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From: Terry S. Singeltary Sr. (216-119-136-100.ipset16.wt.net)
Subject: Extraneural Pathologic Prion Protein in Sporadic Creutzfeldt–Jakob Disease [FULL TEXT]
Date: November 6, 2003 at 10:31 am PST
-------- Original Message --------
Subject: Extraneural Pathologic Prion Protein in Sporadic Creutzfeldt–Jakob Disease [FULL TEXT]
Date: Thu, 6 Nov 2003 12:10:42 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@uni-karlsruhe.de
######## Bovine Spongiform Encephalopathy #########
original article
The
new england journal
of
medicine
n engl j med
349;19
www.nejm.org november
6, 2003
1812
Extraneural Pathologic Prion Protein
in Sporadic Creutzfeldt–Jakob Disease
Markus Glatzel, M.D., Eugenio Abela, Manuela Maissen, M.S.,
and Adriano Aguzzi, M.D., Ph.D.
From the Institute of Neuropathology and
National Reference Center for Prion Diseases,
University Hospital of Zurich, Zurich,
Switzerland. Address reprint requests to Dr.
Aguzzi at the Institute of Neuropathology,
University Hospital of Zurich, Schmelzbergstr.
12, CH-8091 Zurich, Switzerland,
or at adriano@pathol.unizh.ch.
Dr. Glatzel and Mr. Abela contributed equally
to the article.
N Engl J Med 2003;349:1812-20.
Copyright © 2003 Massachusetts Medical Society.
background
In patients with sporadic Creutzfeldt–Jakob disease, pathologic
disease-associated prion
protein (PrP
Sc
) has been identified only in the central nervous system and olfactorynerve
tissue. Understanding the distribution of PrP
Sc
in Creutzfeldt–Jakob disease is
important for classification and diagnosis and perhaps even for prevention.
methods
We used a highly sensitive method of detection — involving the
concentration of PrP
Sc
by differential precipitation with sodium phosphotungstic acid, which
increased the
sensitivity of Western blot analysis by up to three orders of magnitude
— to search for
PrP
Sc
in extraneural organs of 36 patients with sporadic Creutzfeldt–Jakob
disease who
died between 1996 and 2002.
results
PrP
Sc
was present in the brain tissue of all patients. In addition, we found PrP
Sc
in 10 of
28 spleen specimens and in 8 of 32 skeletal-muscle samples. Three
patients had PrP
Sc
in both spleen and muscle specimens. Patients with extraneural PrP
Sc
had a significantly
longer duration of disease and were more likely to have uncommon
molecular variants
of sporadic Creutzfeldt–Jakob disease than were patients without
extraneural PrP
Sc
.
conclusions
Using sensitive techniques, we identified extraneural deposition of PrP
Sc
in spleen
and muscle samples from approximately one third of patients who died
with sporadic
Creutzfeldt–Jakob disease. Extraneural PrP
Sc
appears to correlate with a long duration
of disease.
abstract
n engl j med
349;19
www.nejm.org november
6, 2003
extraneural pathologic prion protein in sporadic creutzfeldt–jakob disease
1813
rion diseases are characterized by
degeneration of central nervous tissue associated
with the replication of a transmissible
agent, a prion.
1
Prions are mainly composed
of an abnormal, partially protease-resistant conformer
(PrP
Sc
) of a cellular protein (PrP
C
).
2
Although
tissue damage occurs only in the central nervous
system, the accumulation of PrP
Sc
and prion infectivity
are not necessarily confined to neural tissue
in all prion diseases. In scrapie in sheep, chronic
wasting disease in deer, and numerous animal models
of prion diseases, prions invade the lymphoreticular
system.
3
PrP
Sc
has also been reported in the
skeletal muscle of mice with experimentally induced
prion disease.
