In situ Genetic Complementation Analysis of Cells with Generalized Peroxisomal Dysfunction

Abstract

Most patients with Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and hyperpipecolic acidemia are characterized by a deficiency of peroxisomes. We have developed a simple cytological method for the in situ detection of genetic complementation among and between these patients who are clinically and biochemically defined as having generalized peroxisomal dysfunction. This technique should facilitate both complementation studies in these disorders and investigations into the biogenesis of peroxisomes.

Oxidation of pristanic acid in fibroblasts and its application to the diagnosis of peroxisomal beta-oxidation defects.

“…Although [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]pristanic acid is not available commercially, it is readily synthesized (33) and the product can be stored indefinitely. In addition, there are a number of advantages in using this substrate for the diagnosis of peroxisomal disorders (see below) which easily justify its synthesis and use.…”

“…Using the distribution of catalase immunofluorescence as an indicator of whether complementation had occurred between two cell lines (8), cell line 1 was placed in their complementation group A, cell line 3 in their group C, and cell lines 6,7,8,9, and 10 in their group E (Shimozawa, N., personal communication). Cell lines 11 and 12 belonged to a newly identified complementation group (3).…”

“…For pristanic acid oxidation, fibroblasts in 25-cm 2 flasks, were first incubated for 24 h with 5 ml basal Eagle's medium containing 0.5% FCS. The medium was then replaced with 5 ml fresh medium containing [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]pristanic acid (55 mCi/mmol, prepared essentially as described by Singh et al (33); 120,000 dpm/flask was used in earlier experiments, but this was later increased to 480,000 dpm/flask) for 24 h. The amount of radioactivity in CO 2 was determined as described by Poulos (34) except that the liberated CO 2 was collected for 5 min and trapped in 2 ml 1 M KOH and counted using scintillant (OptiPhase HiSafe 3; Wallac, Gaithersburg, MD). Radioactivity in water-soluble products was determined as described by Singh et al (33).…”

“…Since several biochemical and immunocytochemical markers are affected in this group of disorders, a range of techniques has been used to assess peroxisomal integrity, and hence complementation, in fused heterokaryons. These include the activity of dihydroxyacetonephosphate acyltransferase (DHAPAT) and the percentage of catalase that is in the particulate fraction (6), the immunocytochemical localization of catalase (7,8), the oxidation of phytanic acid (9) and very long chain fatty acids (VLCFA, i.e., fatty acids with more than 22 carbons) (10), plasmalogen biosynthesis (1), and sensitivity to pyrene fatty acid-mediated ultraviolet damage (11).…”

Complementation in Zellweger Syndrome: Biochemical Analysis of Newly Generated Peroxisomes

“…Because of the overlapping clinical features in these patients and the similar bio chemical abnormalities, the relationship be-tween these diseases has been investigated by genetic complementation analysis following somatic cell fusion. Using this approach, we and others [2][3][4][5] have shown genetic hetero geneity among Zellweger syndrome patients and have also shown that some patients clin ically identified with other peroxisomal dis eases (i.e. NALD.…”