Mutation analysis in 54 propionic acidemia patients.

Deficiency of propionyl CoA carboxylase (PCC), a dodecamer of alpha and beta subunits, causes inherited propionic acidemia. We have studied, at the molecular level, PCC in 54 patients from 48 families comprised of 96 independent alleles. These patients of various ethnic backgrounds came from research centers and hospitals in Germany, Austria and Switzerland. The thorough clinical characterization of these patients was described in the accompanying paper (Grünert et al. 2012). In all 54 patients, many of whom originated from consanguineous families, the entire PCCB gene was examined by genomic DNA sequencing and in 39 individuals the PCCA gene was also studied. In three patients we found mutations in both PCC genes. In addition, in many patients RT-PCR analysis of lymphoblast RNA, lymphoblast enzyme assays, and expression of new mutations in E.coli were carried out. Eight new and eight previously detected mutations were identified in the PCCA gene while 15 new and 13 previously detected mutations were found in the PCCB gene. One missense mutation, p.V288I in the PCCB gene, when expressed in E.coli, yielded 134% of control activity and was consequently classified as a polymorphism in the coding region. Numerous new intronic polymorphisms in both PCC genes were identified. This study adds a considerable amount of new molecular data to the studies of this disease.

Propionic acidemia: neonatal versus selective metabolic screening.

BACKGROUND: Whereas propionic acidemia (PA) is a target disease of newborn screening (NBS) in many countries, it is not in others. Data on the benefit of NBS for PA are sparse. STUDY DESIGN: Twenty PA patients diagnosed through NBS were compared to 35 patients diagnosed by selective metabolic screening (SMS) prompted by clinical findings, family history, or routine laboratory test results. Clinical and biochemical data of patients from 16 metabolic centers in Germany, Austria, and Switzerland were evaluated retrospectively. Additionally, assessment of the intelligent quotient (IQ) was performed. In a second step, the number of PA patients who have died within the past 20 years was estimated based on information provided by the participating metabolic centers. RESULTS: Patients diagnosed through NBS had neither a milder clinical course regarding the number of metabolic crises nor a better neurological outcome. Among NBS patients, 63% were already symptomatic at the time of diagnosis, and <10% of all patients remained asymptomatic. Among all PA patients, 76% were found to be at least mildly mentally retarded, with an IQ <69. IQ was negatively correlated with the number of metabolic decompensations, but not simply with the patients' age. Physical development was also impaired in the majority of patients. Mortality rates tended to be lower in NBS patients compared with patients diagnosed by SMS. CONCLUSION: Early diagnosis of PA through NBS seems to be associated with a lower mortality rate. However, no significant benefit could be shown for surviving patients with regard to their clinical course, including the number of metabolic crises, physical and neurocognitive development, and long-term complications.

Rapid quantification of conjugated and unconjugated bile acids and C27 precursors in dried blood spots and small volumes of serum.

The aim of the study was to develop a method for fast and reliable diagnosis of peroxisomal diseases and to facilitate differential diagnosis of cholestatic hepatopathy. For the quantification of bile acids and their conjugates as well as C(27) precursors di- and trihydroxycholestanoic acid (DHCA, THCA), in small pediatric blood samples we combined HPLC separation on a reverse-phase C18 column with ESI-MS/MS analysis in the negative ion mode. Analysis was done with good precision (CV 3,7%-11.1%) and sufficient sensitivity (LOQ: 11-91 nmol/L) without derivatization. Complete analysis of 17 free and conjugated bile acids from dried blood spots and 10 microL serum samples, respectively, was performed within 12 min. Measurement of conjugated primary bile acids plus DHCA and THCA as well as ursodeoxycholic acid was done in 4.5 min. In blood spots of healthy newborns, conjugated primary bile acids were found in the range of 0.01 to 2.01 micromol/L. Concentrations of C(27) precursors were below the detection limit in normal controls. DHCA and THCA were specifically elevated in cases of peroxysomal defects and one Zellweger patient.

Compliance to clinical guidelines determines outcome in glutaric aciduria type I in the era of newborn screening.