4
The most common prion disease in humans is
Creutzfeldt–Jakob disease, which has been classified
as sporadic, familial, iatrogenic, or variant. The
causation of sporadic Creutzfeldt–Jakob disease is
unclear, whereas biochemical, histopathological,
and epidemiologic evidence suggests that the variant
form results from the transmission of bovine
spongiform encephalopathy prions to humans.
5,6
PrP
Sc
accumulates in tonsils
7
and other lymphoreticular
organs
8
of patients with variant Creutzfeldt–
Jakob disease,
9
whereas the sporadic form of the
disease is not thought to target the lymphoreticular
system.
10
The allegedly unique involvement of
the lymphoreticular system in variant Creutzfeldt–
Jakob disease serves as a diagnostic criterion to distinguish
variant from sporadic Creutzfeldt–Jakob
disease and is one of the principal pieces of evidence
that these forms represent two distinct disease entities.
6
Refinements in the sensitivity of the methods
of detection
11
prompted us to reinvestigate
the distribution of PrP
Sc
in humans with sporadic
Creutzfeldt–Jakob disease.
collection of samples
We collected central nervous system, spleen, and
muscle samples at autopsy from a cohort of Swiss
patients who died between 1996 and 2002: 36 patients
with prion disease (36 brain specimens, 32
muscle specimens, and 28 spleen specimens), 10
with Alzheimer’s disease (10 muscle and 8 spleen
samples), and 9 patients without neurologic disorders
(9 muscle and 9 spleen samples). The utmost
care was taken to avoid contamination of extraneural
samples with central nervous system tissue. All
tissues were processed according to established
guidelines regarding safety and ethics.
detection of p
r
p
Sc
in extraneural tissues
All procedures were carried out in biosafety level
3 facilities with strict adherence to safety guidelines.
The method of precipitation with the use of
sodium phosphotungstic acid was adapted from
published protocols.
8
We prepared 10 percent tissue
homogenates (weight per volume) in 2 percent
sarkosyl in sterile phosphate-buffered saline using
a RiboLyser (Hybaid). Cellular debris was removed
by centrifugation at 80¬
g
for one minute in a microcentrifuge.
Occasional residual debris was removed
by additional centrifugation at 2700¬
g
for five minutes.
Supernatants (500 µl) were mixed with equal
volumes of 2 percent sarkosyl in phosphate-buffered
saline and incubated for 10 minutes at 37°C.
Benzonase (Benzon nuclease, Merck) and magnesium
chloride were added (final concentration, 50
units per milliliter and 1 mmol per liter, respectively),
and the mixture was incubated under constant
agitation for 30 minutes at 37°C.
Samples were adjusted to a final concentration
of 0.3 percent phosphotungstate, incubated at 37°C
for 30 minutes under constant agitation, and centrifuged
at 15,800¬
g
for 30 minutes in a microcentrifuge.
The pellets were resuspended in 20 µl of
phosphate-buffered saline with 0.1 percent sarkosyl.
Samples were adjusted to a final concentration
of 20 µg of proteinase K per milliliter and incubated
at 37°C for 30 minutes. Digestion was terminated
by the addition of protease inhibitors (Complete
protease inhibitor cocktail, Boehringer Mannheim).
Samples were boiled in loading buffer (125 mM
TRIS sodium chloride, 4 percent sodium dodecyl
sulfate, 20 percent glycerol, and 0.02 percent bromophenol
blue), subjected to electrophoresis in 12
percent TRIS–glycine gels, and blotted on nitrocellulose
membranes. Membranes were blocked with
5 percent Topblock (Jura) in phosphate-buffered saline
with 0.05 percent Tween and then incubated
overnight with a monoclonal antibody against PrP
(3F4; dilution 1:5000).
