670 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 trans- mission 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 undis- covered 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, 30, 31]. 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 character- ized 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 frag- ments 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]. 123 Acta Neuropathol (2009) 118:659–671 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 pro- vides 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. Acknowledgments We wish to thank Barbara Polischi and Sabrina Boninsegna for her technical assistance. We also thank all the phy- sicians who provided clinical data and helped in the collection of tissues and all family members who consented to the use of tissue for research. This study was funded in the frame of the bilateral Italy (ISS)–USA (NIH, Ofﬁce for Rare Diseases) agreement on joint research on rare diseases, by the European Commission (FOOD-CT- 2004-506579), the Italian Ministry of University, Research and Technology (FIRB-2003-RBNE03FMCJ_006), the Federal Ministry of Health (ZV2-1369-340): grant PHS P30 AG010133, and the Gino Galletti Foundation. Conﬂict of interest statement The authors declare that they have no conﬂict of interest. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References 1. Baron T, Biacabe AG, Arsac JN, Benestad S, Groschup MH (2007) Atypical transmissible spongiform encephalopathies (TSEs) in ruminants. Vaccine 25:5625–5630 2. Barron RM, Thomson V, Jamieson E et al (2001) Changing a single amino acid in the N-terminus of murine PrP alters TSE incubation time across three species barriers. EMBO J 20:5070– 5078 3. Bessen RA, Marsh RF (1994) Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol 68:7859–7868 4. Bolton DC, McKinley MP, Prusiner SB (1982) Identiﬁcation of a protein that puriﬁes with the scrapie prion. Science 218:1309– 1311 5. Brown P, Coker-Vann M, Pomeroy K et al (1986) Diagnosis of Creutzfeldt–Jakob disease by Western blot identiﬁcation of marker protein in human brain tissue. N Engl J Med 314:547–551 6. Bruce ME, McConnell I, Fraser H, Dickinson AG (1991) The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. J Gen Virol 72:595–603