We report on a 4.5-year-old patient diagnosed with Glutaric aciduria type I (GAI), an autosomal recessive inborn error of lysine, hydroxylysine and tryptophan metabolism. Enzymatic assay in cultivated skin fibroblasts revealed complete absence of glutaryl-CoA dehydrogenase activity. All 11 Exons of the GCDH-Gen were sequenced and homozygosity for a yet undescribed mutation was identified. The patient was treated following the recently published guidelines for GA-I. Following this treatment regimen, the child developed normally without any manifest clinical crises. Our patient provides evidence that early commencement and strict adherence to treatment improves clinical outcome even in patients with complete absence of enzyme activity.

Peripheral neuropathy in a patient with D-2-hydroxyglutaric aciduria.

D-2-hydroxyglutaric aciduria (D-2-HGA; OMIM 600721) is a rare autosomal recessive neurometabolic disorder with a wide clinical spectrum. The severe phenotype is homogeneous and is characterized by early infantile-onset epileptic encephalopathy with hypotonia, delayed cerebral visual development, cardiomyopathy and facial dysmorphic features. The mild phenotype has a more variable clinical expression with hypotonia and developmental delay. We present peripheral neuropathy as an additional clinical and electrophysiological feature in a 16-year-old boy with a homozygous missense mutation in exon 3 of the D-2-hydroxyglutarate dehydrogenase gene (D2HGDH) at position c.458T>C. This mutation results in replacement of a methionine residue, which was highly conserved during evolution, by threonine (p.Met153Thr).

The relation of cerebrospinal fluid and plasma glycine levels in propionic acidaemia, a 'ketotic hyperglycinaemia'.

The characteristic elevation of plasma glycine concentrations observed in propionic acidaemia (PA) and other 'ketotic hyperglycinaemias' has been attributed to secondary inhibition of the hepatic glycine cleavage system (GCS) by accumulating CoA derivatives of branched-chain amino acid metabolites. In nonketotic hyperglycinaemia (NKH), cerebrospinal fluid (CSF) and plasma glycine levels and their ratio are increased due to primary deficiency of central nervous system (CNS) as well as hepatic GCS. Whether the GCS in the CNS is also inhibited in PA is unclear, as there are scant data available on CSF glycine levels in this disorder. We studied the relation of CSF and plasma glycine levels in 6 paired samples from 4 PA patients, including one PA patient with bacterial meningitis who underwent ventriculoperitoneal shunting and multiple CSF analyses (n = 26). In contrast to the CSF glycine levels which were generally elevated in all four PA patients, the CSF/plasma glycine concentration ratios in paired samples were normal (0.016-0.029), with the exception of a single sample (0.132) with extremely high CSF protein concentration (2010 mg/L) during the course of meningitis indicating a disturbed blood-brain barrier. This finding of normal CSF/plasma glycine ratio in PA suggests that the observed elevations of CSF glycine levels are a reflection of the concurrent hyperglycinaemia resulting from secondary inhibition of hepatic GCS, but that brain GCS is not affected, in contrast to the situation in NKH. The neurological sequelae in PA are therefore unlikely to be related to disturbed glycine metabolism.

L-2-hydroxyglutaric aciduria: identification of ten novel mutations in the L2HGDH gene.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a metabolic disease with an autosomal recessive mode of inheritance. It was first reported in 1980. Patients with this disease have mutations in both alleles of the L2HDGH gene. The clinical presentation of individuals with L-2-HGA is somewhat variable, but affected individuals typically suffer from progressive neurodegeneration. Analysis of urinary organic acids reveals an increased signal of 2-hydroxyglutaric acid, mainly as the L-enantiomer. L-2-HGA is known to occur in individuals of various ethnic backgrounds, but up to now mutation analysis has been mainly focused on patients of Turkish and Portuguese origin. This led us to confirm the diagnosis on the DNA level and undertake the corresponding mutation analysis in individuals of diverse ethnicity previously diagnosed with L-2-HGA on the basis of urinary metabolites and clinical/neuroimaging data. In 24 individuals from 17 families with diverse ethnic and geographic origins, 13 different mutations were found, 10 of which have not been reported previously. At least eight of the patients were compound heterozygotes. The identification of two mutations (c.751C > T and c.905C > T in exon 7) in patients with different origins supports the view that they occurred independently in different families. In contrast, the mutation c.788C > T was detected in all six Venezuelan patients originating from the same Caribbean island of Margarita, but not in other patients, thus rendering a founder effect likely. None of the mutations was found in the control population, indicating that they are most probably causative. Mutation analysis may improve the quality of diagnosis and prenatal diagnosis of L-2-HGA.

