Genetic basis of Creutzfeldt-Jakob disease in the United Kingdom: a systematic analysis of predisposing mutations and allelic variation in the PRNP gene

Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disorder characterized by the accumulation of aggregates of a cellular protein, PrP, in the brain. In both human and animals, genetic alterations to the gene encoding PrP (PRNP in human) modulate susceptiblity to CJD. The recent epidemic of bovine spongiform encephalopathy in the UK has raised the possibility of transmission from animal produce to humans. To provide a baseline against which to assess possible risk factors, we have determined the frequencies of predisposing mutations and allelic variants in PRNP and their relative contributions to disease. Systematic PRNP genotype analysis was performed on suspected CJD cases referred to the National Surveillance Unit in the UK over the period 1990–1993. Inspection of 120 candidate cases revealed 67 patients with definite and probable CJD, based on clinical and neuropathological criteria. No PRNP mutations were detected in any of the remaining 53 patients assessed as “non-CJD”. A disease-associated mutation in the PRNP gene was identified in nine (13.4%) definite and probable cases of CJD, a reliable estimate of the incidence of PRNP-related inherited CJD based on a prospective epidemiological series. Within the group of sporadic CJD patients (lacking PRNP mutations), we confirmed that the genotype distribution with respect to the common methionine/valine (Met/Val) polymorphism at codon 129 within PRNP was significantly different from the normal Caucasian population. The incidence of Met homozygosity at this site was more than doubled and correlated with increased susceptibility to the development of sporadic CJD. Unlike other recent studies, Val homozygosity was also confirmed to be a significant risk factor in sporadic CJD, with the relative risks for the three genotypes Met/Met:Val/Val:Met/Val being 11:4:1. Received: 18 December 1995 / Revised: 19 January 1996     

BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein

Variant Creutzfeldt-Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrP(Sc) type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.     

Cross-sequence transmission of sporadic Creutzfeldt-Jakob disease creates a new prion strain

The genotype (methionine or valine) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of abnormal isoform of PrP (PrP(Sc)) are major determinants of the clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD). Here we found that the transmission of sCJD prions from a patient with valine homozygosity (129V/V) and type 2 PrP(Sc) (sCJD-VV2 prions) to mice expressing human PrP with methionine homozygosity (129M/M) generated unusual PrP(Sc) intermediate in size between type 1 and type 2. The intermediate type PrP(Sc) was seen in all examined dura mater graft-associated CJD cases with 129M/M and plaque-type PrP deposits (p-dCJD). p-dCJD prions and sCJD-VV2 prions exhibited similar transmissibility and neuropathology, and the identical type of PrP(Sc) when inoculated into PrP-humanized mice with 129M/M or 129V/V. These findings suggest that p-dCJD could be caused by cross-sequence transmission of sCJD-VV2 prions.     

Sporadic—but Not Variant—Creutzfeldt-Jakob Disease Is Associated with Polymorphisms Upstream of PRNP Exon 1

Human prion diseases have inherited, sporadic, and acquired etiologies. The appearance of the novel acquired prion disease, variant Creutzfeldt-Jakob disease (vCJD), and the demonstration that it is caused by the same prion strain as that causing bovine spongiform encephalopathy, has led to fears of a major human epidemic. The etiology of classical (sporadic) CJD, which has a worldwide incidence, remains obscure. A common human prion-protein-gene (PRNP) polymorphism (encoding either methionine or valine at codon 129) is a strong susceptibility factor for sporadic and acquired prion disease. However, a quantitative-trait-locus study of prion incubation periods in mice has demonstrated an important factor that is close to Prnp but is independent of its coding sequence or that of the nearby prion-like doppel gene (Prnd). We have analyzed the PRNP locus for such tightly linked susceptibility factors. Fifty-six polymorphic sites have been identified within 25 kb of the PRNP open reading frame, including sites within the PRNP promoter and the PRNP 3' untranslated region. These have been characterized in 61 Centre d'Etude du Polymorphisme Humain (CEPH) families, demonstrating extensive linkage disequilibrium around PRNP and the existence of 11 major European PRNP haplotypes. Haplotype frequencies estimated in healthy U.K. control individuals were very similar to those deduced in the CEPH families. A common haplotype was overrepresented in patients with sporadic CJD (sCJD). Through use of a log-linear modeling approach to simultaneously model Hardy-Weinberg and linkage disequilibria, a significant independent association was found between sCJD and a polymorphism upstream of PRNP exon 1 (P=.005), in addition to the strong susceptibility conferred by codon 129 (P=2x10(-8)). However, although our sample size was necessarily small, no association was found between these polymorphisms and vCJD or iatrogenic CJD, in keeping with their having distinct disease mechanisms. In addition, there was no evidence of a PRNP founder effect in the first reported geographical cluster of vCJD.     

