Making more amyloid: common diabetic drug
Two prion-inducing regions of ure2p are nonoverlapping
PrP-dependent association of prions with splenic but not circulating lymphocytes
Raccoon encephalopathy resoved: not a TSE
Copper chelators and strain types 1 and 2
Sporadic fatal insomnia? -- making lemonade out of a misdiagnosis
Reuse of angioplasty catheters and risk of CJD: money vs risk
Is scrapie a genetic disease? New Zealand sheep have suspect alleles
Steering committee on sheep,goats, and monitoring
J Biol Chem, Vol. 274, Issue 23, 15990-15995, June 4, 1999 Bernd Bohrmann, ...,Walter Huber, Heinz Dľbeli, and Christer NordstedtComment (webmaster): This article raises a host of questions about the cause of common all sorts of amyloid diseases. Note that prion protein is also briefly considered in the study.
"We report that certain plasma proteins, at physiological concentrations, are potent inhibitors of amyloid -peptide (Abeta) polymerization. These proteins are also present in cerebrospinal fluid, but at low concentrations having little or no effect on Abeta . Thirteen proteins representing more than 90% of the protein content in plasma and cerebrospinal fluid were studied. Quantitatively, albumin was the most important protein, representing 60% of the total amyloid inhibitory activity, followed by 1-antitrypsin and immunoglobulins A and G.
Albumin suppressed amyloid formation by binding to the oligomeric or polymeric Abeta, blocking a further addition of peptide. This effect was also observed when the incorporation of labeled Abeta into genuine beta-amyloid in tissue section was studied. The Abeta and the anti-diabetic drug tolbutamide apparently bind to the same site on albumin.
Tolbutamide displaces Abeta from albumin, increasing its free concentration and enhancing amyloid formation. The present results suggest that several endogenous proteins are negative regulators of amyloid formation. Plasma contains at least 300 times more amyloid inhibitory activity than cerebrospinal fluid.
These findings may provide one explanation as to why beta-amyloid deposits are not found in peripheral tissues but are only found in the central nervous system. Moreover, the data suggest that some drugs that display an affinity for albumin may enhance beta-amyloid formation and promote the development of Alzheimer's disease."
Beta-amyloid displays several important features that distinguish it from other types of amyloid. (i) The peptide forming the amyloid deposits is present at very low concentrations in the circulation. This is in contrast to peripheral amyloid disorders in which the amyloid proteins are present at high concentrations. Examples of such non-central nervous system amyloid proteins include serum amyloid A, myeloma protein, and transthyretin (11). (ii) The levels of Abeta are not higher and the peptide is not structurally different (except in extremely rare cases of familial AD) in individuals with the disease than in healthy controls (for a review, see Ref. 3). (iii) It is well known that most and possibly all nucleated cells in the body produce the Abeta; however, for unknown reasons, beta-amyloid is only deposited in the central nervous system.
The bulk of large proteins do not penetrate the blood-brain barrier efficiently. Thus, the levels of soluble proteins in the central nervous system are much lower than those in peripheral tissues. It has been estimated that the ratio between protein content in the CSF and plasma is approximately 0.004. However, in contrast to large proteins, the Abeta levels are higher in CSF than in plasma, which probably reflects a higher rate of secretion from neuronal cells than from other cell types. Overall, this suggests that a smaller fraction of the Abeta is protein-bound in the central nervous system than in the periphery.
In experiments with the prion protein-derived peptide PrP106-126 (26), similar methodology was used, but with two exceptions. [26.Prusiner, S. B. (1998) PNAS 95, 13363-13383]
Figure legend: HSA Inhibits the Polymerization of a Prion-derived Peptide-- PrP106-126 represents the central core of the prion protein (for a recent review, see Ref. 26) and spontaneously forms amyloid-like fibrils. Similar to the Abeta, prion protein can form amyloid deposits in the CNS and cause neurodegeneration.
We therefore decided to study whether albumin can also prevent polymerization of this peptide. As seen in Fig. 3, HSA dose-dependently inhibited the binding of biotinylated PrP106-126 to immobilized homologous peptide. The IC50 value for HSA in this system was approximately 100 ÁM, 10 times higher than that for Abeta. This concentration represents less then one-sixth of the albumin concentration in blood but is more than 30 times higher than the albumin concentration in CSF. Hence, these findings demonstrate that HSA displays a certain degree of specificity for beta-amyloid.
