Premature Termination Codon Read-Through in the ABCC6 Gene: Potential Treatment for Pseudoxanthoma Elasticum

Abstract

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder manifesting with ectopic connective tissue mineralization, caused by mutations in the ABCC6 gene, ~35% of all mutations being premature termination mutations. In this study, we investigated the therapeutic potential of the nonsense codon read-through-inducing drug, PTC124, in treating PXE. The ability of this drug to facilitate read-through of nonsense mutations was examined in HEK293 cells transfected with human ABCC6 expression constructs harboring seven different PXE associated nonsense mutations, and evaluated by immunofluorescence and In-Cell ELISA. Our data demonstrated that PTC124 did not exhibit cell toxicity in concentrations up to 40 µg/ml, and the facilitated read-through was not only dose dependent but also sequence context dependent. Considering the redundancy of the genetic code, it was postulated that in case of the most common recurrent nonsense mutation, p.R1141X, the read-through may result in substitution of the arginine 1141 by either glycine, tryptophan or cysteine. Their potential pathogenicity was tested in a recently developed zebrafish mRNA rescue assay, and demonstrated that all three mRNA transcripts were able to rescue abcc6a morpholino-induced phenotype of zebrafish. Thus, our results suggest that read-through of nonsense mutations in ABCC6 by PTC124 may have potential for pharmacologic treatment of PXE.

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

“…Multiple experimental approaches with cell lines, patient cells, animal models, and genetic disorder patients have demonstrated that the drug ataluren can restore expression to genes and mRNAs otherwise inactive because of the presence of premature nonsense codons (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)23). Collectively, such experiments have strongly suggested that ataluren promotes nonsense suppression, i.e., the insertion of near-cognate tRNAs at PTCs, but direct evidence for this activity has been lacking.…”

“…1 and SI Appendix, Fig. S3) (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)23); (ii) the effects on luciferase activity could not be independently replicated by others (24) or by us (SI Appendix , Fig. S3C); (iii) ataluren's putative luciferase inhibitory activity depends on a specific enzyme substrate (25); and (iv) most of the hypothetical inhibitory molecule, PTC124-AMP (22), is rapidly converted to the active readthrough molecule PTC124 (ataluren) under conditions of in vitro translation (SI Appendix, Fig.…”

Pseudoxanthoma Elasticum: A Pediatric Case

“…Mineralizasyonun önüne geçmek için diyette magnezyumu arttırmak üzerine deneyler yapılmaktadır, faydalı olduğuna dair yayınlar mevcuttur (11). ABCC6 geninde PTC124 aracılığıyla saçma mutasyonların oluşturulmasını tedavi için öneren çalışmalar bulunmaktadır (14). Moleküler genetik çalışmalar devam etmektedir (8,13 …”

Pseudoxanthoma elasticum: the paradigm of ectopic mineralization disorders – diagnosis and treatment

“…Some of these approaches could be adopted from the progress made in other heritable diseases, including those being developed for other ABC transporter gene defects such as cystic fibrosis. Examples of such approaches include development of small molecules that facilitate the readthrough of premature termination codon-causing mutations, so as to allow synthesis of the full-length protein from the mutant allele [81]. In case of PXE, approximately 30-35% of all mutations are premature termination-causing mutations in the ABCC6 gene, including the most common recurrent mutation p.R1141*, which accounts for approximately 25% of all mutations in caucasian patient populations [11].…”