PRNP contains both intronic and upstream regulatory regions that may influence susceptibility to Creutzfeldt-Jakob Disease

The Prion protein (PrP) plays a central role in Creutzfeldt-Jakob Disease (CJD) and other transmissible spongiform encephalopathies (TSEs). Mutations in the protein coding region of the human PrP gene (PRNP), which have been proposed to alter the stability of the PrP protein, have been linked to a number of forms of TSE. However, the majority of CJD cases are not associated with mutations in the PRNP coding region and alternative mechanisms must therefore underlie susceptibility to these forms of CJD. Transgenic mice, that over- or under-express PrP genes, have shown a correlation between the level of PrP gene expression and the incubation time of disease. Polymorphisms that lead to alterations in human PRNP gene expression, could therefore be candidates for influencing susceptibility of an individual to CJD. In order to investigate this hypothesis, we have defined an upstream and intronic regulatory region of the PRNP gene. Sequencing of these regions in controls, sporadic CJD (sCJD) and variant CJD (vCJD) patients has identified three polymorphisms, all of which are more common in sCJD patients than controls. Our data suggests that polymorphisms in the regulatory region of the PRNP gene may be a risk factor for CJD.     

Molecular genetics of human prion diseases in Germany

Human prion diseases may be acquired as infectious diseases, they may be inherited in an autosomal dominant fashion or occur sporadically. Mutations and polymorphisms in the sequence of the coding region of the prion protein gene (PRNP) have been established as an important factor in all of these three types of prion diseases. Therefore, a total of 578 patients with suspect prion diseases referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit over a period of 4.5 years have been examined for mutations and polymorphisms in the coding region of PRNP. We found 40 cases with a missense mutation previously reported as pathogenic. Amongst these, the aspartate to asparagine change at codon 178 (D178N) was the most common mutation. All of these cases carried the D178N mutation in coupling with methionine at codon 129, resulting in the typical fatal familial insomnia (FFI) genotype. Most cases with pathogenic mutations were not found in the group of clinically "probable" cases according to established clinical criteria, supporting the notion that inherited prion diseases often exhibit atypical features. Two novel missense mutations (T188R and P238S) and several silent polymorphisms were found, demonstrating the quality of our screening procedure based on a modified version of the single-stranded conformational polymorphism technique. In "definite" CJD cases with no pathogenic mutation, the patients clinically classified as "probable" were mostly homozygous for methionine at the common polymorphism at codon 129, whereas there was a marked over-representation of patients homozygous for valine amongst those clinically classified as "possible". This large study on suspect cases of human prion diseases in Germany clearly shows that PRNP genetics is essential for a comprehensive analysis of prion diseases.     

Heterozygous inhibition in prion infection

The human PrP gene (PRNP) has two major polymorphic codons: 129 for methionine (M) or valine (V), and 219 for glutamate (E) or lysine (K). The PRNP heterozygotes appear to be protected from sporadic CJD compared to the PRNP homozygotes. The molecular mechanism responsible for these protective effects of PRNP heterozygosity has remained elusive. In this review, we describe the inhibition of PrP conversion observed in a series of transmission studies using PRNP heterozygous animal models. In vCJD infection, the conversion incompetent human PrP 129V molecules showed an inhibitory effect on the conversion of human PrP 129M molecules in the 129M/V heterozygous mice. Furthermore, though the human PrP 219E and PrP 219K were both conversion competent in vCJD infection, these conversion competent PrP molecules showed an inhibitory effect in the 219E/K heterozygous animals. To explain this heterozygous inhibition, we propose a possible mechanism designated as the stone fence model.     

Novel differences between two human prion strains revealed by two-dimensional gel electrophoresis

The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP(Sc)), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrP(Sc) type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrP(Sc) in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrP(Sc) form is overrepresented and carries glycans that are different from those present in the PrP(Sc) of sFI. Because the altered glycans are detectable only in the PrP(Sc) and not in the normal or cellular PrP (PrP(C)), they are likely to result from preferential conversion to PrP(Sc) of rare PrP(C) glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.     