12
The samples were then incubated with an alkaline-
phosphatase–conjugated secondary antibody
(1:20,000) and examined with use of 2-chlor-
5-(4-methoxyspiro{1,2-dioxetan-3,2'-(5'-chlor)
tricycl[3.3.1.13,7]decan}-4-yl)-1-phenylphosphate
dinatrium salt chemiluminescent substrate (Amersham)
and a VersaDoc digital imager (model 5000,
p
methods
n engl j med
349;19
www.nejm.org november
6
,
2003
The
new england journal
of
medicine
1814
Bio-Rad). Each blot included appropriate positive
and negative controls (10 µg or 100 µg of brain homogenate
from a patient with sporadic Creutzfeldt–
Jakob disease or a patient without a prion disease
added to 50 mg of muscle or spleen specimen from
a patient without a prion disease). All analyses were
carried out and all but six samples were examined at
least twice by independent investigators. Samples
were deemed positive if proteinase K–resistant diglycosylated,
monoglycosylated, and unglycosylated
bands with electrophoretic motility indicative
of PrP
Sc
were unambiguously identified.
detection of p
r
p
Sc
in neural tissues
Western blot analysis was carried out as described
previously.
13
PrP glycoforms were quantified with
use of a Kodak image station (model 440) or a Versa-
Doc digital imager (model 5000), and patients were
typed according to the size of the protease-resistant
core PrP fragment
14
and the prevalence of glycoforms.
15,16
genetic analysis
Genomic DNA was extracted from frozen tissues.
Polymerase chain reaction and analysis of the entire
coding region of the prion protein gene (
PRNP
)
were performed with use of standard techniques
and software (SeqScape, Applied Biosystems). In
addition, codon 129 polymorphisms were identified
by restriction-fragment–length polymorphism
analysis.
17
histologic analysis
Tissue was fixed with 4 percent buffered formalin,
inactivated by exposure to 98 percent formic acid
for one hour, and embedded in paraffin. Sections
(3 µm) were subjected to conventional staining and
to immunostaining for glial fibrillary acid protein
(Dako) and PrP (monoclonal antibody 3F4) after hydrolytic
autoclaving.
We analyzed a total of 45 spleen specimens and 51
skeletal-muscle samples from 36 patients with histopathologically,
biochemically, and genetically confirmed
sporadic Creutzfeldt–Jakob disease (mean
[
±
SD] age, 66.8
±
8.7 years; 20 men and 16 women);
10 patients with Alzheimer’s disease (mean age,
76.2
±
4.6 years; 6 men and 4 women); and 9 patients
without neurologic disease (mean age, 59
±
19.2
years; 5 men and 4 women). The age distribution,
sex ratio, and duration of disease (mean, 5.0
±
4.6
months) of the cohort of patients with sporadic
Creutzfeldt–Jakob disease did not differ significantly
from those in published series.
18
accumulation of p
r
p
Sc
in spleen
and skeletal muscle
Western blot analysis of protease-resistant prion
protein after phosphotungstate precipitation
showed PrP
Sc
in 10 of 28 spleen samples and in 8 of
32 skeletal-muscle samples from the patients with
sporadic Creutzfeldt–Jakob disease (Fig. 1). Three
patients had PrP
Sc
in both spleen and muscle (Table
1). Assays were repeated with independently homogenized
tissue fragments: three spleen and three
muscle samples always tested positive, whereas others
yielded variable results (Table 1), possibly because
of inhomogeneous peripheral distribution of
PrP
Sc
, as described previously.
4,19
Control samples of spleen and muscle were assessed
in parallel: none of them contained PrP
Sc
(data not shown). Patients with PrP
Sc
in spleen and
patients without PrP
Sc
in spleen did not differ significantly
with respect to age (Table 2). However,
patients with PrP
Sc
only in spleen or in spleen or
muscle or both had significantly longer intervals
between the onset of clinical symptoms and death
than did patients without extraneural PrP
Sc
. Patients
with PrP
Sc
in muscle tended to be younger at death
than patients without PrP
Sc
in muscle (Table 2).
None of the subgroups differed significantly with
respect to the ratio of men to women.
