Molecular basis of phenotypic variability in sporadc creudeldt-jakob disease

Molecular Basis of Phenotypic Variability in Sporadc Creudeldt-Jakob Disease

Piero Parchi, MD,* Rudolph Castellani, MD,* Sabina Capellari, MD,* Bernardino Ghetti, MD,t§ Katherine Young, PhD,? Shu G. Chen, PhD,* Martin Farlow, MD,* Dennis W. Dickson, MD,’ Anders A. F. Sima, MD, PhD,** John Q. Trojanowski, MD, PhD,?? Robert B. Petersen, PhD,*

and Pierluigi Gambetti, MD*

We sequenced the prion protein gene and studied the biochemical characteristics and the intracerebral distribution of protease-resistant prion protein with Western blot and immunohistochemistry in 19 cases of sporadic Creutzfeldt-Jakob disease. We identified four groups of subjects defined by the genotype at codon 129 of the prion protein gene, the site of a common methioninelvaline polymorphism, and two types of protease-resistant prion proteins that differed in size and glycosylation. The four Creutzfeldt-Jakob disease groups showed distinct clinicopathological features that corre- sponded to previously described variants. The typical Creutzfeldt-Jakob disease phenotype or myoclonic variant and the Heidenhain variant were linked to methionine homozygosity at codon 129 and to “type 1” protease-resistant prion protein. The atypical and rarer variants such as that with dementia of long duration, the ataxic variant, and the variant with kuru plaques were linked to different genotypes at codon 129 and shared the “type 2” protease-resistant prion protein. Our data indicate that the sporadic form of Creutzfeldt-Jakob disease comprises a limited number of variants. The methionine/valine polymorphism at codon 129 of the prion protein gene and two types of protease-resistant prion proteins are the major determinants of these variants. These findings suggest the existence of prion strains in humans and provide the molecular basis for a novel classification of sporadic Creutzfeldt-Jakob disease.

Parchi P, Castellani R, Capellari S, Ghetti B, Young K, Chen SG, Farlow M, Dickson DW, Sima AAF, Trojanowski JQ, Petersen RB, Gambetti P. Molecular basis of phenotypic variability

in sporadic Creutzfeldt-Jakob disease. Ann Neurol 1996;39:767-778

Jakob in 1921 [I] originally described Creutzfeldt- Jakob disease (CJD) as a progressive dementing illness. It is one of the human prion diseases, which include the Gerstmann-Straussler-Scheinker disease, kuru, and fatal familial insomnia [2-41. Prion diseases are, for the large part, transmissible and are characterized by cerebral deposition of an abnormal protease-resistant isoform of a membrane-bound glycoprotein called prion protein (PrP) [2-41. Gerstmann-Straussler- Scheinker disease and fatal familial insomnia are familial diseases linked to a mutation in the PrP gene (PRNP); kuru is acquired only by transmission; and CJD includes sporadic, familial, and iatrogenically transmitted forms [2-41. The sporadic form is the most frequent, accounting for approximately 85% of all human prion diseases [2-4].

Sporadic CJD is typically characterized by rapidly progressive dementia, myoclonus, periodic sharp-wave

electroencephalographic (EEG) activity, and widespread spongiform degeneration. However, variations in clinical presentation, disease duration, as well as type and distribution of lesions have been consistently observed [4-71. Visual disturbances precede the cognitive decline in the Heidenhain variant [8-lo], and cerebellar signs characterize the onset of the cerebellar variant [ 1 1 - 171. The duration of the disease also varies greatly, ranging from a few weeks to several years [18, 191. Variations in the type and distribution of histopathology that cannot be ascribed to disease duration have also been observed. They include the presence of Congo red-positive, sharply demarcated amyloid plaques [20-241 and prominent involvement of the white matter, characteristic of the panencephalopathic variant [25-281.

The molecular basis of the phenotypic variability of sporadic CJD is largely unknown. Codon 129 of

From the *Division of Neuropathology, Institute of Pathology, Received Nov 8, 1995, and in revised form Dec 19, 1995, and Jan Case Western Reserve University, Cleveland, OH; the $Department 16, 1996. Accepted for publication Jan 16, 1996. Of and Genetics, eDepartment Of Address correspondence to Dr Gambetti, Division of Neuropathol-

ogy, Institute of Pathology, Case Western Reserve University, and §Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN; the ’Department Cleveland, OH 44 106, of Pathology, Albert Einstein College of Medicine, Bronx, NY; the **Department of Pathology, University of Michigan Medical Cen- ter, Ann Arbor, M1; and the ??Department of Pathology and Labo- ratory Medicine, University of Pennsylvania, Philadelphia, PA.

Copyright 0 1996 by the American Neurological Association 767

PRNP, the site of a common methionine/valine ( M N ) polymorphism, affects the clinical and pathological features of familial prion diseases [29, 301 a n d probably also plays a role in the phenotypic expression of sporadic CJD [31-331. Moreover, in experimental scrapie, an animal prion disease, the phenotype is not determined by the host genotype alone. Different pr ion strains transmit diseases to syngeneic animals that differ in topography, type oflesion, a n d intracerebral distribution of the protease-resistant PrP (PrPres) [34-361. Strain diversity is at- tributed to different conformations of PrPrer that can be replicated by nongenetic mechanisms [37]. PrPres heterogeneity, independent of the PRNP genotype, may con- tribute to the phenotypic variability of sporadic CJD.

