Eveline M Hogenhout

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.

Reinvestigation of peroxisomal 3-ketoacyl-CoA thiolase deficiency: Identification of the true defect at the level of D-bifunctional protein

In this report, we reinvestigate the only patient ever reported with a deficiency of peroxisomal 3-ketoacyl-CoA thiolase (THIO). At the time when they were described, the abnormalities in this patient, which included accumulation of very-long-chain fatty acids and the bile-acid intermediate trihydroxycholestanoic acid, were believed to be the logical consequence of a deficiency of the peroxisomal beta-oxidation enzyme THIO. In light of the current knowledge of the peroxisomal beta-oxidation system, however, the reported biochemical aberrations can no longer be explained by a deficiency of this thiolase. In this study, we show that the true defect in this patient is at the level of d-bifunctional protein (DBP). Immunoblot analysis revealed the absence of DBP in postmortem brain of the patient, whereas THIO was normally present. In addition, we found that the patient had a homozygous deletion of part of exon 3 and intron 3 of the DBP gene, resulting in skipping of exon 3 at the cDNA level. Our findings imply that the group of single-peroxisomal beta-oxidation-enzyme deficiencies is limited to straight-chain acyl-CoA oxidase, DBP, and alpha-methylacyl-CoA racemase deficiency and that there is no longer evidence for the existence of THIO deficiency as a distinct clinical entity.

Molecular basis of rhizomelic chondrodysplasia punctata type I: High frequency of the Leu-292 Stop mutation in 38 patients

chondrodysplasia punctata (RCDP; Mckusick 215100 is an autosomal Rhizomelic recessive disease characterized by a disproportionate stature, typical facial appearance, congenital contractures, eye abnormalities and severe growth and mental retardation, although patients have been described with a much milder clinical presentation (see for instance et al Apart from the clinical heterogeneity Smeitink 1992). there is also biochemical heterogeneity, suggesting the involvement of at least three distinct genes. In most RCDP patients ([90%) there is a tetrad of biochemical abnormalities including a deÐciency of dihydroxyacetonephosphate acyltransferase (DHAPAT), alkyldihydroxyacetonephosphate synthase (alkyl DHAP synthase), phytanoyl-CoA hydroxylase, and peroxisomal thiolase I (see et al for Wanders 1996 review). A minority of patients lack this tetrad of abnormalities and su†er from an isolated deÐciency of DHAPAT or alkyl DHAP synthase deÐciency. We propose to call these three biochemical phenotypes RCDP type I, type II and type III, respectively. Genetic complementation studies have revealed that all type I RCDP patients characterized by the tetrad of biochemical abnormalities belong to a single complementation group, suggesting the involvement of a single gene et al (Heikoop Obviously, the product of this gene is of crucial importance, being required 1992). for the correct expression of all four enzymes. The gene involved, PEX7, was recently identiÐed simultaneously by three groups of investigators including our own et al et al et al It encodes the per(Braverman 1997 ; Motley 1997 ; Purdue 1997). oxisomal PTS2 receptor which is involved in the recognition of peroxisomal proteins containing a certain peroxisome targeting signal (PTS2) in the cytosol and

Identification and characterization of the mouse cDNA encoding acyl-CoA:dihydroxyacetone phosphate acyltransferase

We used the amino acid sequence of human acyl-CoA:dihydroxyacetone phosphate acyltransferase (DHAPAT) as bait to screen the database of expressed sequence tags (dbEST) and identified several partial mouse cDNA clones showing high identity. Primers were selected based on the dbEST sequences and used for amplification of this transcript from cDNA prepared from mouse skin fibroblasts. The complete nucleotide sequence was then determined and revealed an open reading frame (ORF) of 2034 bp encoding a protein consisting of 678 amino acids with a calculated molecular mass of 76870. The deduced amino acid sequence showed high identity (80%) with that of human DHAPAT and also revealed a typical peroxisomal targeting signal type 1 (PTS1) at its extreme carboxy-terminus (alanine-lysine-leucine, AKL). Definitive evidence that this cDNA indeed codes for DHAPAT was obtained by heterologous expression in the yeast Saccharomyces cerevisiae. Northern blot analysis revealed high expression of DHAPAT especially in mouse heart, liver and testis.