One patient with splenic PrP
Sc
had received a cadaveric
dura mater transplant more than 22 years
before the onset of dementia. This case was deemed
unlikely to represent iatrogenic transmission, since
the incubation period would be longer than that in
any other documented case of dura mater–associated
transmission.
20
genetic analysis of
prnp
We sequenced the entire open reading frame of
PRNP
from all patients with extraneural PrP
Sc
and
found no disease-associated mutations. The common
methionine–valine polymorphism at codon
129 was analyzed in all 10 patients with splenic
PrP
Sc
and all 8 patients with muscle PrP
Sc
as well as
in 15 patients without splenic PrP
Sc
and 21 patients
without muscle PrP
Sc
. We observed a tendency toward
overrepresentation of heterozygosity for methionine
and valine and homozygosity for valine
among patients with splenic PrP
Sc
, whereas the maresults
n engl j med
349;19
www.nejm.org november
6, 2003
extraneural pathologic prion protein in sporadic creutzfeldt–jakob disease
1815
jority of patients with muscle PrP
Sc
were homozygous
for methionine (Table 2).
histologic and biochemical analyses
Typical histopathological features of sporadic
Creutzfeldt–Jakob disease, consisting of spongiform
changes, neuronal loss, and gliosis, were present in
all patients (Fig. 2). The electrophoretic mobility of
unglycosylated, protease-digested PrP
Sc
(also called
the core fragment size, which can be 21 kD for type
1 or 19 kD for type 2)
14
allows for the stratification
of Creutzfeldt–Jakob disease into subgroups with
specific clinical features, which may be caused by different
prion strains.
6
A finer degree of stratification
is attained by including information on the codon
129 genotype and patterns of cerebral deposition
of PrP
Sc
; accordingly, we assigned each patient in
our series to one of seven proposed groups.
18
This
analysis indicated an overrepresentation of uncommon
variants of sporadic Creutzfeldt–Jakob disease
such as MM2C (homozygous for methionine, type 2,
with cortical deposition) and MV2 (heterozygous,
type 2) in patients with extraneural PrP
Sc
(Table 2),
suggesting that peripheral deposition of PrP
Sc
may
identify a biochemically or genetically unique subgroup
of patients.
* Up to four individual fragments of each organ were independently
homogenized,
subjected to phosphotungstate precipitation, and analyzed by Western
blotting. Variability in detection may be due to inhomogeneous
distribution of
PrP
Sc
in extraneural organs. ND denotes not done.
Table 1. Age at Onset and Duration of Disease in 15 Patients with Sporadic
Creutzfeldt–Jakob Disease and Extraneural Pathologic Prion Protein (PrP
Sc
).
Patient
No. Age
Duration
of Disease
Location of
Extraneural PrP
Sc
Muscle* Spleen*
yr mo no. with PrP
Sc
/total no.
1 47 7 Spleen 0/2 1/4
2 48 6 Muscle 2/2 ND
3 52 6 Spleen ND 1/2
4 55 8 Spleen 0/2 1/3
5 60 5 Spleen and muscle 1/2 1/1
6 62 3 Muscle 1/2 0/1
7 65 29 Muscle 1/3 ND
8 66 3 Muscle 1/2 0/2
9 67 7 Spleen and muscle 1/2 3/3
10 68 4 Muscle 2/2 0/2
11 68 7 Spleen 0/2 2/4
12 73 5 Spleen and muscle 1/1 2/2
13 77 3 Spleen 0/2 2/3
14 77 4 Spleen 0/2 1/3
15 81 5 Spleen 0/2 1/3
Figure 1. Western Blot Analysis of Phosphotungstate-Precipitated
Pathologic Prion Protein (PrP
Sc
) from Spleen and Muscle Samples
from Patients with Sporadic Creutzfeldt–Jakob Disease (sCJD).
Lanes 1 and 2 show Western blot (total protein) analysis of 50 µg of
brain homogenate from a patient without Creutzfeldt–Jakob disease.