To elucidate the phenotypic heterogeneity of CJD, we examined the PRNP genotype a n d the characteristics and the intracerebral distribution of PrPrer i n 19 subjects with sporadic CJD. The correlation of the molecular data with those obtained from the clinical and pathological examinations led us to propose a novel classification of the disease.

Materials and Methods Patients Nineteen subjects were selected from a group of patients diagnosed as having CJD, possible prion disease, or atypical dementia. The criteria for selection were (a) presence of PrPrea on Western blots of brain homogenates; (b) lack of mutation in the PRNP coding sequence; (c) no family history of the disease and no known exposure to prion contamination; and (d) availability of medical records and tissues for clinical, pathological, and biochemical evaluation.

PRNP Genotype Determination Genomic DNA was extracted from frozen brain tissue [38]. The PRNP coding sequence was amplified and sequenced according to Khorana and colleagues [33], using previously described primers and reagents [38].

Tissue Preparation Brains were removed at autopsy and either one half or selected pieces of tissue were immediately frozen and stored at -80OC. The remaining tissue was fixed in formalin and used for neuropathological examination and PrP immunohistochemistry. The tissue blocks used for histopathological examination included the cerebral cortex from each of the lobes, the hippocampus and entorhinal cortex, basal ganglia, thalamus, hypothalamus, midbrain, pons, medulla, and cerebellum. For PrP immunohistochemistr, blocks of tissue from the frontal and occipital cortices, striatum, thalamus, midbrain, and cerebellum were used. For the biochemical studies, frozen coronal sections from one hemisphere, the brainstem, and the cerebellum were available from 12 CJD subjects. Samples of gray matter were obtained from the following regions: frontal (middle frontal gyrus), parietal (infe- rior parietal lobule), and temporal (middle temporal gyrus) cortices (considered also as the anterior cortex); occipital or posterior cortex (calcarine cortex and lateral occipital gyrus);

hippocampus (CAI and subiculum); limbic cortex (entorhinal cortex and anterior cingulate gyrus); striatum (caudate nucleus and putamen); thalamus (mediodorsal and ventrolat- era1 nuclei); hypothalamus; brainstem (substantia nigra, midbrain periaqueductal gray matter, locus ceruleus, medullar periventricular gray matter); and cerebellum (hemisphere and vermis). White matter from the cerebral (subcortical) and the cerebellar hemispheres was also obtained. In 5 other subjects, all of the above-cited brain samples except those from the brainstem were obtained. Finally, in 2 subjects, only small frozen blocks of tissue from the cerebral cortex were available and in these subjects, the protein studies (see below) were limited to the analysis of the biochemical characteristics of PrPrc'.

Histopathological Examination A semiquantitative evaluation of spongiosis, neuronal loss, and gliosis was carried out in the same brain regions sampled for the biochemical studies by blindly comparing hematoxylin and eosin-stained sections from the affected subjects with corresponding sections from 5 age-matched subjects with no history of neurological disorders and no histopathological changes.

Prio n Protein Im m u nohistochemistry Paraffin sections were processed for PrP"' immunostaining after hydrolytic autoclaving [ 401. The sections were deparaf- finized, rehydrated, and immersed in 38% formic acid for 1 hour at room temperature. Endogenous peroxidase was blocked by immersion in 8% hydrogen peroxide in methanol for 10 minutes. Sections were completely immersed in 1.5 mM hydrochloric acid and autoclaved at 121°C for 10 minutes. After rinsing, they were incubated with the mouse monoclonal antibody 3F4 [41] (1 :ZOO), which recognizes the human PrP residues 103-112; washed; and incubated with bridge antibody (goat antimouse, Cappel, 1 : 50) fol- lowed by incubation with mouse PAP complex (Sternberger, Meyer Immunocytochemicals, 1 : 250). Diaminobenzidine tetrahydrochloride was used to visualize the immunoreactivity.

Western Blot o f Brain Extracts Quantitative Western blot analysis was carried out as previously described 1421. Briefly, from each brain sample approximately 100 mg of tissue (or less in case of small anatomical structures) was homogenized in 9 volumes of lysis buffer, and aliquots after proteinase K (PK) or PK and PNGase digestion (equivalent to 0.3 mg of wet tissue) were resolved on 12% polyacrylamide gels and immunoblotted. The monoclonal 3F4 (1 : 50,000) was used as primary antibody. The blots were developed by the enhanced chemilumines- cence system (ECL, Amersham). Quantitative analysis was carried out with a computer-assisted laser scanner (LKB, UI- troscan XL) as previously described [42]. Serial dilution of one of the samples, chosen as standard, was included in each blot. Using the linear curve generated from the standard sample and multiple exposure times, we obtained quantitative data for each sample relative to the standard.