Topical retinaldehyde increases skin content of retinoic acid and exerts biologic activity in mouse skin.

Retinaldehyde, a natural metabolite of beta-carotene and retinol, has been proposed recently for topical use in humans. Because retinaldehyde does not bind to retinoid nuclear receptors, its biologic activity should result from enzymatic transformation by epidermal keratinocytes into ligands for these receptors, such as all-trans retinoic acid and 9-cis-retinoic acid. In this study, we analyzed by high performance liquid chromatography the type and amounts of tissue retinoids as well as several biologic activities resulting from topical application of either retinaldehyde or all-trans retinoic acid on mouse tail skin. Biologic activities of all-trans retinoic acid and retinaldehyde were qualitatively identical in metaplastic parameters (induction of orthokeratosis, reduction of keratin 65-kDa mRNA, increase in filaggrin and loricrin mRNAs) and hyperplastic parameters (increase in epidermal thickness, increase in bromodeoxyuridine (BrdU)-positive cells, increase in keratin 50-kDa mRNA, and reduction in keratin 70-kDa mRNA). Some quantitative differences, not all in favor of all-trans retinoic acid, were found in several indices. Cellular retinoic acid-binding protein II and cellular retinol-binding protein I mRNAs were increased by both topical retinaldehyde and all-trans retinoic acid. Whereas all-trans retinoic acid, 9-cis-retinoic acid, and 13-cis-retinoic acid were not detectable (limit 5 ng/g) in vehicle-treated skin, 0.05% retinaldehyde-treated skin contained 13 +/- 6.9 ng/g wet tissue of all-trans retinoic acid (mean +/- SD), 12.6 +/- 5.9 ng/g 13-cis-retinoic acid, and no 9-cis-retinoic acid. In contrast, 9-cis-retinoic acid was detectable in 0.05% of all-trans retinoic acid-treated skin, which also contained 25-fold more all-trans retinoic acid and 5-fold more 13-cis-retinoic acid than retinaldehyde-treated skin. Our results show that topical retinaldehyde is transformed in vivo into all-trans retinoic acid by mouse epidermis. The small amounts of ligand for retinoic acid nuclear receptors thus produced are sufficient to induce biologic effects similar to those resulting from the topical application of the ligand itself in much higher concentration.

Retinoid signaling by all-trans retinoic acid and all-trans retinoyl-beta-D-glucuronide is attenuated by simultaneous exposure of human keratinocytes to retinol.

Retinol and retinyl esters are converted with time to slowly increasing amounts of all-trans retinoic acid (RA) in cultured human keratinocytes. Exogenous RA has been shown to limit retinol oxidation and to increase retinol esterification. Because significant amounts of retinol are present in biologic systems, we examined whether RA and all-trans-retinoyl-beta-D-glucuronide (RAG) interact with retinol in exhibiting their activities on HaCaT keratinocytes maintained in a retinoid-free culture system. RA was more potent than RAG and retinol in inducing ultrastructural changes attributed to retinoids, inhibiting cell proliferation as well as enhancing keratin 19 expression. In addition, retinoids were able to induce cellular retinoic acid-binding protein II mRNA levels in the cultures, whereas early RA and late RAG activity was detected. The described biologic effects of RA and RAG were diminished by simultaneous cell exposure to retinol. HaCaT cells quickly metabolized retinol to retinyl esters and consequently to low amounts of RA. RA treatment led to an early high peak of cellular RA followed by reduction to trace amounts. Treatment with RAG resulted in constantly high cellular RAG and low RA levels. Under the combined RA and retinol treatment retinyl esters were increased and RA was reduced in HaCaT cells, whereas extracellular RA levels were similar to those obtained by RA alone. On the other hand, the combination of RAG and retinol resulted in higher extracellular RAG, similar cellular RAG, and lower cellular RA levels than those obtained by RAG alone without any change in retinyl esters. This study demonstrates that retinoid signaling by RA and RAG is attenuated by simultaneous exposure of HaCaT keratinocytes in vitro to retinol. The presence of retinol in the medium alters the rate of RA or RAG metabolism and thus cellular RA concentrations. The intensity of retinoid signal is probably dependent on cellular RA levels. The resulting "antagonism" among retinoids is consistent with the presence of an auto-regulatory mechanism in human keratinocytes offering protection against excessive accumulation of cellular RA.

