A. H. van Gennip

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.

Disorders of mitochondrial fatty acyl-CoA β-oxidation

In recent years tremendous progress has been made with respect to the enzymology of the mitochondrial fatty acid β-oxidation machinery and defects therein. Firstly, a number of new mitochondrial β-oxidation enzymes have been identified, including very-long-chain acyl-CoA dehydrogenase (VLCAD) and mitochondrial trifunctional protein (MTP). Secondly, the introduction of tandem MS for the analysis of plasma acylcarnitines has greatly facilitated the identification of patients with a defect in fatty acid oxidation (FAO). These two developments explain why the number of defined FAO disorders has increased dramatically, making FAO disorders the most rapidly growing group of inborn errors of metabolism. In this review we describe the current state of knowledge of the enzymes involved in the mitochondrial oxidation of straight-chain, branched-chain and (poly)unsaturated fatty acyl-CoAs as well as disorders of fatty acid oxidation. The laboratory diagnosis of these disorders is described, with particular emphasis on the methods used to identify the underlying enzyme defect and the molecular mutations. In addition, a simple flowchart is presented as a guide to the identification of mitochondrial FAO-disorders. Finally, treatment strategies are discussed briefly.

Quantitative plasma acylcarnitine analysis using electrospray tandem mass spectrometry for the diagnosis of organic acidaemias and fatty acid oxidation defects

Electrospray tandem mass spectrometry (ESI-MS/MS) of acylcarnitines has been successfully applied in newborn screening for defects in fatty acid oxidation and organic acidaemias (Millington et al 1990; Rashed et al 1995a, 1997). In addition, acylcarnitine analysis has also been applied for the selective screening for these disorders (Chace et al 1997; Van Hove et al 1993, 1995), for post-mortem diagnosis using bile fluid (Rashed et al 1995b) and for prenatal diagnosis of organic acidaemias and defects of fatty acid oxidation (Nada et al 1996; Shigematsu et al 1996). Since for selective screening for inborn errors of metabolism, apart from urine samples, mostly serum or plasma samples are sent in, we developed a quantitative ESI-MS/MS acylcarnitine analysis in plasma and included the analysis of free carnitine in the same assay. The results show that this analysis is highly sensitive and reproducible and therefore suitable for selective screening of fatty acid oxidation defects, organic acidaemias and secondary carnitine deficiency.

Heterozygosity for a point mutation in an invariant splice donor site of dihydropyrimidine dehydrogenase and severe 5-fluorouracil related toxicity

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases and it catalyses the reduction of thymine and uracil to 5,6-dihydrothymine and 5,6-dihydrouracil, respectively. DPD is also responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. 5FU is one of the few drugs that shows some antitumour activity against various otherwise untreatable tumours including carcinomas of the gastrointestinal tract, breast, ovary and skin. Although the cytotoxic effects of 5FU are probably directly mediated by the anabolic pathways, the catabolic route plays a significant role since more than 80% of the administered 5FU is catabolised by DPD. The important role of DPD in the chemotherapy with 5FU has been shown in cancer patients with a complete or near-complete deficiency of this enzyme. These patients suffered from severe (neuro)toxicity including death, following 5FU chemotherapy.1,2 It has been suggested that patients suffering from 5FU toxicities due to a low activity of DPD are genotypically het-erozygous for a mutant allele of the gene encoding DPD.3 Furthermore, the frequency of heterozygotes in the normal population has been estimated to be as high as 3%.3 In this study we investigated the cDNA and a genomic region of the DPD gene of a cancer patient experiencing severe toxicity following 5FU treatment, for the presence of mutations.