768 Annals of Neurology Vol 39 N o 6 June 1996

+ + + + - - - - PNGas e 39.5 - 1 7 . 5 - 18.5 -

1 2 3 4 5 6 7 8

CODON 129 WM MM MV W MM MV W

Fig 1. Protease-resistant prion protein f i a p e n t s ( P r P ) from brain extracts (cingulate cortex) o f 4 subjects with sporadic Creutzfeldt-jakob disease (CJD) representative of the four groups. A j e r proteinase K treatment, P r P migrates as three major isoforms (lanes 1-4). N-Deglycosylation results in a single PrP-immunoreactive band (lanes 5-8) that corresponds in mobility to the fdstest migrdting isoform before deglycosylation. Therefore, the three PrP" isoforms are accounted f o r by the degree of glycosylation, with the most rapidly migrating form being unglycosylated. Two types of P r P that dzffer in size and glycosylation (see Table I ) were detected P r P type 1 (lanes 1 and 5) was seen in I 1 subjects homozygous fDr methionine a t PRNP codon 129, whereas P r P gpe 2 (lanes 2-4 and 6-8) was observed in 2 methionine homozygotes and in all the heterozygotes and valine homozygotes.

Results PRNP Genotype Determination Thirteen subjects were methionine (MIM) homozygous at codon 129, 3 were heterozygous (MIV), and 3 were valine (VIV) homozygous. Together, codon 129 homozygotes accounted for 83% of all cases. One subject carried one copy of a silent polymorphism at codon 117, and another showed a 24-bp deletion polymorphism in the octapeptide repeat region.

Protease-Resistant Prion Protein Characteristics Two types of PrPres (type 1 and type 2) were detected and differed in electrophoretic mobility and ratio of the differently glycosylated P r P isoforms (Fig 1, Table 1). PrPres type 1 was found in 11 of the 13 MIM homozygous subjects (MIM1); the smaller PrP"' form, type 2, was found in the other 2 MIM homozygotes (MlM2) and in all MIV (MlV2) and VIV (VlV2) subjects. No significant variation in the pattern of electrophoretic mobility of each type of PrPrrs was seen either among the different brain regions or between the early (e.g., at biopsy) and terminal stages of the disease (data not shown). Thus, this characteristic of PrPrrs is highly consistent, not influenced by the severity of histopathology and not due to partial proteolysis.

Types of PrPTes differing in size and glycosylation have been found in animal [37, 431 and familial human prion diseases [44]. The size variation follows different patterns of N-terminal trimming by proteases [43, 441. The findings are consistent with the distinct

Table I . Characteristics of Protease-Resistant Prion Protein Q p e 1 and Type 2"

Size (kd)" 20.5 +- 0.3 (n = 25) Glycoform'

Upper 22.8 I 3 (n = 110) (19.3 t 3'-27 i 3')

Lower 47.1 t 3 40.9 5 2d (43.2 Z 2'-51.5 2 3')

Unglycosylated 30.1 i 3 25.1 i 5" (28.9 t 4'-33.6 2 3')

18.7 F 0.2" (n = 25)

34 i 4' (n = 80) (30.6 ? 3g-39 ? 4h)

(39.7 t 4"-41.5 2 3')

(20.4 5 3"-27.6 t 2')

Range

Range

Range

"Areas examined are described in Methods. hMean ? SD following deglycosylation performed as previously described 1441. 'Mean ? SD of percent of total accounted for by each form. ' p < 10 ' (Mann-Whitney test). 'Midbrain (periaqueductal gray). fCingulate gyrus. SOccipital cortex. hLocus ceruleus. 'Hippocampus. 'Cerebellum.

isoforms having different protein conformations or specific ligand interactions. The different glycoform ratios suggest an uneven conversion of the three PrP glyco- forms into those of the two PrP"'s types.

Clinical Findings All 11 MIM homozygotes with PrPrCs type 1 had symptoms for less than 6 months and 9 of the 11 had cognitive impairment as the first symptom (Table 2). Severe visual loss of central origin (posterior visual pathway) preceded dementia in 1 patient and accompanied the cognitive decline in another. These patients were clini- cally diagnosed as having the Heidenhain variant of CJD. In 2 patients, gait ataxia was also observed ar onset and in 2 others it occurred later. Myoclonus or periodic sharp waves on EEG were present in all subjects.

The 2 MIM homozygotes with PrPre' type 2 had a slower course than did the MIM homozygotes with PrPres type 1 (see Table 2). Both patients showed progressive dementia (diagnosed as possible Alzheimer's disease in l), without sustained myoclonus or periodic sharp waves on EEG. Cerebellar signs were mild or absent.

The 3 heterozygotes had symptoms of variable duration (see Table 2). In all, signs at onset and during the disease progression implicated both cortical and subcortical, including cerebellar, structures. Periodic sharp waves on EEG were absent.

All 3 VIV homozygous subjects had variable symptom duration and presented with cerebellar signs, whereas dementia appeared later (see Table 2). Periodic sharp waves on EEG were observed in only 1 subject late in the course.

