Previous studies have addressed the issue of PrPSc
types 1 and 2 co-occurrence in sCJD. Most of them raised the question of the influence of the number of cases and brain areas analyzed and emphasized the possibility that the co-occurrence of PrPSc
types 1 and 2 is underestimated [13
]. On the other hand, the use of a novel, potentially very sensitive approach, later shown to have pitfalls related to the detection of unspecific bands generated by partially digested PrPSc
], likely led other investigators to overestimate the incidence of the concurrent PrPSc
]. Thus, the overall results on the phenomenon of the coexistence of molecular and clinico-pathological sCJD subtypes are at present inconclusive with respect to incidence, effect on disease phenotype and criteria for disease classification. To contribute to the full understanding of these issues, in the present study, we combined a systematic analysis of several brain regions in a large series of case including all codon 129 genotypes and the rarest phenotypes with the use of a refined methodology for the detection of the PrPSc
type concurrence, which provides good sensitivity combined with high specificity [25
After screening about 4,200 samples from a largely consecutive series of 200 cases, we estimated that PrPSc
types 1 and 2 coexist in about 35% of sCJD cases, which is overall consistent with figures from some of the previous studies [13
] in which the number of cases and areas analyzed were significantly lower. This finding supports the idea that PrPSc
types co-occurrence involves a relevant but limited group of sCJD subjects and indicates that the incidence of the phenomenon had not been significantly underestimated.
As far as the characteristics of the CJD population with mixed phenotypes are concerned, our data show that the PrPSc
types 1 and 2 co-occur more frequently in the MM than in the MV and VV genotypes. More specifically, the large majority of sCJD cases with concurrent PrPSc
types combines features of the MM and MM 2C sCJD subtypes, in variable proportions. Most commonly, in such cases, the MM1 phenotype is predominant over the MM 2C phenotype, but the opposite situation also rarely occurs. The latter results significantly differ from those obtained in most previous studies. Indeed, Head et al. [13
] mainly found a focal type 1 co-occurrence in MM and MV subjects with dominant type 2, Schoch et al. [40
] detected the mixed protein types mostly in MV2 cases showing the type 1 only focally in subcortical areas, and Uro-Coste et al. [43
] mainly detected a random co-occurrence of type 1 in MV or VV cases with dominant type 2. Given that only our study was based on a large series of consecutive cases, we attribute such heterogeneity of previous results to case selection biases, although methodological differences may also have contributed [43
Since subjects with mixed PrPSc types represent a significant proportion of the sCJD population, show distinctive phenotypic features, and potentially represent a distinct subtype in terms of biological relevance, it is important that they are properly identified and are added as new subtypes in the current sCJD classification (Table ).
Nomenclature and classification of sCJD subtypes
Despite the emphasis on molecular features of current sCJD classification, it has become increasingly clear that PrPSc
typing alone, when limited to a single or even a few brain samples, fails to provide an accurate classification in a significant proportion of cases. This is mainly related to the focal nature of the “mixed features” in many sCJD cases with PrPSc
types 1 and 2 concurrence. Indeed, we would have misclassified the disease subtype in about 27.5% (using 3F4) of cases with MM or MV genotype, if we had analyzed PrPSc
only in the frontal cortex, the area more commonly used for typing worldwide. For the same reason, discrepancies may arise when PrPSc
-typing and PrP immunohistochemistry are performed from individual samples taken from opposite hemispheres or even adjacent cortical gyri. However, our study shows that the regional deposition of either type 1 or type 2 when concurrent is not random and that a relatively limited number of critical brain structures must be assessed to reach an accurate classification. Furthermore, our results further underline the importance of applying both molecular and neuropathological assessment for sCJD subtype classification. In this regard, the lack of detection of PrPSc
type 2 in a minority of MM subjects, despite the presence of a mixed synaptic and perivacuolar pattern of PrP deposition, indicates that when type 2 is very focal or limited in amount, histopathologic examination is more sensitive in identifying such cases than PrPSc
typing, at least when only the 3F4 antibody is used. Given the very strong correlation in MM subjects between PrPSc
type 2 detection and the large “grape-like” vacuoles and the perivacuolar pattern of PrP deposition on histopathologic examination, which is in line with results previously obtained in other studies [18
], we propose that these cases are classified as MM 1+2C or MV 1+2C even without the final proof of type 2 detection by western blot. Alternatively, PrPSc
typing using the antibody 1E4 was in our hand as sensitive as the histopathologic examination in the detection of cases with very focal type 2.