Lanes 3 and 4 show brain homogenate from a patient with sporadic
Creutzfeldt–Jakob disease diluted with spleen homogenate from a patient
without a prion disease (control); lanes 5 and 6 brain homogenate from a
control diluted with spleen homogenate from a control; lanes 7 and
8 spleen homogenate from a patient with sporadic Creutzfeldt–Jakob
disease; and lanes 9, 10, 11, 12, and 13 muscle homogenate from a patient
with sporadic Creutzfeldt–Jakob disease. PrP
Sc
is present in two samples (lanes 7 and 10). Lanes 2, 4, and 6 through 13
show results
after proteinase K digestion.
kD
37 —
29 —
20 —
—
—
—
1 2 3 4 5 6 7 8 9 10 11 12 13
Control brain before
Control brain after
Brain from patient with sCJDdiluted with control spleen
Brain from patient with sCJDdiluted with control spleen
Control brain with control spleen
Control brain with control spleen
Spleen from patient with sCJD
Spleen from patient with sCJD
Muscle from patient with sCJD
Muscle from patient with sCJD
Muscle from patient with sCJD
Muscle from patient with sCJD
Muscle from patient with sCJD
n engl j med
349;19
www.nejm.org november
6
,
2003
The
new england journal
of
medicine
1816
Quantification of the relative prevalence of diglycosylated,
monoglycosylated, and unglycosylated
PrP
Sc
is a further instrument for the biochemical
characterization of PrP
Sc
, which allows one to
discriminate sporadic from variant Creutzfeldt–
Jakob disease.
16
This analysis did not uncover any
unorthodox PrP
Sc
glycotypes in patients with peripheral
PrP
Sc
(Fig. 3): the distribution of brain glycotypes
in patients with peripheral PrP
Sc
was similar
to that in other cohorts of patients with sporadic
Creutzfeldt–Jakob disease.
13
distribution of p
r
p
Sc
in skeletal-muscle
groups
Earlier studies in mice have shown that the PrP
Sc
content may vary among muscle groups.
4
In our
series, the presence of PrP
Sc
was not limited to specific
muscle groups: PrP
Sc
was found in four samples
of pectoral muscle, two samples of psoas muscle,
and two samples of intercostal muscle. It was
absent in 10 pectoral-muscle specimens, 5 psoasmuscle
specimens, 2 biceps-muscle specimens, and
4 intercostal-muscle specimens. In three patients
without extraneural PrP
Sc
, no information on the
anatomical origin of muscle samples could be obtained.
quantification of p
r
p
Sc
in extraneural
organs
In order to estimate the relative concentration of
PrP
Sc
in extraneural organs, we compared the intensity
of proteinase K–digested Western blot signals
with calibration curves obtained by diluting brain
homogenate from a patient with Creutzfeldt–Jakob
disease with muscle or spleen homogenate from a
patient without a prion disease. Despite considerable
variations in individual spleen and muscle specimens,
the signal intensity of peripheral PrP
Sc
was
generally similar to that obtained when 10 µg of
brain homogenate from a patient with sporadic
Creutzfeldt–Jakob disease was diluted with 50 mg
of spleen or muscle from a patient without a prion
disease (Fig. 1). Therefore, we estimate that the levels
of PrP
Sc
in extraneural organs are lower by a factor
of approximately 1¬10
¡4
than those typically
found in the central nervous system of patients with
sporadic Creutzfeldt–Jakob disease.
Previous studies did not detect PrP
Sc
in the tonsils,
spleen, lymph nodes,
10 or appendix of patients
with sporadic Creutzfeldt–Jakob disease.21 Howdiscussion
* Plus–minus values are means ±SD. Data on classification are from
Parchi et al.14,18 Mann–Whitney tests were used for
all comparisons of age and the duration of illness. Sex ratios were
compared with use of chi-square tests. Only significant
differences between groups are indicated.