Parchi et al: Molecular Basis of Sporadic CJD 769

Table 2. Clinical Features o f the Four Groups of Subjects with Sporadic Creutzfeldt-jakob Disease

Subject Age : ~ t On5et Ilur.ition (mu)' Signs at Onsrt Evolution uf Signs Electroencci,balograph~ GW"p No. Sex (yr)

M I M with PIP"' type I 1-1 1 6Ml5F Mat i : 66 Mean: 3 Llenirntia," 9111; Dementia, 1111 I ; myoilonus, I I l l I ; Periodic sharp waves, Rmgc: 44-Rh Range: 1.5-5.0 visual,L L / l I ; 10/11

ataxic gait. 21 1 I 411 I , ceizurrs, 2/11; stuporlcoma, 11111

decreased speech; mild gait disturbance

occarivndl rnyoclnnus; later rigidity and seizurei; no cerebellar sign,

MIM with PrP"' type 2 12 F 77 12 Dementia" TL)cmentia, rigidity, dysphagid, and Diffuse 40wing, tr.tnsienr \pika

13 F 75 36 I)emcnria" TI>emcntia. apraxia, and anomia; Diffuse slowing

M I V with I'rP"" type 2 14 M 61 5 ~ c m m i i a ~ ataxic ?'Ataxia, dysinIma, dmmrnria, Dilfusc slowing

15 F 76 11 Dementia, visuorpaiial deieit, Tataxia Uiffiisr slawlng I 6 F 5 5 18 AI Suhjecr 14 arid myoclonus Diffuse slowng

V/V with I'rP"' type 2 17 F: 67 4 Ar. 'LU' :. gall ?Ataxia. dysarrhria, mild dementia Difhsc slowing 18 M 52 8 Visual,' ataxic gait TAraxia, dysarrhria, dysphagia, Difiusr slowing

nystagmus, dementia I 9 F 5 0 20 Ataxic gait As Subject I8 and vcgrrativr stare Uiffiisr slowing,

( a h 10 mo) prriodic sharp

giii liallucinntioiis

waveh (at 10 n o )

'((:alculated from the timr of presrntanon uf signc mdic.itivr uf an org:inic disensc of the nervous system "lncludcs conforion, diroiienratiun, and niemo~y loss. 'Reduccd visual acuiry of c r n t r ~ l origin. "Subjective signiticnnt reduciron of toral slcrp rime. 'Diplopia.

f = increase in severity; PIP'' = prutcasc-resistant p m n prorein.

Histopnthological Fentures All MIM homozygotes with PrP'" type 1 showed mild to moderate spongiosis and gliosis with mild or no vi- sually detectable neuronal loss in the cerebral cortex, basal ganglia, thalamus, and molecular layer of the cerebellum (Fig 2A, Table 3). In the cerebral neocortex, vacuolation involved all layers and was more pro- nounced in the occipital lobe in 8 subjects. In contrast, the hippocampus, hypothalamus, and brainstem were virtually spared. The white matter was unremarkable.

In the 2 M/M homozygotes with PrPrCs type 2, the lesions were similar in distribution to those of the first group but, with the exception of the cerebellum, were more severe (see Fig 2C, Table 3). In the subject with symptoms for the longest duration, the spongiform degeneration was replaced by status spongiosus to the ex-

tent that the pathology was not easily recognized as that of CJD.

The 3 heterozygotes had widespread lesions in the hippocampus and subcortical structures, including the brainstem and hypothalamus. The involvement of the cerebral cortex appeared to be a function of symptom duration as the neocortex was almost unaf- fected in Subject 14 after having symptoms for 5 months, whereas it was severely involved in those with symptoms for longer than 10 months. Cerebellar pathology was distinctive in this group, with kuru plaques in the superficial granular cell layer and only mild spongiosis or astrogliosis regardless of duration (see Fig 2E, Table 3) .

The 3 VIV homozygotes were similar to the heterozygotes in demonstrating widespread subcortical in-

b Fig 2. Histopatholoa and patterns qf prion protein (PrP) immunoreactivity in the four C/O groups (see Discussion for classifca- tion of the groups). (A) Moderate spongijorm degeneration with astrogliosis (hematoxylin and eosin, X 25) and (B) j n e , punctate pattern of PrP imhunoreactivity (x 31.5) in the cerebral cortex . f a CJDM/Ml subject. (C) Status spongiosus o f the superficial layers with severe neuronal loss and astrogliosis (hernatoxylin and eosin, X 12.5) and ( 0 ) coarse immunoreactivity, ofen located at the rim o f the large vacuoles (X 31.5) in the cerebral cortex . f a CJDMIM2 subject. Note the diference between t h e j n e vacuoles that characterize the spongiform degeneration (A) and the course and confluent vacuoles of the status spongiosus (C). (E) Kuru-like plaque at the edge of the granular cell layer (Congo red, X 50) and (fl plaquelike pattern of immunoreactivity largely conjined to the granular cell layer of the cerebellum of a CJDM/V2 subject (X 31.5). The majoriv of the plaquelike deposits stain negatively for hematoxylin and eosin and Congo red. (G) Moderate spong;form degeneration in the molecular layer associated with granular cell loss and severe astrogliosis in the cerebellum (hematoqlin and eosin, X 12.5) and (H) laminar pattern of PrP immunoreactivity in the deep layers of the cerebral cortex (X 31.5) in a C]DV/VZ subject. (All magnifications are be@re 2096 reduction.)

