Phenotypic Heterogeneity of Variably Protease-Sensitive Prionopathy: A Report of Three Cases Carrying Different Genotypes at PRNP Codon 129

Abstract

Variably protease-sensitive prionopathy is an exceedingly rare, likely underestimated, sporadic prion disease that is characterized by heterogeneous and often non-specific clinical and pathological features posing diagnostic challenges. We report the results of a comprehensive analysis of three emblematic cases carrying different genotypes at the methionine (M)/valine (V) polymorphic codon 129 in the prion protein gene (PRNP). Clinical, biochemical, and neuropathological findings highlighted the prominent role of the host genetic background as a phenotypic modulator. In particular, the PRNP codon 129 showed a remarkable influence on the physicochemical properties of the pathological prion protein (PrP^Sc), especially on the sensitivity to proteinase K (PK) digestion (VV > MV > MM), which variably affected the three main fragments (i.e., of 19, 17, and 7 kDa, respectively) comprising the PrP^Sc profile after PK digestion and immunoblotting. This, in turn, correlated with significant differences in the ratio between the 19 kDa and the 7 kDa fragments which was highest in the MM case and lowest in the VV one. The relative amount of cerebral and cerebellar PrP mini-plaques immunohistochemistry showed a similar association with the codon 129 genotype (i.e., VV > MV > MM). Clinical manifestations and results of diagnostic investigations were non-specific, except for the detection of prion seeding activity by the real-time quaking-induced conversion assay in the only cerebrospinal fluid sample that we tested (from patient 129VV).

Keywords: CJD; Creutzfeldt-Jakob disease; PRNP; RT-QuIC; VPSPr; amyloid; prion disease; protein misfolding; real-time quaking-induced conversion; scrapie.

Figure 1

Neuropathological features of VPSPr 129MM (Case#1). (A,B) Spongiform change with vacuoles of “intermediate” size in the cerebellum molecular layer and occipital cortex. (C) Activated astrocytes in the occipital cortex. (D,E) Marked microglial activation involving both the occipital gray and white matter. (F) PrP deposits in the occipital cortex: on the left, the upper left box shows delicate, patchy granular PrP deposits in the superficial cortical layers, while the lower box small plaque-like PrP deposits in the deep layers. (G) Granular PrP deposits surrounding the vacuoles (occipital cortex). (H,I) Small plaque-like PrP deposits in the entorhinal cortex and cerebellar molecular layer. Haematoxylin-eosin staining (A,B), and immunochemistry for GFAP (C), HLA (D,E), and PrP (F–I). Legend: s, superficial; d, deep.

Figure 2

Neuropathological features of VPSPr 129MV (Case#2). (A) Mild spongiform change in the occipital cortex. (B) Lack of spongiform change in the cerebellum. (C–E) Severe astrocytic (the lower box shows a detail at higher magnification) and microglial activation in the parietal gray (C,D) and white matter (E). (F–H) PrP dot-like and mini-plaque deposits in the occipital cortex (F,G) and thalamus (H). (G) PrP mini-plaque that was surrounded by a cluster of fine granules. (I) Aβ core plaque in the frontal cortex. (K) Cerebral Aβ angiopathy in the meningeal vessels of occipital lobe. Haematoxylin-eosin staining (A,B), and immunochemistry for GFAP (C), HLA (D,E), PrP (F–H), and Aβ (I,K).

Figure 3

Neuropathological features of VPSPr 129VV (Case#3). (A,B) Mild spongiform change that was characterized by a mixture of small and intermediate size vacuoles in the occipital cortex and cerebellum. (C,D) Astro- and microglial activation in the temporal gray and occipital white matter, respectively. (E,F) PrP granular and mini-plaque deposits in the occipital cortex. (G) Clusters of dot-like PrP deposits in the medial thalamus. On the left, the lower box shows a detail at higher magnification. (H) Diffuse Aβ deposits in the frontal cortex. (I) Tau-positive globular and flame-shape neurofibrillary tangles and neuropil threads in the entorhinal cortex. (K) Fuzzy tau-immunoreactive astrocytes in the occipital cortex. Haematoxylin-eosin staining (A,B), and immunochemistry for GFAP (C), HLA (D), PrP (E–G), Aβ (H), and p-tau (I,K).

Figure 4

Immunoblotting of proteinase K (PK)-untreated and -treated PrP from VPSPr affected brains. (A) Comparison of the electrophoretic profile of full-length PK-untreated PrP in total homogenate (TH) and purified (P3) samples from the VPSPr cases carrying 129VV, 129 MM, and 129 MV. The membrane was probed with the polyclonal PrP 23-40 antibody. (B) The electrophoretic profiles of proteinase K-treated TH from VPSPr cases before (left) and following PNGase treatment (right), which revealed the different pattern of unglycosylated fragments among the three cases. The membranes were probed with mAb 3F4. A sCJD VV2 case was included as a control. Approximate molecular masses are in kilodaltons. M, methionine; V, valine; D, diglycosylated; M, monoglycosylated; U, unglycosylated; Ctrl, control.

Figure 5

Analysis of PrP^Sc protease-resistance by PK titration. Brain homogenates from 129VV, 129MM, and 129MV cases were run after digestion with increasing amounts of PK to highlight the variable PK resistance of the PrP^Sc fragments that are associated with the three codon 129 genotypes. In the 129VV case, we used lower PK concentrations than in 129MM and 129MV because of the higher sensitivity of PrP^Sc to PK digestion, as demonstrated by the more significant signal loss in the three points of the curves with identical PK levels (i.e., 0.250 U/mL, 0.500 U/mL, 1.00 U/mL). Note the striking difference in the degree of PK resistance of the 19 kDa fragment among the three cases. The membranes were probed with mAb 3F4. Approximate molecular masses are in kilodaltons. Lane 1 sCJD VV2; Lane 12 sCJD MM1; Lane 18 sCJD VV2. M, methionine; V, valine.