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The peroxisomal diseases can be divided into three categories: 1) diseases in which morphologically distinguishable peroxisomes are virtually absent (Zellweger syndrome; infantile Refsum disease; Hyperpipecolic Acidaemia; neonatal Adrenoleukodystrophy); 2) diseases in which peroxisomes are present but several peroxisomal functions are impaired (rhizomelic Chondrodysplasia punctata; Zellweger-like syndrome?); and 3) diseases in which a single peroxisomal function is impaired. We have used complementation analysis after somatic cell fusion in order to investigate the genetic relationship between diseases in category 1. The activity of acyl-CoA: dihydroxyacetonephosphate acyltransferase, which is deficient in these diseases and in rhizomelic Chondrodysplasia punctata, was used as an index of complementation. The cell lines studied, all of which complemented with rhizomelic Chondrodysplasia punctata, could be divided into at least 4 and possibly 5 complementation groups. This indicates that at least 5 and possibly 6 genes are involved in the assembly of peroxisomes. One of the complementation groups is comprised of cell lines from patients with the Zellweger syndrome, infantile Refsum disease and Hyperpipecolic Acidaemia. Thus mutations in the same gene can lead to clinically distinguishable diseases. On the other hand, the Zellweger cell lines studied fall into 3 complementation groups and the infantile Refsum disease cell lines into 2 groups. Thus mutations in different genes can lead to the same clinical phenotype. Fusion of complementary cell lines lacking morphologically distinguishable peroxisomes leads to assembly of peroxisomes, which can be monitored by measuring particle-bound catalase biochemically or by immunofluorescence. In two combinations of cell lines assembly of peroxisomes was rapid and cycloheximide insensitive. Thus the components required for peroxisome assembly must be present in a stable form in the parental cell lines, at least one of which must contain peroxisomal ghost-like structures.

The Zellweger syndrome is a rare inborn error of metabolism characterized by the absence of morphologically distinguishable peroxisomes. As a consequence tissues and cells from Zellweger patients contain severely reduced levels of ether phospholipids. These are replaced by diacylphospholipids while keeping the polar headgroup composition of the cellular phospholipids constant. Both peroxisomal enzymes involved in glycero-ether lipid bond formation appear to be deficient. The experiments clearly establish that peroxisomes are indispensible for ether lipid biosynthesis. The peroxisomal deficiency in de novo ether lipid biosynthesis in fibroblasts and amniotic fluid cells can be applied in diagnostic assays. The mutation can be by-passed by feeding the cells with alkylglycerol. Similar characteristics as found for plasmalogen biosynthesis in Zellweger syndrome were assessed in other diseases with a general impairment of peroxisomal functions such as infantile Refsum disease and neonatal adrenoleukodystrophy as well as in rhizomelic chondrodysplasia punctata, a disease characterized by the absence of some, but not all, peroxisomal functions. Complementation analysis after somatic cell fusion has revealed that at least three genes must be involved in the biogenesis of fully functional peroxisomes.