Glucuronidation and isomerization of all-trans- and 13-cis-retinoic acid by liver microsomes of phenobarbital- or 3-methylcholanthrene-treated rats.

Glucuronidation and isomerization of all-trans-retinoic acid (tr-RA) and 13-cis-retinoic acid (13-cis-RA) were investigated in an in vitro system using liver microsomes of differently pretreated rats. In agreement with their thermodynamic stability, more retinoic acid was isomerized from the 13-cis form to the all-trans form than vice versa. Also some 9-cis-retinoic acid (9-cis-RA) could be found. Isomerization was reduced, but in contrast to glucuronidation was still important if boiled microsomes were used. This supports the view that isomerization can proceed as a non-enzymatic process. 3-Methylcholanthrene (MC) pretreatment of the rats increased the microsomal glucuronidation of 13-cis-RA and tr-RA and the formation of 13-cis-retinoyl-beta-glucuronide was enhanced up to 7-fold by MC-induced rat microsomes. The rates of glucuronidation by uninduced and phenobarbital-induced rat microsomes differed only slightly. In addition to glucuronides of the applied retinoic acid isomers (13-cis-RA and tr-RA), 9-cis-RA and its glucuronide were found. Induction of retinoid glucuronidation by pretreatment with MC indicates that this metabolic reaction is catalysed by a MC-inducible UGT isozyme. After two recently described pathways (conversions of retinol to retinal and of retinyl methyl ether to retinol) this is a third step of retinoid metabolism, induced by pretreatment with MC. With human microsomes no more than traces of glucuronides were detected; also, incubations with human microsomes resulted in a lower degree of isomerization than with rat microsomal fractions.

9-cis-retinoyl-beta-D-glucuronide is a major metabolite of 9-cis-retinoic acid.

The in vivo metabolism of 9-cis-retinoic acid (9-c-RA), an endogenous ligand of retinoid X receptors (RXRs), which can also bind to retinoic acid receptors (RARs), was examined in pregnant mice and rats following a single oral dose of 100 mg 9-cis-retinaldehyde (9-c-RAL)/kg body weight. 9-Cis-retinoyl-beta-D-glucuronide (9-c-RAG), a metabolite not found in vivo before, was a major metabolite of 9-c-RA in mouse plasma and was also present in all mouse tissues examined as well as in rat plasma. In both species putative oxidation products of retinoic acids and high levels of retinyl esters were found. Concentrations of retinoic acid isomers and retinoyl-beta-D-glucuronides in the mouse plasma greatly exceeded those of the rat plasma. The finding of high levels of 9-c-RAG underlines the importance of glucuronidation in the metabolism of retinoids.

Single-run analysis of isomers of retinoyl-beta-D-glucuronide and retinoic acid by reversed-phase high-performance liquid chromatography.

Reversed-phase HPLC methods capable of separating several retinoic acid isomers are generally not designed for simultaneous analysis of isomers of other classes of retinoids. A reversed-phase HPLC method is presented which allows the separation of at least four retinoic acid (RA) isomers (13-cis-RA, 9,13-dicis-RA, 9-cis-RA, all-trans-RA)and of all isomers of retinoyl-beta-D-glucuronide (RAG), which have been observed in vivo (13-cis-RAG, 9-cis-RAG,all-trans-RAG). The recovery of retinoids was generally between 80 and 90%. Intra-day reproducibility (expressed as relative standard deviation) was less than or equal to 7%. As little as 0.25 ng of RA isomers and of all-trans-RAG could be detected. This method allowed the study of the metabolism of 9-cis-retinoids, where isomerization reactions play a predominant role.

Quantitation of total homocysteine in human plasma by derivatization to its N(O,S)-propoxycarbonyl propyl ester and gas chromatography-mass spectrometry analysis.

