Janet Haasjes

Identification of PEX7 as the Second Gene Involved in Refsum Disease

Patients affected with Refsum disease (RD) have elevated levels of phytanic acid due to a deficiency of the peroxisomal enzyme phytanoyl-CoA hydroxylase (PhyH). In most patients with RD, disease-causing mutations in the PHYH gene have been identified, but, in a subset, no mutations could be found, indicating that the condition is genetically heterogeneous. Linkage analysis of a few patients diagnosed with RD, but without mutations in PHYH, suggested a second locus on chromosome 6q22-24. This region includes the PEX7 gene, which codes for the peroxin 7 receptor protein required for peroxisomal import of proteins containing a peroxisomal targeting signal type 2. Mutations in PEX7 normally cause rhizomelic chondrodysplasia punctata type 1, a severe peroxisomal disorder. Biochemical analyses of the patients with RD revealed defects not only in phytanic acid α-oxidation but also in plasmalogen synthesis and peroxisomal thiolase. Furthermore, we identified mutations in the PEX7 gene. Our data show that mutations in the PEX7 gene may result in a broad clinical spectrum ranging from severe rhizomelic chondrodysplasia punctata to relatively mild RD and that clinical diagnosis of conditions involving retinitis pigmentosa, ataxia, and polyneuropathy may require a full screen of peroxisomal functions.

Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1.

Rhizomelic chondrodysplasia punctata (RCDP) is a genetically heterogeneous, autosomal recessive disorder of peroxisomal metabolism that is clinically characterized by symmetrical shortening of the proximal long bones, cataracts, periarticular calcifications, multiple joint contractures, and psychomotor retardation. Most patients with RCDP have mutations in the PEX7 gene encoding peroxin 7, the cytosolic PTS2-receptor protein required for targeting a subset of enzymes to peroxisomes. These enzymes are deficient in cells of patients with RCDP, because of their mislocalization to the cytoplasm. We report the mutational spectrum in the PEX7 gene of 78 patients (including five pairs of sibs) clinically and biochemically diagnosed with RCDP type I. We found 22 different mutations, including 18 novel ones. Furthermore, we show by functional analysis that disease severity correlates with PEX7 allele activity: expression of eight different alleles from patients with severe RCDP failed to restore the targeting defect in RCDP fibroblasts, whereas two alleles found only in patients with mild disease complemented the targeting defect upon overexpression. Surprisingly, one of the mild alleles comprises a duplication of nucleotides 45-52, which is predicted to lead to a frameshift at codon 17 and an absence of functional peroxin 7. The ability of this allele to complement the targeting defect in RCDP cells suggests that frame restoration occurs, resulting in full-length functional peroxin 7, which leads to amelioration of the predicted severe phenotype. This was confirmed in vitro by expression of the eight-nucleotide duplication-containing sequence fused in different reading frames to the coding sequence of firefly luciferase in COS cells.

Reduced 5-FU clearance in a patient with low DPD activity due to heterozygosity for a mutant allele of the DPYD gene.

5-fluorouracil pharmacokinetics, dihydropyrimidine dehydrogenase-activity and DNA sequence analysis were compared between a patient with extreme 5-fluorouracil induced toxicity and six control patients with normal 5-fluorouracil related symptoms. Patients were treated for colorectal cancer and received chemotherapy consisting of leucovorin 20 mg m−2 plus 5-fluorouracil 425 mg m−2. Blood sampling was carried out on day 1 of the first cycle. The 5-fluorouracil area under the curve0 → 3h in the index patient was 24.1 mg h l−1 compared to 9.8±3.6 (range 5.4–15.3) mg h l−1 in control patients. The 5-fluorouracil clearance was 520 ml min−1 vs 1293±302 (range 980–1780) ml min−1 in controls. The activity of dihydropyrimidine dehydrogenase in mononuclear cells was lower in the index patient (5.5 nmol mg h−1) compared to the six controls (10.3±1.6, range 8.0–11.7 nmol mg h−1). Sequence analysis of the dihydropyrimidine dehydrogenase gene revealed that the index patient was heterozygous for a IVS14+1G>A point mutation. Our results indicate that the inactivation of one dihydropyrimidine dehydrogenase allele can result in a strong reduction in 5-fluorouracil clearance, causing severe 5-fluorouracil induced toxicity.

Dihydropyrimidine Dehydrogenase Deficiency and 5-Fluorouracil Associated Toxicity

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is responsible for the breakdown of the widely used chemotherapeutic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. Because 5FU has a relative narrow therapeutic index, toxicity increases as the dose is escalated. Since more than 80% of 5FU is degraded by DPD a partial DPD deficiency is increasingly recognized as an important pharmacogenetic syndrome resulting in severe 5FU associated toxicity. Thus, patients with a low DPD activity and those heterozygous for a mutant DPD allele might be at risk for developing severe toxicity after the administration of 5FU. To evaluate the importance of this specific type of inborn error of pyrimidine metabolism in the aetiology of 5FU toxicity we have determined the DPD activity in peripheral blood mononuclear cells (PBM cells) of 37 cancer patients suffering from severe grade 3 and 4 toxicity after the administration of 5FU. In addition, the prevalence of mutant DPD alleles in patients with a low DPD activity has been investigated.

Dihydropyrimidine dehydrogenase (DPD) deficiency: novel mutations in the DPD gene.

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases and it catalyzes the reduction of uracil and thymine to 5,6-dihydrouracil and 5,6-dihydrothymine, respectively. In children, a deficiency of DPD is often accompanied by a neurological disorder but a considerable variation in the clinical presentation among these patients has been reported1. In these patients, a large accumulation of uracil and thymine has been detected in urine, blood and in cerebrospinal fluid whereas no activity of DPD could be detected in fibroblasts and mononuclear cells . The detection of more than 30 patients of various nationalities with a (partial) DPD deficiency within 15 years in The Netherlands alone suggest that this type of inborn error is less rare than previously assumed 1, 2 The recent cloning of the cDNA coding for human DPD and the sequence of the entire human DPD gene3 (DPYD) has allowed the detection of the defects at the molecular level. Identification of disease-causing mutations in the DPD gene will allow rapid pre-screening of patients at risk.