The autosomal recessive Zellweger syndrome spectrum (ZSS) disorders comprise a main subgroup of the peroxisome biogenesis disorders and can be caused by mutations in any of 12 different currently identified PEX genes resulting in severe multisystemic disorders. To get insight into the spectrum of PEX gene defects among ZSS disorders and to investigate if additional human PEX genes are required for functional peroxisome biogenesis, we assigned over 600 ZSS fibroblast cell lines to different genetic complementation groups. These fibroblast cell lines were subjected to a complementation assay involving fusion by means of polyethylene glycol or a PEX cDNA transfection assay specifically developed for this purpose. In a majority of the cell lines we subsequently determined the underlying mutations by sequence analysis of the implicated PEX genes. The PEX cDNA transfection assay allows for the rapid identification of PEX genes defective in ZSS patients. The assignment of over 600 fibroblast cell lines to different genetic complementation groups provides the most comprehensive and representative overview of the frequency distribution of the different PEX gene defects. We did not identify any novel genetic complementation group, suggesting that all PEX gene defects resulting in peroxisome deficiency are currently known.

Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD) are clinically overlapping syndromes, collectively called "peroxisome biogenesis disorders" (PBDs), with clinical features being most severe in ZS and least pronounced in IRD. Inheritance of these disorders is autosomal recessive. The peroxisome biogenesis disorders are genetically heterogeneous, having at least 12 different complementation groups (CGs). The gene affected in CG1 is PEX1. Approximately 65% of the patients with PBD harbor mutations in PEX1. In the present study, we used SSCP analysis to evaluate a series of patients belonging to CG1 for mutations in PEX1 and studied phenotype-genotype correlations. A complete lack of PEX1 protein was found to be associated with severe ZS; however, residual amounts of PEX1 protein were found in patients with the milder phenotypes, NALD and IRD. The majority of these latter patients carried at least one copy of the common G843D allele. When patient fibroblasts harboring this allele were grown at 30 degrees C, a two- to threefold increase in PEX1 protein levels was observed, associated with a recovery of peroxisomal function. This suggests that the G843D missense mutation results in a misfolded protein, which is more stable at lower temperatures. We conclude that the search for the factors and/or mechanisms that determine the stability of mutant PEX1 protein by high-throughput procedures will be a first step in the development of therapeutic strategies for patients with mild PBDs.

In the past few years, many patients have been described who have a defect of unknown origin in the peroxisomal beta-oxidation pathway. Complementation analysis has been done by various groups to establish the extent of the genetic heterogeneity among the patients. These studies were based on the use of two established cell lines, one with a deficiency of acyl-CoA oxidase and one with a deficiency of l-bifunctional protein (l-BP), and they showed that most patients belong to the l-BP-deficient group. However, molecular analysis of the cDNA encoding l-BP in patients failed to show any mutations. The recent identification of a new d-specific bifunctional protein (d-BP) prompted us to reinvestigate the original patient with presumed l-BP deficiency. In a collaborative effort, we have now found that the true defect in this patient is at the level of the d-BP and not at the level of the l-BP. Our results suggest that most, if not all, patients whose condition has been diagnosed as l-BP are, in fact, d-BP deficient. We tested this hypothesis in nine patients whose condition was diagnosed as l-BP deficiency on the basis of complementation analysis and found clear-cut mutations in the d-BP cDNA from all patients.

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