666 Fig. 4 a Western blot proﬁles of PrPSc (3F4 antibody) from different brain regions of the sCJD VV 2?1 subject with the more signiﬁcant type 1 deposition. Areas shown include the middle frontal gyrus (MFG), middle temporal gyrus (MTG), parietal cortex (PC), lateral occipital cortex (OCC2), insular cortex (INS), putamen nucleus (PUT), medial thalamic nuclei (TH1), and cerebellum (CE). Approximate molecular masses are in kilodaltons. b Western blot proﬁles of PrPSc doublets in sCJD VV2, sCJD MM 1?2 and sCJD MV 2K. Lanes 1 and 2 show the association of a *18.5 kDa fragment with the typical 19 kDa type 2 band in sCJD VV2 cerebellum (CE, V = vermis, H = hemisphere). Approximate molecular masses are in kilodaltons Fig. 5 Relative severity of lesions (i.e. spongiform degeneration and astrogliosis) in the cerebral cortex (mean of scores in neocortical and limbic cortical areas) and cerebellum: comparison between MM/MV 1 (n = 65), MM/MV 1?2C (n = 63), MM/MV 2C?1 (n = 9), and MM/MV 2C (n = 4) sCJD groups. The line slope expresses the ratio between the degree of cortical and cerebellar pathology. Group MM/ MV 1?2C* includes only the cases within the MM/MV 1?2C group showing type 2 in at least 5 areas (n = 17) affected cortical areas (Fig. 7). Among subcortical struc- tures, the thalamus most often showed the perivacuolar staining, whereas the brainstem and to a greater extent the cerebellum (Fig. 7) were the areas with the lowest frequency. The pathological phenotype of MM/MV 2C?1 largely overlapped to that of MM/MV 2C subjects. However, as a 123 Acta Neuropathol (2009) 118:659–671 distinctive feature from the pure MM/MV 2C phenotype, all these cases had a synaptic pattern of PrP staining in the cerebellum, which well correlated with the presence of PrPSc type 1 on western blots. The lesion proﬁle and the pattern of PrP staining in all 6 VV subjects with concurrent PrPSc types (VV2?1) were very similar to those of typical VV2 sCJD cases. As the only distinctive feature, the two cases with the most sig- niﬁcant type 1 accumulation showed a less consistent laminar pattern of spongiform degeneration in the cerebral cortex and a milder cerebellar pathology when compared to VV2 cases with similar disease duration. Lastly, the two MV 2K?1 cases had no clear distin- guishing pathological features although they both showed a relatively mild pathology and the lack of a laminar pattern of spongiform degeneration in the cerebral cortex. Of notice, however, we observed a perivacuolar pattern of PrPSc deposition co-localizing with the presence of larger vacuoles in 6 out of 25 of the sCJD MV 2K subjects (Fig. 6c, d) as well as in one MM2-thalamic (MM 2T) case. The regional distribution of the perivacuolar staining was not distinguishable from that observed in MM/MV 1?2C and mainly involved samples from the cerebral cortex (data not shown). Molecular-phenotypic correlations: clinical features MM/MV 1?2C and MM/MV 2C?1 subjects showed a signiﬁcant difference in both disease duration and fre- quency of symptoms at onset according to the relative load