PMC:4596894 / 17443-19478 JSONTXT

{"project":"2_test","denotations":[{"id":"26387595-12402331-2046289","span":{"begin":1691,"end":1693},"obj":"12402331"}],"text":"PEX-cDNA-Transfection Complementation Assay\nTo confirm that the variants found in PEX1 and PEX6 are the cause of the aberrant peroxisomal phenotype, we performed a genetic complementation assay. We cultured fibroblasts of individuals F1-II:3 and F5-II:2 at 40°C and then transfected these with control PEX1 and PEX6 cDNAs. The introduction of PEX1 cDNA rescued the impaired peroxisome biogenesis in cells from individual F1-II:3, and PEX6 cDNA rescued peroxisome biogenesis in cells from individual F5-II:2 (complementation in 14% or 20% of cells, respectively; Figure S4). These results confirm that the variants in PEX1 and PEX6 are the cause of the peroxisomal defects in the individuals with HS.\nTo determine whether, and to which degree, each of the identified PEX1 and PEX6 variants affect peroxisome biogenesis, we co-transfected cDNAs harboring the different variants with fluorescent peroxisomal marker EGFP-SKL into cells completely deficient in PEX1 or PEX6. We compared the capability of these cDNAs to functionally complement the peroxisome-deficient cells to the complementation capability of the control cDNAs (Figure 4). Because PEX1 c.1239+1G\u003eT causes a splicing defect and consequently does not produce a functional protein, we did not test it in the complementation assay. Transfection with the constructs containing the previously reported pathogenic variants PEX1 c.2097dup and PEX6 c.821C\u003eT, as well as the variant PEX6 c.1841del, resulted in no (0%) to minimal (1.3%) functional complementation. These results are consistent with the severe clinical presentation and complete absence of import of peroxisomal matrix protein in individuals homozygous for these variants31 (data not shown). In contrast, transfection with the other variants resulted in rescue of peroxisomal biogenesis in between 23% and 58% of cells, indicating that these variants are associated with significant residual activity. Thus, all affected individuals possess at least one PEX variant with residual activity in peroxisomal biogenesis."}