770 Annals of Neurology Vol 39 No 6 June 1996


T,zble 3. Histopatbology in Four Sporadic Cseutzfeldt-]akob Disease (CJD) Subjects Representative o f the Four Groupsa

Group, Subject No., Symptom Duration

Brain Region CJDMIMl, 7, 4 mo CJDMIM2, 13, 36 mo CJDMIV2, 14, 5 mo CJDVIV2, 17, 4 mo -

Frontotemporal cortex Occipital cortex Limbic cortex Hippocampus Striatum Thalamus Hypothalamus Brainstem Cerebellum

SSIG SS/N/CG SS/N/GG 0 SSIG S/N/GG 0 0 S I C ,

~

SS/NN/GG SSINNIGG SS/ NN/ GG S/G SSSINIG SSINNIGG G G G

S (> D) S (D) SS/N/GG (> D) SSIG SSSIG SSIGG SS/GG SS/GG S/G (KP)

s (D) 0 SS/N/GG (> D) SS/G SSSIG SSlGG SSIGG SS/GG SS/ NN / GG

'Severity of lesions is expressed as mild (one symbol), moderate (two symbols), and severe (three symbols). See Discussion for explanation of group classifications.

0 = no significant pathology; S, N, and G = spongiosis, neuronal loss, and astrocytic gliosis, respectively; SS = status spongiosus; (KP) = amyloid, kuru-like plaques; (D) = pathology limited or (> D) prevalent in the deep cortical layers.

volvement and, in the subject with symptoms for the shortest duration, relative sparing of the cerebral neocortex. However, they showed more severe cerebellar pathology with moderate to severe spongiosis of the molecular layer and prominent neuronal loss and astrogliosis in the granular layer (see Fig ZG, Table 3). They also lacked Congo red-positive plaques.

The V/V homozygotes and heterozygotes also differed from the MIM homozygotes in the following features: (a) Spongiosis in the cerebral cortex was located predominantly in the deep layers with laminar distribution in the subjects with symptoms of short or intermediate duration (up to 13 months) (see Table 3) and (b) the occipital cortex was relatively spared compared to the other lobes.

Prion Protein hnmunobistocbemist~ Overall PrP immunoreactivity showed a clear parallel with the distribution of the spongiform degeneration, with the only exception of the granular cell layer of cerebellum which displayed immunoreactivity in the absence of spongiosis. All MIM homozygotes with PrP"' type 1 showed a fine, punctate pattern of staining with occasional enhancement around the perikaryon of small neurons (see Fig 2B). Rarely, the immunostaining was coarse (see below).

The MIM homozygotes with PrP"' type 2 displayed a predominantly coarse immunoreactivity, often located at the rim of large vacuoles, in regions with the most severe pathology (see Fig 2D). Plaques were absent in both groups of M/M homozygotes.

In the cerebrum of the 3 heterozygotes, PrP immunostaining revealed numerous plaquelike deposits of PrP that were Congo red negative, except for a few in the subject with the symptoms for the longest duration. The punctate pattern was also present with strong en-

hancement around the perikaryon and dendritic arbori- zations of neurons. In the cerebellum, immunostaining was unique, consisting of only plaquelike deposits predominantly in the granular layer (see Fig 2F).

In the 3 VIV homozygotes, PrP immunostaining of the cerebrum showed predominantly the punctate pattern with strong neuronal peridendritic and perikaryal enhancement, although plaquelike foci were also seen. In the cerebellum, the molecular and granular cell layers displayed intense and widespread punctate immunoreactivity and the granular cell layer showed also the plaquelike pattern, although kuru plaques were absent (see results on histopathology).

Finally, PrP immunoreactivity in both heterozygotes and VIV homozygotes with symptoms for a short or intermediate duration (up to 13 months) showed a laminar distribution in the deep layers of the cerebral cortex which colocalized with the spongiform degeneration (see Fig 2H).

Proteuse-Resistmt Prim Protein Regional Distribution In all subjects, PrPrrs was detected in all gray matter samples examined and the amount of the protein corre- lated with the severity of the histopathology, particularly with that of spongiosis (Figs 3, 4; see Table 3).

The MIM homozygotes with PrPrcs type 1 had the highest amount of PrP"' in the cerebral cortex and slightly less in the striatum, thalamus, and cerebellum, while the protein was barely detectable in the hippocampus, hypothalamus, and brainstem or in the white matter (see Figs 3A, 4A). The M/M homozygotes with PrPre' type 2 showed a similar distribution but almost twice the amount of PrPreS, with the exception of the cerebellum, which on the contrary displayed less PrPrCs (see Figs 3A, 4B).


CJD-MIM1 (n = 10, disease duration 3 t 1.5 mos.) CJD-MIM2 (n = 2, disease duration 12 and 36 mos.)