In the light of the present results, the most important regions to be assessed pathologically include the cerebral cortex from each of the 4 lobes, the striatum, hippocampus, thalamus and cerebellum. The cerebellum, in particular, is critical for the recognition of the synaptic pattern of PrP deposition as marker of PrPSc type 1 concurrence in the cases with dominant type 2.
Taken together, our data indicate that a protocol including the neuropathologic assessment of the eight brain regions mentioned above and PrPSc typing in four critical regions such as the temporal, parietal and occipital neocortices, and medial thalamus is strongly recommended for a reliable sCJD group classification addressing the issue of mixed phenotypes. Indeed, by applying this protocol instead of examining all 21 brain regions, we would have reached the same classification of cases in the present series.
We also wish to underline the importance of identifying correctly the sCJD cases with mixed features for transmission purposes. Indeed, the question of whether the concurrence of PrPSc
types 1 and 2 in CJD reflects a co-infection by two prion strains related to specific undiscovered human genotypes, or determined by epigenetic factors remains unanswered and will largely rely on transmission studies in which the careful selection of samples will be of critical importance. Concerning this critical question, we find intriguing that the large, confluent vacuoles and the perivacuolar pattern of PrPSc
deposition, we originally linked to sCJD MM 2C are also found in a subgroup of MV 2K subjects in addition to MM/MV 1+2C. In addition, we have described here the same morphological features in one case of fatal insomnia (i.e. the MM2-thalamic subtype or MM 2T) which adds to two previously reported cases [19
]. Thus, it seems that large confluent vacuoles and the perivacuolar pattern of PrPSc
deposition may be found in sCJD associated with all phenotypes linked to MM or MV at codon 129. Although this observation remains difficult to interpret at present, it appears relevant for our future understanding of the molecular basis and the extent of strain variation in sCJD. In any case, our observation strongly suggests that the phenomenon of mixed phenotypes in sCJD goes beyond PrPSc
types 1 and 2 coexistence and also involves subtypes which shares the same PrPSc
type. This, in turn, further underlines the importance of combining histopathological assessment and biochemical PrPSc
typing for sCJD subtype characterization.
The present data also show that the association of two PrP27-30 fragments, which does not represent a bona-fide type 1 and 2 concurrence, may also be a feature of some sCJD cases. Thus, the PrP27-30 profile in VV2 cases in the cerebellum, thalamus and midbrain is sometime characterized by a doublet comprising a 18.5 kDa in addition to the typical 19 kDa band, while the western blot profile of PrP27-30 in the MV 2K cases appears almost invariably characterized by the association of two PrPSc
core fragments including a classic 19 kDa type 2 band and a slower migrating band of about 20 kDa. Although these profiles truly represent concurrent PrPSc
fragments, and the 20 and 18.5 kDa fragments likely reflect specific PK cleavage sites, the 20 and 18.5 kDa bands are distinguished from the type 1 and type 2 fragments because, at least to date, they were never detected independently from types 1 and 2, and are not markers of specific clinico-pathological phenotypes. Knowledge of these regional variations is nonetheless important to avoid misinterpreting a PrPSc
profile as novel when only one brain region is analyzed [21
Finally, the results obtained from the analyses of lesion profiles and clinical features in the subgroups of sCJD cases with mixed features deserve further comment. By showing that the relative “load” of each of the two PrPSc types significantly correlates with disease duration, the relative frequency of certain symptoms, and the ratio between cortical and cerebellar pathology, our study provides further strong evidence for the PrPSc type being a major biological determinant in human prion disease.
In conclusion, the present data add to our knowledge of the prevalence and phenotypic spectrum of the sCJD variants with mixed molecular and pathological features, provide an updated molecular classification of the disease subtypes and will serve for future epidemiologic and transmission studies aimed at disclosing the etiology and extent of strain variation in sCJD.