† Patients were classified according to the size of the
protease-resistant fragment of PrPSc (type 1 [21 kD] or type 2 [19 kD]),
whether they were homozygous for methionine (MM1, MM2C, or MM2T) or
valine (VV1P or VV2) at codon 129 of the
prion gene or were heterozygous (MV1 or MV2), and histologic
characteristics such as plaque or plaque-like lesions (P),
cortical or synaptic deposits (C), and thalamic deposits (T).
‡ P=0.004.
§ Patients with no available information on codon 129 were not classified.
¶P=0.003.
Table 2. Demographic Characteristics and Classification of Sporadic
Creutzfeldt–Jakob Disease in Patients
with and Those without Pathologic Prion Protein (PrPSc) in Spleen and
Muscle Specimens.*
Status of Peripheral PrPSc Disease Phenotype† Sex Age
Duration
of Disease
MM1 MV1 VV1P MM2C MM2T MV2 VV2
no. of patients M/F yr mo
Spleen sample
PrPSc present
PrPSc absent§
3
12
11
1 11
21
2 5/5
11/7
65.9±6.9
67.9±6.9
5.7±1.6‡
4.0±2.2‡
Muscle sample
PrPSc present
PrPSc absent§
5
14 2 1
2
3
11
4/4
15/9
63.6±7.4
67.6±8.8
7.7±8.7
4.4±2.3
Spleen or muscle sample
PrPSc present
PrPSc absent§
7
15
11
1 2 21
2 7/8
13/8
64.4±10.5
68.6±7.0
6.8±6.3¶
3.8±2.2¶
n engl j med 349;19 www.nejm.org november 6, 2003
extraneural pathologic prion protein in sporadic creutzfeldt–jakob disease
1817
ever, after a systematic search for PrPSc in extraneural
organs of patients referred to the Swiss National
Reference Center for Prion Diseases, we found that
extraneural deposits of PrPSc are much more frequent
in patients with sporadic Creutzfeldt–Jakob
disease than previously thought. There may be several
reasons why this fact was not previously recognized.
First, our series of patients is much larger than
previous ones. Second, repeated homogenization of
distinct fragments of extraneural organs identified
substantial variations in the PrPSc content of these
tissues. Generic problems related to the technical
reproducibility of our results are unlikely to account
for this variation, since control samples consisting
of brain homogenate from a patient with sporadic
Creutzfeldt–Jakob disease diluted with muscle
or spleen homogenate from a patient without
a prion disease did not have such variations (data
not shown). Instead, the presence of variability suggests
that the distribution of PrPSc is not homogeneous
in single muscle fibers and single splenic germinal
centers. This finding parallels the finding that
prion infectivity titers vary in spleen22 and muscle4
samples from experimentally infected mice.
Third, PrPSc levels in spleen and muscle were
lower than those in brain by a factor of 1¬10¡4: extraneural
PrPSc was never detectable by plain Western
blot analysis (data not shown) and could be visualized
only after sodium phosphotungstic acid
precipitation, an approach that increases the sensitivity
of Western blot analysis by three orders of
magnitude by preferentially precipitating PrPSc from
tissue homogenates.8,11 Although this method is
not suitable for use with very small biopsy specimens
(less than 100 mg), it is ideal for situations in
which sample size is not a limiting factor.
Previous studies suggested the presence of abnormal
PrP in the skeletal muscle of patients with
inclusion-body myositis.23 However, this may represent
denervation-dependent up-regulation of PrPC
transcription.24 In one patient with coincident sporadic
Creutzfeldt–Jakob disease and inclusion-body
myositis, we found PrPSc levels that were 103 times
as high as the levels in our study cohort, possibly
because the overexpression of PrPC in muscle facilitates
the deposition of PrPSc (Kovacs G: personal
communication).
