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 8year-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.

Congenital DPD deficiency was originally described in 1984 in a child with psychomotor retardation and seizures (Bakkeren et al., 1984).Until now, approximately 50 cases of the disease have been reported.Most of the patients suffered from seizures, mental retardation, microcephally, and muscle tone disorders whereas some individuals were asymptomatic (Van Kuilenburg et al., 1999).
Human DPD gene (DPYD) is present as a single copy gene on chromosome 1p22 and con-sists of 23 exons (Wei et al., 1998).The estimated number of individuals homozygous for the most common IVS14+1G>A mutation is 1.2 in 10 000 (Van Kuilenburg et al., 2001).The G to A mutation in IVS14+1G>A leads to the skipping of exon 14 immediately upstream of the mutated splice donor site in the process of DPD pre-mRNA splicing.As a result, the mature DPD mRNA lacks a 165-nucleotide (nt) segment encoding the amino acids 581-635 (Wei et al., 1998;Van Kuilenburg et al., 2001).
No clear correlation between the genotype and phenotype could be established in 17 families presenting 22 patients with complete deficiency of DPD (Van Kuilenburg et al., 1999).In this group of patients, seven different mutations were identified, including two deletions, one splice site mutation, and four missense mutations.
DPD is also the main enzyme catalyzing the degradation of 5-fluorouracil which is used in oncologic chemotherapy.Patients with DPD deficiency are prone to develop severe 5-fluorouracil-associated toxicity, and its usage in such patients may even re-
So, decreased DPD activity due to mutation heterozygosity usually is asymptomatic, but causes increased toxicity of 5-fluorouracil, while deficiency of DPD activity due to pathogenic mutations (in homozygous or compound heterozygous forms) results in a rare disease with an autosomal recessive genetic trait.
In this paper, the first case of a Polish patient with a DPD deficiency, presenting with mental retardation and speech disabilities, is reported.

MATERIALS AND METHODS
GC-MS urinary organic acids analysis was performed by the method described by (Chalmers & Lawson, 1975) with modification.
Analysis of pyrimidine bases and DPD activity.The concentrations of pyrimidine bases and their degradation products in urine and plasma were determined using reversed-phase HPLC combined with electrospray tandem mass spectrometry, and detection was performed by multiple-reaction monitoring.Stable-isotope-labeled reference compounds were used as internal standards (Van Lenthe et al., 2000).
The activity of DPD was determined in peripheral blood mononuclear (PBM) cells using radiolabeled thymine followed by separation of radiolabeled thymine from radiolabeled dihydrothymine using reversed-phase HPLC (Van Kuilenburg et al., 2000).
Mutation analysis of DPYD.DNA was isolated from leukocytes using the Wizard Genomic DNA Purification Kit.PCR amplification of all 23 coding exons and flanking intronic regions was carried out by using intronic primer sets, as described before (Van Kuilenburg et al., 2000).Sequence analysis of genomic fragments amplified by PCR was carried out on an Applied Biosystems model 3100 automated DNA sequencer using the dye-terminator method.

Case Report
The girl was born after a healthy non-complicated pregnancy and spontaneous labour, her brith mass was 3800 g, and after clinical examination her clinical condition was rated at 8 points on the Apgar scale.The family history is unsignificant, parents are young and unrelated, and her brothers (6 and 2 year-old) are healthy.
The patient's early history revealed psychomotor retardation; she sat when 9 months old, walked at the age of 16 months and spoke when 2 years old, and her speech was retarded from the very beginning.At the age of 2 years, at a clinical neurologic examination she was moderately hypertonic, showed signs of visual-motor incoordination, small manual disabilities, and mental retardation of moderate severity.At present at the age of 8 years, she is suffering from mild mental retardation without neurological symptoms.MRI scan of the head did not show any

Dihydropyrimidine dehydrogenase deficiency
abnormalities.An organic acid profile in the urine was established, using GC/MS, and it showed abnormal secretion of uracil and thymine which suggested DPD deficiency.

DISCUSSION
The most frequent manifestation of DPD deficiency are neurological symptoms such as seizures, psychomotor retardation, microcephaly, hypotonia and autistic features.Dysmorphy and ocular symptoms are sometimes observed.Asymptomatic cases have also been reported (Bakkeren et al., 1984;Diasio et al., 1988).
In our patient with a complete DPD deficiency a mild course of history involving mental retardation was observed from infancy till present.Additionally, increased muscle tone and disorders of visual-motor coordination were found.Enns et al. (2004) showed that children with DPD deficiency might present with white matter abnormalities.In our patient, an MRI scan of the brain revealed no abnormalities.
It is estimated that 1-3% of the Caucasian race might be carriers for a mutation in the gene encoding DPD (Van Kuilenburg et al., 2001).As for now it is not clear why in some individuals with a complete DPD deficiency no symptoms are present.
Recently, from among eight patients with a DPD deficiency, none have presented with seizures, which is considered to be the most frequent clinical manifestation.However, all those patients showed motor-developmental retardation, and the majority -mental retardation (Van Kuilenburg et al., 2002).To date, no clear genotype-phenotype correlations have been established (Van Kuilenburg et al., 1999).
DPD is also an essential enzyme in the metabolism of 5-fluorouracil, which is used in the chemotherapy of colon, breast, kidney, and ovarian cancer, and neoplasms of the central nervous system.In case of a DPD deficiency, 5-fluorouracil can not be degraded any more and will be converted into toxic 5-fluoronucleotides.Thus, patients with a DPD deficiency are at risk of developing severe 5-fluorouracil-associated toxicity (Van Kuilenburg et al., 2000;Ciccolini et al., 2006).The most prevalent mutation in patients suffering from DPD deficiency is the IVS14+IG>A mutation.The incidence of this mutation in different populations is shown in Table 1.There are no such data concerning the Polish population.
The detection of the IVS14+1G>A mutation in Poland, clearly indicates that also in this country there is a necessity of broadening diagnostic investigations in order to detect DPD deficiency, especially in the case of patients diagnosed with cancer, who might be eligible for the treatment with 5-fluorouracil-containing chemotherapy.