Inborn errors of pyrimidine degradation: clinical, biochemical and molecular aspects

The pyrimidines, uracil and thymine, are degraded in four steps. The first three steps of pyrimidine catabolism, controlled by enzymes shared by both pathways, result in the production of the neurotransmitter amino acid β-alanine from uracil and the nonfunctional (R)-(-)-β-aminoisobutyrate from thymine. The fourth step is controlled by several aminotransferases, which have different affinities for β-alanine, β-aminoisobutyrate and GABA. Defects concerning the first three steps all lead to a reduced production of β-alanine; defects of the transaminases involving the metabolism of β-alanine and GABA lead to accumulation of these neurotransmitter substances. In addition, other metabolites will accumulate or be reduced depending on the specific enzyme defect. Analysis of the abnormal concentrations of these metabolites in the body fluids is essential for the detection of patients with pyrimidine degradation defects. Clinically these disorders are often overlooked because symptomatology is highly aspecific. The growth in our knowledge concerning inborn errors of pyrimidine degradation has emphasized the importance of the clinical awareness of these defects as a possible cause of neurological disease and a contraindication for treatment of cancer patients with certain pyrimidine analogues. The various defects are discussed and attention is paid to clinical, genetic and diagnostic aspects.

Clinical features of galactokinase deficiency: a review of the literature.

Summary: Galactokinase deficiency (McKusick 230200) is a rare autosomal recessive inborn error of galactose metabolism. Cataract and, rarely, pseudotumor cerebri caused by galactitol accumulation seem to be the only consistently reported abnormalities in this disorder. We performed a literature search to obtain information on the clinical spectrum of galactokinase deficiency. A total of 25 publications were traced describing 55 galactokinase-deficient patients. Cataract was reported in most patients. Clinical abnormalities other than cataract were reported in 15 (35%) out of 43 cases on which information was available. However, all symptoms were reported infrequently and a causal relationship with the galactokinase deficiency is unlikely. As cataract and pseudotumor cerebri appear to be the sole complications of galactokinase deficiency, the outcome for patients with galactokinase deficiency is much better than for patients with classical galactosaemia (McKusick 230400), a more common autosomal recessive disorder of galactose metabolism caused by galactose-1-phosphate uridyltransferase (GALT; EC 2.7.7.12) deficiency. Long-term follow-up of patients with this disorder has shown that, in spite of a severely galactose-restricted diet, most patients develop abnormalities such as a disturbed mental and/or motor development, dyspraxia and hypergonadotropic hypogonadism. Endogenous production of galactose has been considered an important aetiological factor. Although damage may well occur inutero, available evidence suggests that damage will continue after birth. Inhibition of galactokinase may then be a promising approach for controlling damage in GALT-deficient patients.

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

Nomenclature for human DPYD alleles.

To standardize DPYD allele nomenclature and to conform with international human gene nomenclature guidelines, an alternative to the current arbitrary system is described. Based on recommendations for human genome nomenclature, we propose that each distinct allele be designed by DPYD followed by an asterisk and an Arabic numeral. The number specifies the key mutation and, where appropriate, a letter following the number indicates an additional mutation on the mutant allele. Criteria for classification as a distinct allele are also presented.

Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass spectrometry.

A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography-mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.

Rapid analysis of conjugated bile acids in plasma using electrospray tandem mass spectrometry: application for selective screening of peroxisomal disorders

The final step in bile acid biosynthesis takes place in peroxisomes and involves oxidative cleavage of the side-chain of C 27 -β-cholestanoic acids, leading to the formation of the primary bile acids cholic and chenodeoxycholic acid. Therefore, in a number of peroxisomal disorders, including both peroxisomal biogenesis defects and isolated defects of peroxisomal β-oxidation, the accumulation of serum C 27 -5β-cholestanoic acids can be observed (Bjorkhem 1994; Clayton et al 1987; Libert et al 1991; Wanders et al 1995). Since routine gas chromatography mass-spectrometry (GC-MS) analysis of plasma bile acids requires laborious sample preparation including enzymatic and/or chemical deconjugation of plasma bile acids prior to derivatization and GC-MS analysis, we have developed a rapid HPLC electrospray tandem mass spectrometry (ESI-MS/MS) method for analysing taurine- and glycine-conjugated bile acids.