Abstract
Prion diseases are a family of unique fatal transmissible neurodegenerative diseases that affect humans and many animals. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease in humans, accounting for 85–90% of all human prion cases, and exhibits a high degree of diversity in phenotypes. The etiology of sCJD remains to be elucidated. The human prion protein gene has an octapeptide repeat region (octarepeats) that normally contains 5 repeats of 24–27 bp (1 nonapeptide and 4 octapeptide coding sequences). An increase of the octarepeat numbers to six or more or a decrease of the octarepeat number to three is linked to genetic prion diseases with heterogeneous phenotypes in humans. Here we report that the human octarepeat region is prone to either contraction or expansion when subjected to PCR amplification in vitro using Taq or Pwo polymerase and when replicated in wild type E. coli cells. Octarepeat insertion mutants were even less stable, and the mutation rate for the wild type octarepeats was much higher when replicated in DNA mismatch repair-deficient E.coli cells. All observed octarepeat mutants resulting from DNA replication in E.coli were contained in head-to-head plasmid dimers and DNA mfold analysis (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form) indicates that both DNA strands of the octarepeat region would likely form multiple stable hairpin structures, suggesting that the octarepeat sequence may form stable hairpin structures during DNA replication or repair to cause octarepeat instability. These results provide the first evidence supporting a somatic octarepeat mutation-based model for human sCJD etiology: 1) the instability of the octarepeat region leads to accumulation of somatic octarepeat mutations in brain cells during development and aging, 2) this instability is augmented by compromised DNA mismatch repair in aged cells, and 3) eventually some of the octarepeat mutation-containing brain cells start spontaneous de novo prion formation and replication to initiate sCJD.
Highlights
Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a unique family of fatal neurodegenerative diseases that affect both humans and animals
We showed that the wild type 5-repeat octarepeat region (Oct5) in the human PRNP gene is unstable during PCR amplifications with a mutate rate of 0.9%
In the XL-1 Red E.coli cells where all three major DNA mismatch repair genes were defective, Oct5 had a mutation rate of 0.8%, 11 times higher than that in DH5a; Oct11b had a mutation rate of 3.0%, which is higher than the 1.3% mutation rate found in DH5a the difference did not reach statistical significance
Summary
Transmissible spongiform encephalopathies (TSEs), are a unique family of fatal neurodegenerative diseases that affect both humans and animals. A large number of point mutations in the PRNP coding region have been linked to inherited prion diseases with diverse phenotypes: familial CJD (fCJD), GSS, fatal familial insomnia (FFI), and mixed phenotypes [1]. Some of the insertion mutants contain novel variant repeats (Figure 1A) that may have resulted from recombination between wild type repeats [3]. These pathogenic PRNP mutations are believed to cause prion diseases by rendering the corresponding mutant PrP protein more prone to adopting a prion-associated conformation [4,5]
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