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CJD-MIV (n = 2) and CJD-VIV (n = 1) with long duration (5 and 4 mos.)

(13 2 5 mos.)

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Fig 3. Regional brain distribution o f protease-resistant prion protein (PrPres, PrP' ) in 17 patients with sporadic CJD. The areas analyzed are described in the Methods section. The amounts of PrPr" are expressed in arbitrary units obtained from densitometric analysis as previously described [42]. The value of 100 refers to the highest amountpreviously obtained in FFI subjects [42]. Two major patterns of PrP" distribution were observed: In the C]DM/M1 0) and CJDMM2 groups (see Discussion f i r group classijcations), P r P preferen- tially accumulated in the cerebral cortex, striatum, and thalamus. In addition, the CJDM/MI group showed a signijcant accumulation in the cerebellum. (B) The C]DM/V2 and the C]DV/V2 subjects with symptoms f i r less than 6 months, instead, had a more widespread accumulation o f the abnormal protein that affected all gray matter areas except the cerebral neocortex. PrP" was present in signijcant amounts in the neocortex in the subjects with longer symptom duration, indicating that in these subjects, contrary to the M/M patients, PrP'" accumulated in the neocortex late in the disease course.

PrPreC distribution was similar in M/V heterozygotes and V/V homozygotes and it was much affected by symptom duration (see Fig 3B). The subjects with symptoms for less than 6 months, in contrast to the M/M homozygotes, had the largest amounts of PrPre' in the subcortical regions and little in the neocortex, where PrPres was seen more in the frontal lobe than in the occipital cortex (see Figs 3B, 4C). With a symptom duration longer than 6 months, PrPres also accumulated in relatively large amounts in the neocortex (see Figs 3B, 4D), suggesting a different timing of PrPre' formation between subcortical and cortical areas.

Discussion Previous classifications of sporadic CJD were based on clinical and pathological features [4-71. We have now applied, for the first time, data derived from analysis of PRNP codon 129 and PrPres and showed that in sporadic CJD, distinct clinical and pathological features and the pattern of PrPres deposition correlate with the PRNP codon 129 haplotype and the size of the PrPres fragment that accumulates in the brain. We propose a novel classification of the disease based on these criteria. The classification includes four groups: (1) 129M/M homozygotes with PrP"' type 1, desig- nated CJDM/Ml; (2) 129M/M homozygotes with PrPrea type 2 (CJDM/M2); (3) 129M/V heterozygotes with Pipres type 2 (CJDM/V2); and (4) 129V/V homozygotes with PrP'" type 2 (CJDVN2).

The main clinical features of the CJDM/M 1 phenotype are rapid course, early dementia, myoclonus, and periodic sharp waves on EEG (Table 4). Pathologically, this group is characterized by mild to moderate spongiosis and astrogliosis in the cerebral cortex, especially the occipital lobe, striatum, thalamus, and cerebellar cortex, while the brainstem, hippocampus, and hypothalamus are virtually spared. The pattern of PrP immunostaining is predominantly of the finely punctate or "synaptic" type and the highest amount of PrPrCs is present in the areas showing spongiform degeneration. This group, the most common in our series, overlaps with typical CJD of the myoclonic type [5-71 and also includes the Heidenhain variant. The similarities between the Heidenhain variant and the typical CJD phenotype were previously noted [5-71. Our findings extend these observations and indicate that the two groups also share the same molecular determinants and the pattern of intracerebral deposition of PrPres. The apparent lack of visual disturbances in most CJDMI M 1 patients, despite the prominent occipital pathology and PrP'" accumulation, might be explained by the early dementia [5, 71.

The CJDM/M2 phenotype is associated with symptoms of longer duration, without sustained myoclonus or periodic sharp waves on EEG (see Table 4). Other differences include more severe histopathology, more


Amount 300 430 470 260 340 25 so zoo 3 2 . 5 -

2 7 . 5 -

18.5 -

A r e a FC OC EC CA TH HYP PAG CE A

Amount 90 30 20 480 400 520 680

32.5 - 27.5 -

18.5 -

Area FC TC OC EC CA SN CE C

~~

Amount 730 650 770 580 400 80 so 7 5

33.5 - 2 7 . 5 -

18.5 - Area FC OC EC CA TH HYP PAG CE

B

Amount 7 0 0 750 600 740 600 400 520

32.5 -

2 7 . 5 -

18.5 -

Area FC TC OC EC CA SN CE D

Fig 4. Western immunoblots o f gray matter homogenates after proteinase K treatment, fiom different brain regions of 4 subjects representative of the four sporadic CJD groups (see Discussion for group cLassi5cations): fiontal cortex (FC), temporal cortex (TC), occipital cortex (OC), entorhinal cortex (EC), caudate nucleus (CA), mediodorsal thalamic nuclew (TH), hypothalamus (HYP), substantia nigra (SN), periaqueductal gray matter (PAG), and cerebellum (CE). Each lane was loaded with a volume of sample equivalent to 0.3 mg o f wet brain tissue. Molecular mass standards (in kilodaltons) are indicated at the left, and the amounts of the proteae-resistant prion protein, calculated afier densitometric analysis (see Methods section), are indicated on the top of each immunoblot. (A) Patient 7 of the CJDMMI group (gmptoms f o r 4 months). (B) Patient 13 o f the CJDM/M2 group (symptoms for 36 months). (C) Patient 17 of the CJDV/V2 group (symptoms f i r 4 months). ( 0 ) Patient 16 o f the CJDM/V2 group (rymp- toms for I8 months).