Plasma and CSF proteins with affinity to A serve as carriers for the peptide. This study and previous studies have demonstrated that Abeta-binding proteins in plasma and CSF may also have a function in regulating beta-amyloid formation. The most abundant plasma protein, albumin, is present in a concentration more than 60-fold higher than its IC50 in the Abeta polymerization assay used here (see Table I). Albumin is the most abundant protein in CSF, but it is present at a concentration below its IC50 value at which only a partial inhibition of Abeta polymerization is obtained (see Fig. 1). Plasma also contains significant levels of other proteins, such as 1-antitrypsin, IgG, and IgA, that are capable of inhibiting Abeta polymerization. The other studied proteins capable of inhibiting polymerization are also present in CSF in concentrations substantially below their IC50 values (see Table I).
These results point to a dramatic difference between plasma and CSF: the former contains large quantities of inhibitory proteins, whereas the latter contains small quantities of inhibitory proteins. For unknown reasons, -amyloid deposits are not formed outside the central nervous system (34). The present results suggest that the high concentrations of inhibitory proteins in plasma prevent the formation of -amyloid in peripheral tissues, but the low levels in CSF do not block beta-amyloid formation in the central nervous system. This conclusion is also supported by previous experimental data (35), showing that CSF only partially inhibits the formation of thioflavin-binding amyloid from synthetic Abeta 1-40.
Pathologically reduced levels of albumin might promote beta-amyloidosis and possibly also AD. In clinical studies, it was observed that anti-inflammatory drugs may have beneficial effects on AD. Levels of albumin are often reduced in association with inflammation and, hence, the antiamyloidogenic activity in plasma and CSF is also reduced. However, even heavily reduced plasma levels of albumin are probably still sufficiently high to prevent amyloid formation in peripheral tissues. It may be different in the central nervous system. Because albumin (and other inhibitory proteins) is present in low concentrations having limited effects on amyloid formation, even small reductions in albumin levels in association with inflammation may lead to increased amyloid formation.
The structural background as to why A binds albumin and other proteins is not known. However, it is reasonable to assume that hydrophobic interactions are involved. It was surprising that monomeric Abeta did not display binding to albumin when studied by surface plasmon resonance spectroscopy, considering the findings of Biere et al. (14) showing that soluble Abeta binds albumin and lipoproteins. One explanation may be that Abeta molecules rapidly form small, soluble, oligomers with an affinity to albumin.
Tolbutamide is a drug used to regulate blood glucose levels in diabetes mellitus. It also displays a high affinity for albumin. As a result, its clinical use is often associated with interactions with other drugs when the compounds compete for the same binding site on the albumin molecule (17). Here, we found that tolbutamide, at concentrations corresponding to therapeutic levels, enhanced amyloid formation in the presence but not in the absence of HSA. A reasonable explanation is that tolbutamide and Abeta bind to the same site on albumin. Tolbutamide may therefore displace Abeta from albumin and generate higher free Abeta fractions that can participate in amyloid formation. Drugs that can penetrate into the central nervous system, bind to the Abeta site(s) on albumin, and increase the free fraction of the peptide may thus be capable of enhancing amyloid formation in vivo.
Mutations affecting proteins capable of binding Abeta may promote the development of AD. It is therefore possible that mutations affecting the proteins studied here may also have an impact on the development of AD through a similar mechanism.
In conclusion, the present data suggest a novel and possibly important physiological role for albumin and other plasma/CSF proteins in controlling amyloidogenesis in the central nervous system and possibly also in peripheral tissues. The data also suggest that drugs with certain pharmacokinetic properties may be capable of enhancing amyloidogenesis. Moreover, the reduced levels of albumin seen in association with inflammatory reactions may provide an opportunity for the Abeta to polymerize and thereby more easily form amyloid in the central nervous system. "
Mol Cell Biol 1999 Jun;19(6):4516-24 Maddelein ML, Wickner RBComment (webmaster): TThe yeast prionlike system Ure2p is moving along very rapidly past convoluted genetic inference to actual studies of the protein.. While the prionlike domain had seemed more reminiscent of a poly-glutamine type conformational disease, here we see evidence for significant interaction with a distal portion of the peptide. Note codons 66-80 could have been considered an integral part of the first domain from the get-go based on primary sequence.
