Characterisation of regulatory regions of the PRNP gene and their effect on susceptibility to sporadic and variant Creutzfeldt-Jakob Disease

• Main Author: McCormack, James E.
• Published: University of Edinburgh 2001
• Subjects: 616.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.666128

Based on analysis of PrP gene regulatory regions in other species, 4.8 Kb of human genomic sequence containing 1.5Kb of upstream sequence the first exon and 3.1Kb of intron was cloned from a human genomic lambda library into a CAT reporter gene. Transient transfections in human neuroblastoma cells of this construct and of nested deletions of this construct identified two regulatory regions, one between -43 and -9 bases upstream of the transcription initiation site and one within the intron between +292 and +622 basepairs. Having identified regulatory regions in neuroblastoma cells the effect of the two regulatory regions on expression <i>in vivo</i> was examined by making three constructs where expression of a LacZ reporter gene was driven by the 4.8Kb human fragment, the upstream and exon sequence or the exon and intron sequence. A construct which contained 3.4Kb of equivalent murine sequence was used as a control. Although all four constructs were successfully injected into fertilised eggs giving rise to transgenic embryos and adults, no lacZ expression was detected suggesting that additional sequences may be required for full expression of <i>PRNP.</i> The upstream and intronic regions were sequenced in sCJD, vCJD and control individuals to determine if any polymorphism or mutations exist in the regulatory regions which cause or affect susceptibly to either form of CJD. Three polymorphisms were identified, a C to G transversion at position - 101 relative to the start of exon one was found at an allele frequency of 13% in control individuals, a G to C transversion at +310 found at a frequency of 6% in controls and a T to C transition at position +385 found at a frequency of 5% in controls. Analysis of the frequency of these polymorphisms in CJD patients and controls showed that the - 101G and +310C alleles each independently increase an individual's risk of developing sporadic CJD. The effect of -101G allele was strongest in PrP ORF codon 129 methionine homozygotes even when the linkage between -101G and codon 129 methionine was accounted for. the +385C allele increased susceptibility to vCJD but this was not significant.