Table 4. Genotypic and Phenotypic Features of the Four Creut.fildt-/akob Disease Groups

Codon PrI'"' Symptom Symptoms EEG 129 Type Duration (mo) at Onset PSW Neuropathology

Immunomining I'rP" Distributioo Pattern

M / M 1 1.5-5.0 Dementia, 9/11; 10/11 Spongiosis in all layers of the cerebral visual, 2 / 11; ataxia, 2 / 1 I

cortex, in srriatum, thalamus, and molecular layer of the cerebellum

M / M 2 12-36

M/V 2 5-18

v/v 2 4-20

Llemenria 012 Spongiosis, gliosis, and neuronal loss in all layers of the cerebral correx, in the striatum and thalamus

Araxddementia 013 Widespread spongiosis and gliosis in limbic lobe, striatum, diencephalus, brainstem, and cerebellum with relative sparing of the neocorrex in short-duration (4 6-S ma) cases; kuru plaques in the cerehellum

As in the MIV group but with more severe spongiosis, gliosis, and granular cell loss in the cerebellum and no kuru plaques

113 (after 10 mo)

Ataxia

Highest amount in the cerebral correx, srriatum, thalamus, and cerebellum

Highest amounr in the cerebral cortex, stria- turn, and thalamus

High amounts in all brain regions with the exception of the neocortex in short- duration cases (< G mo)

As in M/V group

Punctatr and coarse (focally)

Coarse

Plaquelike and punctate; laminar distribution in the deep layers of neocortex in short-duration cases

As in M / V group

1%'"' = proteasc-resistant prion protein: EEG = elecrrornceplialograpl~y; PSW = periodic sharp wiives


PrPres, and a pattern of PrPrri immunostaining that is predominantly coarse rather than punctate. Although some of these features may be secondary to the long duration [45], the different clinical phenotype and the smaller size of the PrPre' justify the separation of this group from the CJDM/MI group.

Both the CJDMIV and the CJDV/V groups differ from the two CJDM/M groups in the topography of histopathology and PrPres accumulation, which affect all the deep cerebral nuclei, the brainstem, and the cerebellum with a relative sparing of the neocortex in the subjects with symptoms for less than 6 months (see Table 4). These differences are more conspicuous in VIV homozygotes than in heterozygotes due to the more severe cerebellar pathology in the V/V homozygotes. This, in turn, influences the clinical phenotype at onset: The heterozygotes show both cognitive and cerebellar signs whereas the VIV homozygotes have cerebellar signs and dementia occurs later. The CJDV/ V phenotype coincides with the ataxic or cerebellar variant, while the CJDMIV phenotype identifies with the CJD variant with kuru plaques. These phenotypic differences justify a separation of the heterozygotes from the V/V homozygotes.

None of our subjects had primary white matter lesions, a diagnostic criterion proposed for the panencephalopathic variant. Because the white matter was involved in the V/V homozygous and heterozygous subjects with symptoms for a long duration, and because reported panencephalopathic cases have demon- strated an attenuated course associated with a clinicopathological cerebellar syndrome, it is likely that this variant fits into either the CJDV/V or the CJDM/V group. Other previously described variants may not have been encountered by us because they are rare.

The availability of molecular markers that correlate with the clinical manifestations and duration of the disease may be important in the future assessment of diagnostic and therapeutic strategies in patients with suspected CJD. The early recognition of PrPrCS type and of PRNP genotype allows a prognosis for the duration of the disease in the majority of patients. Brain biopsy, required to establish the presence and type of PrPrrs, is frequently performed in patients with rapid or atypical dementia. With our method, PrPrCb can be detected early in the disease by needle biopsy with as little as 5 mg of brain tissue [46]. Moreover, with the discovery of in vitro inhibitors of PrP conversion [47], trials with pharmacological agents will become more likely. Therefore, a timely and accurate molecular diagnosis of the subtype of CJD will be increasingly important. The classification that we propose must now be tested prospectively to assess whether indeed all cases of sporadic CJD fall within the four groups that we have identified.