Methionine 129 variant of human prion protein oligomerizes more rapidly than the valine 129 variant: implications for disease susceptibility to Creutzfeldt-Jakob disease

The human PrP gene (PRNP) has two common alleles that encode either methionine or valine at codon 129. This polymorphism modulates disease susceptibility and phenotype of human transmissible spongiform encyphalopathies, but the molecular mechanism by which these effects are mediated remains unclear. Here, we compared the misfolding pathway that leads to the formation of beta-sheet-rich oligomeric isoforms of the methionine 129 variant of PrP to that of the valine 129 variant. We provide evidence for differences in the folding behavior between the two variants at the early stages of oligomer formation. We show that Met(129) has a higher propensity to form beta-sheet-rich oligomers, whereas Val(129) has a higher tendency to fold into alpha-helical-rich monomers. An equimolar mixture of both variants displayed an intermidate folding behavior. We show that the oligomers of both variants are initially a mixture of alpha- and beta-rich conformers that evolve with time to an increasingly homogeneous beta-rich form. This maturation process, which involves no further change in proteinase K resistance, occurs more rapidly in the Met(129) form than the Val(129) form. Although the involvement of such beta-rich oligomers in prion pathogenesis is speculative, the misfolding behavior could, in part, explain the higher susceptibility of individuals that are methionine homozygote to both sporadic and variant Creutzfeldt-Jakob disease.     

Protease-sensitive conformers in broad spectrum of distinct PrPSc structures in sporadic Creutzfeldt-Jakob disease are indicator of progression rate

The origin, range, and structure of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD), are largely unknown. To investigate the molecular mechanism responsible for the broad phenotypic variability of sCJD, we analyzed the conformational characteristics of protease-sensitive and protease-resistant fractions of the pathogenic prion protein (PrP(Sc)) using novel conformational methods derived from a conformation-dependent immunoassay (CDI). In 46 brains of patients homozygous for polymorphisms in the PRNP gene and exhibiting either Type 1 or Type 2 western blot pattern of the PrP(Sc), we identified an extensive array of PrP(Sc) structures that differ in protease sensitivity, display of critical domains, and conformational stability. Surprisingly, in sCJD cases homozygous for methionine or valine at codon 129 of the PRNP gene, the concentration and stability of protease-sensitive conformers of PrP(Sc) correlated with progression rate of the disease. These data indicate that sCJD brains exhibit a wide spectrum of PrP(Sc) structural states, and accordingly argue for a broad spectrum of prion strains coding for different phenotypes. The link between disease duration, levels, and stability of protease-sensitive conformers of PrP(Sc) suggests that these conformers play an important role in the pathogenesis of sCJD.     

Beyond PrP9res) type 1/type 2 dichotomy in Creutzfeldt-Jakob disease

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrP(res)) identified on Western blotting (type 1 or type 2). These biochemically distinct PrP(res) types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrP(res) in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain areas from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrP(res) and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrP(Sc)) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrP(res) were identified. Despite this, the other two biochemical assays found that PrP(Sc) from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrP(Sc) subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrP(res) pattern. The identification of four different PrP(Sc) biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrP(res) isoform provides an alternative biochemical definition of PrP(Sc) diversity and new insight in the perception of Human TSE agents variability.     

vCJD prion acquires altered virulence through trans-species infection

Variant Creutzfeldt-Jakob disease (vCJD) appears to be caused by infection with the bovine spongiform encephalopathy (BSE) agent. To date, all patients with vCJD are homozygous for methionine at codon 129 of the PrP gene. To investigate the relationship between polymorphism at codon 129 and susceptibility to BSE or vCJD prions, we performed splenic follicular dendritic cell assay with humanized knock-in mice through peripheral infection. All humanized knock-in mice showed little or no susceptibility to BSE prions. Only the subset of humanized knock-in mice with codon 129 Met/Met genotype showed weak susceptibility by Western blotting. Surprisingly, we succeeded in the transmission of vCJD prions to humanized knock-in mice not only with codon 129 Met/Met but also with codon 129 Met/Val. Humanized knock-in mice with codon 129 Val/Val were not susceptible. The results suggest that human heterozygotes at codon 129 are also at risk for secondary infection with vCJD.     

Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type

The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrPSc) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two-dimensional immunoblot profiles of detergent insoluble and proteinase K-resistant PrP species in a type of sporadic Creutzfeldt-Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full-length and truncated PrP forms present in the insoluble fractions were also separately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrPSc, but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent-insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two-dimensional blots of the proteinase K-resistant PrP fraction suggesting that different PrP species are selected for conversion to proteinase K-resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N-linked glycans. We also show that the over-representation of the highly glycosylated forms distinctive of the proteinase K-resistant PrPSc of vCJD in one-dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases.     

Molecular genetic studies of Creutzfeldt-Jakob disease

Genetic study of over 200 cases of Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI), and kuru have brought a reliable body of evidence that the familial forms of CJD and all known cases of GSS and FFI are linked to germline mutations in the coding region of the PRNP gene on chromosome 20, either point substitutions or expansion of the number of repeat units. No pathogenic mutations have so far been found in sporadic or infectious forms of CJD, although there are features of genetic predisposition in iatrogenic CJD and kuru. In FFI and familial CJD, clinically and pathologically distinct syndromes that are both linked to the 178^Asp→Asn substitution, phenotypic expression is dependent on a polymorphism at codon 129. Synthetic peptides homologous to several regions of PrP spontaneously form insoluble amyloid fibrils with unique morphological characteristics and polymerization tendencies. Peptides homologous to mutated regions of PrP exhibit enhanced fibrilogenic properties and, if mixed with the wild-type peptide, produce even more abundant and larger fibrous aggregates. A similar process in vivo may lead to amyloid accumulation and disease, and transmission of “baby fibrils” may induce disease in other hosts.     

Human prion diseases in the Netherlands (1998-2009): clinical, genetic and molecular aspects

Prion diseases are rare and fatal neurodegenerative disorders that can be sporadic, inherited or acquired by infection. Based on a national surveillance program in the Netherlands we describe here the clinical, neuropathological, genetic and molecular characteristics of 162 patients with neuropathologically confirmed prion disease over a 12-year period (1998-2009). Since 1998, there has been a relatively stable mortality of Creutzfeldt-Jakob disease (CJD) in the Netherlands, ranging from 0.63 to 1.53 per million inhabitants per annum. Genetic analysis of the codon 129 methionine/valine (M/V) polymorphism in all patients with sporadic CJD (sCJD) showed a trend for under-representation of VV cases (7.0%), compared with sCJD cohorts in other Western countries, whereas the MV genotype was relatively over-represented (22,4%). Combined PrP(Sc) and histopathological typing identified all sCJD subtypes known to date, except for the VV1 subtype. In particular, a "pure" phenotype was demonstrated in 60.1% of patients, whereas a mixed phenotype was detected in 39.9% of all sCJD cases. The relative excess of MV cases was largely accounted for by a relatively high incidence of the MV 2K subtype. Genetic analysis of the prion protein gene (PRNP) was performed in 161 patients and showed a mutation in 9 of them (5.6%), including one FFI and four GSS cases. Iatrogenic CJD was a rare phenomenon (3.1%), mainly associated with dura mater grafts. Three patients were diagnosed with new variant CJD (1.9%) and one with variably protease-sensitive prionopathy (VPSPr). Post-mortem examination revealed an alternative diagnosis in 156 patients, most commonly Alzheimer's disease (21.2%) or vascular causes of dementia (19.9%). The mortality rates of sCJD in the Netherlands are similar to those in other European countries, whereas iatrogenic and genetic cases are relatively rare. The unusual incidence of the VV2 sCJD subtype compared to that reported to date in other Western countries deserves further investigation.     

Association between the PRNP 1368 polymorphism and the occurrence of sporadic Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare transmissible neurodegenerative disorder. The etiology of sporadic form of CJD remains unsolved. In addition to the codon 129 polymorphism, polymorphisms in the non-coding region of PRNP are considered as important factors in sCJD development. To assess a possible association between PRNP 1368 SNP and sCJD, we compared the genotype, allele and haplotype frequencies of the 1368 SNP among 46 sCJD patients of Dutch origin with the respective frequencies in healthy controls. We detected a significant association between sCJD and 1368T/T genotype. A significant difference was also observed in 1368 alleles’ distribution. In the haplotype analysis, haplotype 1368C-129G was associated with decreased risk of sCJD in Dutch population. Our findings support the hypothesis that genetic variations in the regulatory region of the PRNP gene may influence the pathogenesis of sCJD.     

