Drosophila melanogaster indicated that the point mutations p.G235R, p.R236W and p.S264R lead to amino acid exchanges in the active site and therefore affect substrate binding and catalysis. The mutations L13S, R326Q and T359M resulted most likely in folding defects and oligomer assembly impairment. Two mutations were identified in several unrelated ß-ureidopropionase patients, indicating that ß-ureidopropionase deficiency may be more common than anticipated.

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.

Analysis of severely affected patients with dihydropyrimidine dehydrogenase deficiency reveals large intragenic rearrangements of DPYD and a de novo interstitial deletion del(1)(p13.3p21.3).

Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14-16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3-1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.

Dihydropyrimidine dehydrogenase deficiency presenting with psychomotor retardation in the first Polish patient.

Dihydropyrimidine dehydrogenase (DPD) deficiency is a rare defect of the first step of the pyrimidine catabolic pathway. Patients with a complete enzyme deficiency may be clinically asymptomatic or suffer from neurological abnormalities of various severity. We report a case of an 8-year-old girl with psychomotor retardation and mild course of the disease. Analysis of urine showed strongly elevated levels of uracil and thymine, and no DPD activity could be detected in peripheral blood mononuclear cells. Sequence analysis of the DPD gene (DPYD) revealed that our patient was homozygous for the common splice-site mutation IVS14+1G > A, which suggest that the carrier status for this mutation may be not rare in the Polish population.

Altered dihydropyrimidine dehydrogenase activity associated with mild toxicity in patients treated with 5-fluorouracil containing chemotherapy.

Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5-fluorouracil (5FU). In patients treated with capecitabine or 5FU combined with other chemotherapeutic drugs, DPD activity in peripheral blood mononuclear cells was increased in patients experiencing grade I/II neutropenia. In contrast, decreased DPD activity proved to be associated with grade I/II dermatological toxicity, including hand-foot syndrome. Thus, patients with a low-normal or high-normal DPD activity proved to be at risk of developing mild toxicity upon treatment with 5FU-based chemotherapy, demonstrating the important role of DPD in the etiology of toxicity associated with 5FU and the catabolites of 5FU.

Paradoxical elevated thiopurine S-methyltransferase activity after pancytopenia during azathioprine therapy: potential influence of red blood cell age.

There is an increased risk of developing bone marrow depression and infections during azathioprine therapy for inflammatory bowel disease. Patients with low or absent thiopurine S-methyltransferase (TPMT) activity have an increased risk of developing myelotoxicity. We describe a patient who developed pancytopenia combined with cytomegalovirus pneumonia after several years of azathioprine use. The bone marrow depression was probably caused by the viral infection, as all others causative factors were unlikely. Surprisingly, we observed grossly elevated TPMT activity (182 nmol/g/h) during the recovery phase, following the pancytopenic period. After complete recovery of the bone marrow suppression, TPMT activity returned to usual reference activity (43 nmol/g/h). This remarkable change in enzymatic activity of TPMT may be explained by differences in the age of red blood cells, as younger erythrocytes have a higher TPMT activity. Determination of a patient's TPMT status by phenotyping should therefore not be performed just after bone marrow depression or in cases of activated erythropoieses.

Arts syndrome is caused by loss-of-function mutations in PRPS1.

Arts syndrome is an X-linked disorder characterized by mental retardation, early-onset hypotonia, ataxia, delayed motor development, hearing impairment, and optic atrophy. Linkage analysis in a Dutch family and an Australian family suggested that the candidate gene maps to Xq22.1-q24. Oligonucleotide microarray expression profiling of fibroblasts from two probands of the Dutch family revealed reduced expression levels of the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1). Subsequent sequencing of PRPS1 led to the identification of two different missense mutations, c.455T-->C (p.L152P) in the Dutch family and c.398A-->C (p.Q133P) in the Australian family. Both mutations result in a loss of phosphoribosyl pyrophosphate synthetase 1 activity, as was shown in silico by molecular modeling and was shown in vitro by phosphoribosyl pyrophosphate synthetase activity assays in erythrocytes and fibroblasts from patients. This is in contrast to the gain-of-function mutations in PRPS1 that were identified previously in PRPS-related gout. The loss-of-function mutations of PRPS1 likely result in impaired purine biosynthesis, which is supported by the undetectable hypoxanthine in urine and the reduced uric acid levels in serum from patients. To replenish low levels of purines, treatment with S-adenosylmethionine theoretically could have therapeutic efficacy, and a clinical trial involving the two affected Australian brothers is currently underway.

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.

HPLC-electrospray tandem mass spectrometry for rapid determination of dihydropyrimidine dehydrogenase activity.

BACKGROUND: Patients with a partial dihydropyrimidine dehydrogenase (DPD) deficiency have an increased risk of developing severe 5-fluorouracil-associated toxicity. We developed a rapid and specific method to measure the DPD activity in peripheral blood mononuclear cells using HPLC tandem-mass spectrometry (HPLC-MS/MS). METHODS: The activity of DPD was measured with thymine as the substrate, followed by reversed-phase HPLC combined with electrospray ionization MS/MS and detection of the product dihydrothymine with multiple-reaction monitoring. Stable-isotope labeled dihydrothymine was used as the internal standard. RESULTS: Dihydrothymine was measured within an analytical run of 10 min, with a lower limit of quantification of 54 microg/L (0.4 micromol/L). The intraassay and interassay variations of the DPD activity assay were both <7%. A linear correlation (R(2) = 0.980; P <0.001) was observed between the HPLC-MS/MS data and those obtained with a reference method using radiolabeled thymine. There were no systematic differences between the 2 methods, and both methods yielded similar results. CONCLUSION: The analysis of the DPD activity with HPLC-MS/MS is rapid, accurate, and sufficiently sensitive to be used as a screening method for patients with a DPD deficiency.