Our data provide new insight into the pathogenesis

of CJD. Considerable attention has focused on the role of the polymorphism at codon 129 of PRNP in human prion diseases. Although the influence of the codon 129 polymorphism on the clinicopathological phenotype of several familial prion diseases has been clearly documented [29, 30, 481, its role in the phenotypic expression of sporadic CJD is still controversial [31- 33, 49, 501. We found that different genotypes, as determined by the PRNP codon 129, codistribute with distinct disease phenotypes in sporadic CJD. However, the presence of two types of PrPres linked to different phenotypes in PRNP syngeneic subjects indicates that PRNP genotype is not the only determinant of phenotypic expression in sporadic CJD. In experimental prion diseases there is good evidence for multiple prion strains that carry information independent from the host genotype [ 3 , 34-36]. Recent data indicate that self-propagation of PrP'" polymers with distinct conformations might be the molecular basis of prion strains [37]. When passaged in syngeneic animals, each strain or isolate shows highly preserved characteristics such as incubation time, distribution and intensity of spongiform degeneration, and pattern of intracerebral PrPLLS deposition. These strain-specific properties, however, may drastically change if the same strain of prion is inoculated in animals with a different PRNP genotype. The animal may become resistant to infection, even with a single amino acid change in PrP, or may show a different incubation time and clinicopathological phenotype. Therefore, compatibility between agent strain and PRNP genotype, often drastically modified by a single amino acid substitution, regulates the inci- dence (susceptibility), incubation time, and phenotypic expression in experimental prion diseases [3, 5 11. Con- sidering all these lines of evidence, our results might be interpreted as evidence of, at least, two major prion strains in sporadic CJD. The most common form is associated with PrP"' type 1 and affects the subjects homozygous for methionine at codon 129, which ap- pears to be the only genotype permissive to this prion strain. This would explain the increased risk of devel- oping sporadic CJD in the population carrying this PRNP genotype [50, 521. In contrast, the rarest form, linked to PrP'" type 2, affects all genotypes at codon 129. The polymorphism, in this case, would modulate the intracerebral distribution and pattern of deposition of PrPrrs and, in turn, determine the three different clinicopathological phenotypes associated with PrP'" type 2.

The origin of different p ion strains in a sporadic, naturally occurring disease remains difficult to explain. It is currently believed that an unfavorable event, such as a somatic mutation or an aberrant posttranslational modification of normal PrP, may occur at random to convert normal PrP into PrPrCs 131. If so, this event is likely to vary and result in a variety of PrP"' types.

Parchi er al: Molecular Basis of Sporadic CJD 775

Although additional PrPres types may be discovered as more subjects with CJD are examined, our finding of only two types of PrPreS and four distinct variants in 19 CJD subjects suggests that the events leading to the formation of PrPres and to CJD are few, stereotyped, and not random. Therefore, an age-related abnormality of PrP metabolism or even an infection by exogenous prions seems to be a more likely explanation of the etiology of sporadic CJD.

As in a previous study [42], we found that PrPrra has a more widespread distribution than the histological lesion. We detected small amounts of PrP" in brain regions lacking structural abnormalities. The amount of PrPres increased in areas displaying lesions and showed a direct correlation with severity, particularly with regard to spongiform degeneration. Therefore, in CJD, PrPres accumulates before the histological lesions ensue. These observations are in agreement with previous data obtained in experimental scrapie [3] and from brain biopsy specimens from CJD subjects at the early stage of the disease [46].

On the other hand, the type more than the amount of PrPres seems to determine the tempo of the disease. The subjects in the CJDMlMl group who had type 1 PrPres and the shortest clinical course had milder structural changes and significantly less PrPrr' than did the subjects in the other three groups with type 2 PrP" and symptoms for a longer duration. Thus, the degree of cell dysfunction causing clinical signs is more severe in the presence of type I than type 2 PrPres. The finding adds new complexity to the physiopathology of the disease and further underlines the differences between the CJD variants associated with PrPres type 1 and type 2.

Our data also have implications for the pathogenesis of iatrogenic CJD. Valine homozygosity at codon 129 of the PRNP enhances susceptibility to iatrogenic CJD via peripheral contamination [53, 541. This form typically manifests with a cerebellar syndrome similar to that of kuru (a disease that is also due to peripheral infection and affects people with a high prevalence of V/V homozygosity [55]), and often shows larger PrPrrs accumulation in the cerebellum than in the cerebral cortex [56]. Our finding that VIV homozygotes with sporadic CJD had similar clinical and pathological features suggests that this phenotype is not specific to the iatrogenic form but is linked to the valine genotype at codon 129 or to the selection of a specific prion strain, or to both. Sporadic CJD V/V homozygotes and, to a lesser extent, the heterozygotes accumulate much more PrP"' in the hypothalamus than do the M/M homozygotes. In view of the hypothalamic-pituitary anatomical connections and the notion that infectivity is strictly related to the amount of PrP"' [43, 571, it is reasonable to speculate that the homozygous VIV and heterozygous subjects are the donors of contaminated pituitary

extracts, and that recipients who are 123V/V homozygotes are more likely to develop the disease. This con- jecture is consistent with the experimental observation that amino acid sequence homology facilitates the conversion of PrP': into PrP'" [58] .

We are indebted to Dr Chris Clarke and Dr Murray Grossman for providing clinical information about 2 patients; to Diane Kofskey, Caroline Cole, Brenda Dupree, and Rosemarie Funkhouser for technical assistance; and to Sandy Bowen for typing the manuscript. W e also thank Prof Elio Lugaresi and Prof Lewis P. Rowland for their helpful suggestions.

This work was supported by Public Health Service grants AG08372, AG08155, AG10133, and NS29822 and the Brirton Fund.

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