Association between the PRNP 1368 polymorphism and the occurrence of sporadic Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare transmissible neurodegenerative disorder. The etiology of sporadic form of CJD remains unsolved. In addition to the codon 129 polymorphism, polymorphisms in the non-coding region of PRNP are considered as important factors in sCJD development. To assess a possible association between PRNP 1368 SNP and sCJD, we compared the genotype, allele and haplotype frequencies of the 1368 SNP among 46 sCJD patients of Dutch origin with the respective frequencies in healthy controls. We detected a significant association between sCJD and 1368T/T genotype. A significant difference was also observed in 1368 alleles’ distribution. In the haplotype analysis, haplotype 1368C-129G was associated with decreased risk of sCJD in Dutch population. Our findings support the hypothesis that genetic variations in the regulatory region of the PRNP gene may influence the pathogenesis of sCJD.     

Familial fatal insomnia with atypical clinical features in a patient with D178N mutation and homozygosity for Met at codon 129 of the prion protein gene

ABSTRACT. Familial fatal insomnia (FFI) is fatal disorder characterized by damage to select thalamic nuclei, together with progressive insomnia and dysautonomia. In subjects carrying the D178N prion protein (PRNP) mutation, distinct phenotypes can be observed, depending on the methionine (Met) /valine (Val) codon 129 polymorphism. We report here a Chinese case of FFI with a D178N/Met129 genotype of the PRNP gene, who exhibited rapidly progressive dementia combined with behavioral disturbances and paroxysmal limb myoclonus. Our patient did not show refractory insomnia early in the disease course, nor demonstrate typical MRI and EEG alterations. There was remarkable family history of similar symptoms.KEYWORDS: D178N, Familial fatal insomnia, Met129, prion protein      

Polymorphisms of the prion protein gene (PRNP) in Hanwoo (Bos taurus coreanae) and Holstein cattle

Polymorphisms in the prion protein gene (PRNP) in humans and sheep correlate with susceptibility to transmissible spongiform encephalopathies (TSEs). Bovine spongiform encephalopathy (BSE) has been reported in British and Japanese cattle; it has occurred thus far in Holstein cattle. BSE in Hanwoo (Bos taurus coreanae) cattle has not been diagnosed up to now. To characterize the bovine PRNP polymorphisms in Korean cattle, we analyzed the open reading frame (ORF) of PRNP in 120 Hanwoo (beef) cattle and 53 Holstein (dairy) cattle. Three polymorphisms were found, the third position of codon 78 (G-->A), the third position of codon 192 (C-->T), and the deletion of a single octa-repeat. An analysis of codon 78 revealed no difference in the genotype (P = 0.2026) or allele (P = 0.7180) frequencies between Hanwoo and Holstein animals. However, there were significant differences in the genotype (P < 0.0001) and allele (P < 0.0001) frequencies at PRNP codon 192 between Hanwoo and Holstein animals. The rate of Holstein animals with deletion of a single octa-repeat was 91.5% undeleted homozygotes, 8.5% heterozygotes (with R3 deletion), and 0% deleted homozygotes. However, none of the 120 Hanwoo animals had any octa-repeat deletions. The genotype (P < 0.0001) and allele (P < 0.0001) frequencies of a single octa-repeat-deletion were also significantly different between Hanwoo and Holstein animals.     

Cellular isoform of the prion protein PrPc in human intestinal cell lines: genetic polymorphism at codon 129, mRNA quantification and protein detection in lipid rafts

The cellular isoform of the normal prion protein PrP(c), encoded by the PRNP gene, is expressed in human intestinal epithelial cells where it may represent a potential target for infectious prions. We have sequenced the PRNP gene in Caco-2 and HT-29 parental and clonal cell lines, and found that these cells have a distinct polymorphism at codon 129. HT-29 cells are homozygous Met/Met, whereas Caco-2 cells are heterozygous Met/Val. The 129Val variant was also detected in Caco-2 mRNAs. Real-time PCR quantifications revealed that PrP(c) mRNAs were more expressed in HT-29 cells than in Caco-2 cells. These data were confirmed by studying the expression of PrP(c) in plasma membranes and lipid rafts prepared from these cells. Overall, these results may be important in view of using human intestinal cell lines Caco-2 and HT-29 as cellular in vitro models to study the initial steps of prion propagation after oral inoculation.