Much evidence supports the hypothesis that mild or moderate hyperhomocysteinaemia represents an important and independent risk factor for occlusive vascular diseases. Therefore, the accurate and reliable determination of total plasma homocysteine has gained major importance for risk assessment. Furthermore, it can help in the detection of folate and vitamin B12 deficiency. This has prompted us to develop a sensitive gas chromatography-mass spectrometry (GC-MS) method in order to quantify total homocysteine in human plasma. Prior to chromatography, reduced homocysteine was released from disulfide bonds by incubation with excess dithiothreitol and converted into its N(O,S)-propoxycarbonyl propyl ester by derivatization with n-propyl chloroformate. Aminoethylcysteine served as internal standard. The method proved to be highly linear over the entire concentration range examined (corresponding to 0-266 microM homocysteine) and showed intra-assay and inter-assay variation (relative standard deviations) of approximately 5 and 5-10%, respectively. External quality control by comparison with duplicate analysis performed on a HPLC-based system revealed satisfactory correlation. The newly developed GC-MS based method provides simple, reliable and fast quantification of total homocysteine and requires only inexpensive chemicals, which are easy to obtain.

Trichothiodystrophy: quantification of cysteine in human hair and nails by application of sodium azide-dependent oxidation to cysteic acid.

The term "trichothiodystrophy" (TTD) covers several autosomal recessive diseases whose diagnostic hallmark is short, brittle hair low in sulfur and cystine because of impaired synthesis of high-sulfur matrix protein. Clinical symptoms associated with TTD represent a variable range of abnormalities in organs derived from ectoderm and neuroectoderm. Important laboratory tests of the hair for the diagnosis of TTD comprise polarizing microscopy ("tiger-tail" pattern), electron microscopy, and amino acids analysis of hydrolyzed hair with a special focus on cystine. However, only very few institutions determine the amino acid composition of human hair and nail clippings, which requires special sample preparation including hydrolysis. If no special precautions are taken, quantification of cysteine and cystine becomes inaccurate because of decomposition of these residues during hydrolysis. We therefore performed the sample work-up with azide-dependent oxidation which we have for the first time adapted for analysis of hair and nail clippings. With our control and parent data resembling published data on hair and nail samples, we obtained a decreased proportion of cysteine (half cystine, determined as cysteic acid) in materials obtained from a boy with TTD. Clearly, the method for the quantification of cysteine following sodium azide-dependent oxidation is a suitable and rather convenient approach to the quantification of cyst(e)ine and other amino acids in hair and nail proteins, and is a valuable contribution to the diagnosis of TTD.

Biological effects and metabolism of 9-cis-retinoic acid and its metabolite 9,13-di-cis-retinoic acid in HaCaT keratinocytes in vitro: comparison with all-trans-retinoic acid.

9-cis-Retinoic acid (9cRA), a geometric isomer of all-trans-retinoic acid (atRA), is an endogenous high-affinity ligand for retinoid X receptors and retinoic acid receptors activating them with high potency. 9,13-di-cis-Retinoic acid (9,13dcRA) has been described as a major plasma metabolite of 9cRA. In this study, the biological activity and the metabolism of 9cRA and 9,13dcRA were investigated and compared with those of atRA in a retinol-free culture system of HaCaT keratinocytes. 9cRA exhibited a slightly weaker activity overall than atRA in inhibiting cell proliferation, inducing cellular retinoic acid binding protein II (CRABP II) mRNA levels and upregulating cytokeratin 19 expression. 9,13dcRA regulated HaCaT keratinocyte activity only at the highest concentration tested (10(-6) M). In cultures of HaCaT keratinocytes with atRA and 9cRA, rapid intracellular accumulation of atRA was observed within 2 h, and atRA levels were higher with atRA treatment than with 9cRA treatment. 9,13dcRA remained relatively stable in the medium with intracellular 9,13dcRA levels below the level of detection. Taken together, 9cRA seems to be slightly less potent than atRA in regulating the biological activity of HaCaT keratinocytes, while its metabolite 9,13dcRA is effectively inactive at biologically relevant concentrations. Our data suggest a prodrug/drug relationship between 9cRA and atRA in human keratinocytes. 9,13dcRA seems to be a weaker prodrug of atRA or an inactive metabolic derivative.

Transplacental pharmacokinetics of a synthetic retinoid which is not bound by mouse embryonic cellular retinoic acid-binding protein.