Does the presence of peripheral PrPSc correlate
with any specific genetic, biochemical, or clinical
Figure 2. Histopathological Findings in the Cerebellum of Patients with
Sporadic Creutzfeldt–Jakob Disease and Peripheral Pathologic Prion
Protein (PrPSc).
Deposition of PrPSc in the cerebellum was diffuse, granular, and
synaptic in a patient who was homozygous for methionine at codon 129 of the
prion protein gene (MM1 and MM2C [Panels A and D]) and in one who was
heterozygous (MV1 [Panel B]). Deposition of PrPSc was in the
form of plaques and plaque-like lesions in patients who were homozygous
for valine at codon 129 (VV1P [Panel C] or VV2 [Panel F]) and in one
who was heterozygous (MV2 [Panel E]).18 IGL denotes internal
granule-cell layer, PCL Purkinje-cell layer, and ML molecular layer.
IGL
PCL
ML
20 µm MV1 MM1
MV2 VV2
VV1P
MM2C
A B C
D E F
n engl j med 349;19 www.nejm.org november 6, 2003
The new england journal of medicine
1818
factors? When plotting the incidence of codon 129
polymorphisms (MM, MV, or VV [homozygous for
valine]), PrPSc core-fragment sizes (type 1, 21 kD,
and type 2, 19 kD), and histologic characteristics
(plaque and plaque-like lesions [P], cortical or synaptic
deposits [C], or thalamic deposits [T]), we observed
several uncommon phenotypes in patients
with peripheral PrPSc, such as MM2C and MV2. In
addition, one patient with PrPSc in lymphoid organs
who was homozygous for valine at codon 129 could
not be classified according to any of the existing
schemes.18,25 These features may represent a new
phenotype with atypical clinical signs,26 which
we have termed VV1P because of the presence of
plaque-like deposits of PrP in the cerebellum and
a PrPSc core-fragment size of 21 kD.
The relative ratios of PrPSc glycoforms are different
in sporadic and variant Creutzfeldt–Jakob
disease. Glycotype analysis failed to reveal features
specific to patients with peripheral PrPSc, and none
of them had the glycoform distribution associated
with variant Creutzfeldt–Jakob disease. Patients with
splenic PrPSc had significantly longer durations of
disease than did those without splenic PrPSc. Assuming
that prions arise primarily in the central
nervous system of these patients, one might speculate
that protracted disease increases the likelihood
of spillover of cerebral PrPSc to extraneural areas.
Figure 3. Glycoform Profiles of Patients with Sporadic and Variant
Creutzfeldt–Jakob Disease (CJD) According to
the Presence or Absence of Peripheral Pathologic Prion Protein (PrPSc).
The triangular plot correlates the intensities of the diglycosylated
(upper), monoglycosylated (middle), and unglycosylated
(lower) bands of PrPSc. Study patients with sporadic Creutzfeldt–Jakob
disease and PrPSc in spleen, muscle, or both;
study patients with sporadic Creutzfeldt–Jakob disease but no PrPSc in
spleen or muscle; and British control patients with
sporadic Creutzfeldt–Jakob disease (black, dark gray, and light gray
boxes) cluster in the same area of the plot. Instead,
control patients with variant Creutzfeldt–Jakob disease (white square13)
are segregated in a distinct region of the plot.
