Polymorphisms Of The PRNP Gene In Chinese Populations And The Pathogenic Mechanism Research Of A Novel Insertion Mutant Of PrP

Transmissible spongiform encephalopathy(TSE) is a group of rare, sub-acute, fatal neurodegenerative diseases in human beings and animals, including scrapie, mad cow disease, Creutzfeld-Jacob disease(CJD) and so on. Based on the protein only hypothesis, TSE is caused by the conversion of a normal cellular prion protein (PrP~C) into a pathogenic, infectious isoform, which has been commonly referred to as scrapie prion protein (PrP~Sc). Human TSEs or prion diseases are described as infectious, inherited, and sporadic form. Approximately 10-15% of the human prion disease is inherited. The majority of the cases of human prion diseases (85%) occur sporadically by an unknown mechanism. Certain polymorphisms in the PRNP have been associated with the incidence or susceptibility of prion disease, such as the two most common polymorphisms, M129V and E219K. The distributions of M129V, E219K genotypes and alleles in various general populations show clear differences between Western Europeans and East Asian.To understand the genotypes and alleles frequencies of PRNP in Chinese populations and monitor the incidence of prion diseases in China, we screened a total number of 626 individuals, which represent three ethnic populations of China, Han, Hui, and Uyghur for M129V and E219K. The frequencies of Met/Met homozygote in these three groups differ significantly. The Han has a much higher frequency (98%) than Uyghur (85%) and Hui (60%). On the other hand, the frequencies of the Glu/Glu at residue 219 are higher in Uyghur (98%) and Hui (96%) than in Han (90%). This result show the higher frequency of 129Met homozygotes in Chinese than in Europeans, which may suggest that Chinese populations are at increased risk of developing prion diseases. We also investigated two other less common variants of -PRNP, a silent substitution at residue 117 (351A>G: A117A), and one octapeptide-repeat deletion mutation (1-OPRD) in the octapeptide-coding region. We found three Uyghur individuals with silent substitution at residue 117. Four Huiindividuals (2.0%) and one Han (0.5%) donor were found to be heterozygous for 1-OPRD.In the course of these studies, we found a healthy girl in Hui ethinic group with a novel three extra-repeat (72 bp) insertion within the octapeptide repeats coding region of PRNP, and named this mutation with PrP8G Sequencing results show that the most likely sequence of repeats in this case is Rl, R2, R2, R2a, R2, R2, R3, R4, with the extra repeats of R2a, R2, R2 inserted between normal R2 and R3 repeats. Identical mutation is also found in her mother but not in her father or a half-brother with a different mother. Three months later, Grasbon-Frodl et al. reported a similar mutation associated with a CJD patient. We also diagnosed a patient of fatal familial insomnia (FFI) by sequencing PRNP and found the D178N point mutation linked with the 129Met allele in this case. With exception of the Chinese FFI family that had been reported in Canada, this is the first FFI case found in China. Attempts have been made to collect as many blood samples and medical records as possible from these two family. Hopefully, we shall be able to establish a more extensive genealogy and follow the clinical development of each individual with the novel insertion mutation or Dl 78N point mutation.To investigate the pathogenic mechanism of the novel insertion, PCR was used to amplify the PrP23-23i gene from pMD-huPrP and pMD-huPrP8G plasmids, which contain the full length PRNP sequence of wild type and mutation respectively. The amplified fragments were inserted into pET30a and expressed in E.coli. The full-length mature huPrP and huPrP8G proteins were purified and refolded on column by using immobilized metal (Ni) affinity chromatography, based on the metal binding property of their unusual octarepeat sequences containing at least five tandem copies. The identification of purified proteins by SDS-PAGE and mass spectrum shows the purity is more than 95% and the Mr of huPrP and huPrP8G proteins are 22961 and 25292Da respectively. Different biochemical and biophysical properties were found between the wild type and mutant. Firstly, huPrP8G was much easier to aggregate than the wt. When the proteins were diluted from the same concentrations, the OD595 of huPrP8G increased much more than that of huPrP by 9.4 times in the buffer ofpH7.0 and by 17 times in pH7.5. Secondly, by SDS-PAGE without DTT, we found more dimers in huPrP8G than in huPrP. Furthermore, the mutant protein seemed to be more stable than the wt in acid circumstance when incubated in 37°C. These results indicate that huPrP8G mutant protein may be easy to converse into PrPSc-like structure compared with the wild type.