Teratogenicity is a major side effect of retinoids, a class of compounds used in dermatology and oncology. The binding of retinoids to cellular retinoic acid-binding protein (CRABP) has been suggested to be important for the mechanism of retinoid embryopathy. Here data are presented on the transplacental pharmacokinetics of CD394 (4-[3-(1-adamantyl)-4-methoxybenzamido] benzoic acid) which does not bind to murine embryonic CRABP, although it is active in rat whole embryo culture and teratogenic in the rabbit in vivo. A single intragastric dose of CD394 (10 mg/kg) was administered to mice on day 11 of gestation. The extent of placental transfer of CD394, determined by HPLC, resembled more that of 13-cis-retinoic acid which also does not bind to CRABP, than that of the CRABP-binding all-trans-retinoic acid. CMax values of CD394 obtained after 1-2 h were: 1368 +/- 652 ng/ml for plasma, 203 +/- 132 ng/g for embryo and 856 +/- 563 ng/g for placenta. AUC (area-under-the-concentration-time-curve) values (0-12 h) were: 4319 ng x h/ml for plasma, 751 ng x h/g for embryo and 3163 ng x h/g for placenta. Thus, CD394 reached the embryo, although embryonic AUC values were less than one fifth of the maternal plasma AUC values. CD394 did not alter endogenous retinol concentrations in plasma, embryo, yolk sac or placenta. Our results indicate that CD394 reaches the embryo in vivo without binding to CRABP, although embryonic concentrations stayed well below plasma levels. This supports the opinion that binding to embryonic CRABP is not a prerequisite for reaching effective embryo concentrations and for the teratogenicity of retinoids.

Comparative studies on effects of all-trans-retinoic acid and all-trans-retinoyl-beta-d-glucuronide on the development of foetal mouse thymus in an organ culture system.

The thymus is a target organ of retinoid teratogens. Retinoids with a generally reduced teratogenic potency should therefore also exert reduced adverse effects on thymus development. The effects of all-trans-retinoic acid (a-tRA) and all-trans-retinoyl-beta-glucuronide (a-tRAG) on the in vitro development of thymic lobes of 15-day-old mouse foetuses were compared in an organ culture system. Both compounds were added to the medium at concentrations ranging from 10(-7) to 10(-5)m. The culture period was 6 days. The investigations showed a concentration-dependent effect of both substances on the proliferation of the lymphatic cells. At 10(-5)m the number of thymocytes was significantly reduced to values of about 70% of the controls by either of the retinoids (P 0.05). Results of flow cytometry showed significant differences concerning the differentiation markers CD4 and CD8 after the culture period. The presence of 10(-6)m a-tRA induced a significant increase in the percentages of CD4(+)CD8(-) cells and a significant decrease of CD4(+)CD8(+) cells. At 10(-5)m a-tRA an additional significant increase in the percentages of CD4(-)CD8(-) cells was found. In contrast, after treatment with a-tRAG, percentages of these populations were in the same range as the controls. Light and electron microscopic investigations revealed a depletion of lymphatic cells and an increase of intracytoplasmic vacuoles in the thymic epithelial cells at 10(-6) and 10(-5)m of either retinoid. HPLC analyses revealed a remarkable degree of retinoid isomerization and (in the case of a-tRAG) of hydrolysis. Compared with the culture medium, retinoids were accumulated in the thymic lobes. Possibly a-tRAG acts by way of limited hydrolysis to retinoic acid.

Propionic acidemia revisited: a workshop report.

Propionic acidemia (PA) is one of the most frequent organic acidurias, but information on the outcome of individuals with PA is rather limited. We present data of 49 patients with PA, which were gathered from 18 metabolic centers throughout Central Europe on the occasion of an international workshop. All patients were identified by selective metabolic screening, and 86% of them were classified as having early-onset PA owing to their presentation with clinical symptoms within the first 90 days of life. Mortality rate was one third, and details of symptoms and treatment of the surviving patients are discussed. The great variation of phenotypic expression of the disease and different therapeutic strategies (especially in regard to the degree of protein restriction) used at the various institutions involved in this study imply the need for a registry of PA patients and for a multicenter prospective treatment study.

Dihydropyrimidine dehydrogenase deficiency caused by a novel genomic deletion c.505_513del of DPYD.

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine degradation pathway. In a patient presenting with convulsions, psychomotor retardation and Reye like syndrome, strongly elevated levels of uracil and thymine were detected in urine. No DPD activity could be detected in peripheral blood mononuclear cells. Analysis of the gene encoding DPD (DPYD) showed that the patient was homozygous for a novel c.505_513del (p.169_171del) mutation in exon 6 of DPYD.