Unglycosylated Bands
Diglycosylated Bands
Monoglycosylated Bands
0.8 0.2
0.2
0.6 0.4
0.4
0.4 0.6
0.6
0.2 0.8
0.8
Sporadic CJD type 2a
Sporadic CJD type 1
Variant CJD
Spleen and muscle PrPSc
Spleen PrPSc
Muscle PrPSc
No spleen or muscle PrPSc
n engl j med 349;19 www.nejm.org november 6, 2003
extraneural pathologic prion protein in sporadic creutzfeldt–jakob disease
1819
Although the amount of PrPSc found in spleen
and muscle samples from patients with sporadic
Creutzfeldt–Jakob disease was much lower than levels
in lymphoid organs of patients with variant
Creutzfeldt–Jakob disease,7 and the infectivity of
prions from these tissues awaits verification, our
findings arouse concern about the possibility of iatrogenic
transmission of sporadic Creutzfeldt–Jakob
disease. Brown et al. reported that extraneural tissues
of patients with spongiform encephalopathy
(including hereditary forms and kuru) occasionally
transmitted disease when inoculated into nonhuman
primates.27 Infectivity was detected in 2 of
4 lung-tissue samples, 4 of 35 liver specimens, 5 of
28 kidney specimens, 3 of 31 spleen samples, and
3 of 15 lymph-node samples.27 Five attempts to
transmit the disease through the inoculation of skeletal
muscle were not successful, possibly because
the sample size was insufficient, because the infectivity
of the samples was not uniform, or because
transmission from human to nonhuman primates
is not always highly efficient.
The causation of sporadic Creutzfeldt–Jakob disease
is unknown. Some allegedly sporadic cases may
in reality have an iatrogenic origin, and surgery of
any kind was found to constitute a mild risk factor
for sporadic Creutzfeldt–Jakob disease.28 Extensive
epidemiologic surveys did not substantiate any risk
of blood-borne transmission,29 suggesting that the
presence of PrPSc in lymphoid organs whose cellular
constituents migrate to and from blood may not
lead to substantial contamination of blood donations
by prions.
Many patients with sporadic Creutzfeldt–Jakob
disease are subjected to extensive neurologic examinations
in the prodromal stages, often including
electromyography and muscle biopsies. The finding
that PrPSc is prevalent in skeletal muscle reinforces
calls for the use of single-use needle electrodes and
of special protocols for the sterilization of surgical
instruments used for muscle biopsies.
Our results suggest that muscle and lymph-node
biopsies can be used as diagnostic procedures for
sporadic Creutzfeldt–Jakob disease. Because of the
unambiguous and extremely specific nature of the
immunochemical detection of PrPSc, any positive
results would firmly establish the diagnosis of
Creutzfeldt–Jakob disease without the need for more
invasive procedures. However, the diagnostic sensitivity
of the detection of PrPSc in muscle-biopsy
specimens is likely to be low, since less than one
third of our patients were positive for PrPSc in muscle.
We did not detect PrPSc in one muscle-biopsy
specimen obtained because of suspected vasculitis
10 weeks before death in a patient with Creutzfeldt–
Jakob disease (data not shown). The sensitivity of
prion detection is likely to improve soon, in view of
the considerable refinements in the technique that
are being reported.30
In contrast to previous reports, our study focused
on a homogeneous, narrowly defined group
of patients. All the patients had died of sporadic
Creutzfeldt–Jakob disease in Switzerland between
1996 and 2002. On the other hand, there has been an
alarming increase in the incidence of Creutzfeldt–
Jakob disease in Switzerland.13 Although the causes
of this epidemiologic shift are not clear, the etiologic
process of sporadic Creutzfeldt–Jakob disease
in our cohort may differ from that in cohorts in other
countries. It will therefore be important to extend
the present study to additional countries, in order to
determine whether our observations apply to all cases
of sporadic Creutzfeldt–Jakob disease.
The Swiss Reference Center for Prion Diseases is funded by the
Swiss Federal Office of Public Health. Dr. Glatzel is supported by a
career development award (Forschungskredit) of the University of
Zurich.
We are indebted to all the referring physicians; to Ivan Hegyi,
Marco Prinz, and Judith Gottwein for assistance with tissue collection;
to Mauri Peltola for technical help; to Lorenz Amsler for help
with statistical analysis; to Johannes Streffer for sequencing; to
Thorsten Lührs for proposing the use of triangular glycoplots; to
Klaus Hess for critical reading of the manuscript; to Prionics for
providing laboratory space; and to the University Hospital of Zurich
for a generous infrastructural grant enabling the creation of the
Swiss CJD tissue collection.
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