Analyse des PEX1-Gens bei Patienten mit Zellweger-Syndrom: Identifikation einer neuen Deletion und Untersuchung von Polymorphismen in der 5'-untranslatierten Region

Abstract

Zellweger spectrum disorders are autosomal recessive peroxisomal metabolic diseases. Mutations in the PEX1 gene are the most common cause. This thesis deals with the analysis of the PEX1 gene and characterization of two polymorphisms in the 5 UTR of PEX1 deficient patients. Major focus is the identification of the molecular cause for peroxisome biogenesis disorder in two patients (ZS 8, ZS 26). Only one heterozygous PEX1 mutation, c.2528 G>A (p.Gly843Asp), was identified in both patients. The wildtype mRNS of patient ZS 26, determined by allelespecific quantitative real time-PCR, was significantly underrepresented. A novel pathogenic mutation was identified in patient ZS 26 by applying long range PCR based deletion screening. We identified a 3838 bp deletion spanning exons 10 to 12 of the PEX1 gene. The deleted area contained an insertion of 395 bp, a duplicated sequence from intron (16 g.20805_24642delins28829_29223; NCBI Reference Sequence NC_000007.13). The deletion was inherited from the healthy father. Based on reporter assays described in literature, the 5 polymorphism c.-137 T>C of the PEX1 gene leads to reduced PEX1 expression, whereas the 5 polymorphism c.-53 C>G of the PEX1 gene leads to increased expression. If present together, these polymorphisms cause near-normal PEX1 expression. Within 30 PEX1-deficient patients four combinations of 5 polymorphisms could be found in this work: c.-137 TT/c.-53 CC (43 %), c.-137 TC/c.-53 CG (37 %), c.-137 CC/c.-53 GG (10 %) und c.-137 TC/c.-53 CC (10 %). The 5 polymorphism c.-53 C>G has occurred on a c.-137 T>C background, as the latter is found independently of the c.-53 C>G change. The common c.2528G>A (p.Gly843Asp) mutation appears to be independent of a specific upstream polymorphism in contrast to the second most common mutation, c.2097_2098insT (p.Ile700TyrfsX42), that arose on the basis of the c.-137 T>C c.-53 C>G polymorphic constellation. Our results indicate that the exonic PEX1 mutation correlates with patient survival, while the two 5 polymorphisms analysed in this study do not seem to be of diagnostic and/or prognostic value. Both 5 polymorphisms have been described as 5 UTR polymorphisms. The PEX1 5 UTR was reported to extend up to c.-245. To experimentally confirm the transcriptional start point of PEX1, we conducted a 5 RACE. The longest 5 extension of cDNA that could be identified by this approach extended to c.-120. Our study suggests that only the PEX1 5 polymorphic site at c.-53 is part of the 5 UTR while the polymorphism 137 bp upstream of the PEX1 start codon might be part of the PEX1 promoter.