The authors do not have a 3D structure for the monomer and even if they did, they would be stymied like everyone else by lack of methods for fine structure determination of the fibril, though yeast present many advantages for manipulating the sequence to find out what is required for prionlike activity, unlike mammalian prion which has no assay
"Ure2p of Saccharomyces cerevisiae normally functions in blocking utilization of a poor nitrogen source when a good nitrogen source is available. The non-Mendelian genetic element [URE3] is a prion (infectious protein) form of Ure2p, so that overexpression of Ure2p induces the de novo appearance of infectious [URE3]. Earlier studies defined a prion domain comprising Ure2p residues 1 to 64 and a nitrogen regulation domain included in residues 66 to 354. We find that deletion of individual runs of asparagine within the prion domain reduce prion-inducing activity. "
"Although residues 1 to 64 are sufficient for prion induction, the fragment from residues 1 to 80 is a more efficient inducer of [URE3]. In-frame deletion of a region around residue 224 does not affect nitrogen regulation but does eliminate prion induction by the remainder of Ure2p. Larger deletions removing the region around residue 224 and more of the C-terminal part of Ure2p restore prion-inducing ability. A fragment of Ure2p lacking the original prion domain does not induce [URE3], but surprisingly, further deletion of residues 151 to 157 and 348 to 354 leaves a fragment that can do so. The region from 66 to 80 and the region around residue 224 are both necessary for this second prion-inducing activity. Thus, each of two nonoverlapping parts of Ure2p is sufficient to induce the appearance of the [URE3] prion. " [This last sentence is confusing: if 1-80 is considered the original domain, then part of it is still required, ie, deletion of 1-80 cannot be compensated by further downstream deletions -- webmaster]
Biochem J 1999 Mar 1;338 ( Pt 2):403-7 Fernandez-Bellot E, Guillemet E, Baudin-Baillieu A, Gaumer S, Komar AA, Cullin CIn the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.
EMBO J 1999 May 17;18(10):2702-2706 Raeber AJ, Klein MA, Frigg R, Flechsig E, Aguzzi A, Weissmann CAn intact immune system, and particularly the presence of mature B lymphocytes, is crucial for mouse scrapie pathogenesis in the brain after peripheral exposure. Prions are accumulated in the lymphoreticular system (LRS), but the identity of the cells containing infectivity and their role in neuroinvasion have not been determined. We show here that although prion infectivity in the spleen is associated with B and T lymphocytes and to a lesser degree with the stroma, no infectivity could be detected in lymphocytes from blood [threshhold of detectability not stated in abstract -- webmaster].
In wild-type mice, which had been irradiated and reconstituted with PrP-deficient lymphohaematopoietic stem cells and inoculated with scrapie prions, infectivity in the spleen was present in the stroma but not in lymphocytes. Therefore, splenic B and T lymphocytes can either synthesize prions or acquire them from another source, but only when they express PrP.
Neurosci Lett 1999 Apr 16;265(2):135-8 McBride PA, Beekes M Institute for Animal Health, Neuropathogenesis Unit, Edinburgh, UK.Although the ultimate target of infection is the CNS, there is evidence that the peripheral nervous system (PNS) is involved in the pathogenesis of TSEs. We used immunocytochemistry to identify the presence of pathological accumulations of a host protein, PrP, in the CNS and PNS (sensory and autonomic ganglia) of hamsters orally infected with 263K scrapie. All hamsters showed pathological deposition of PrP in most brain areas, along the length of the spinal cord, in nodose (NG) and dorsal root (DRG) ganglia and in the coeliac mesenteric ganglion complex (CMGC). In one case, scant deposition was observed along a few axons of the vagus nerve. This finding suggests that, after oral challenge, TSE infectious agent uses neural pathways and ganglia of the peripheral nervous system to reach target sites in the CNS.
Biophys Chem 1999 Mar 29;77(2-3):139-52 Masel J, Jansen VA, Nowak MA email@example.comThe mechanism of protein-only prion replication is controversial. A detailed mathematical model of prion replication by nucleated polymerisation is developed, and its parameters are estimated from published data. PrP-res decay is around two orders of magnitude slower than PrP-sen decay, a plausible ratio of two parameters estimated from very different experiments. By varying the polymer breakage rate, we reveal that systems of short polymers grow the fastest. Drugs which break polymers could therefore accelerate disease progression. Growth in PrP-res seems slower than growth in infectious titre. This can be explained either by a novel hypothesis concerning inoculum clearance from a newly infected brain, or by the faster growth of compartments containing smaller polymers. The existence of compartments can also explain why prion growth sometimes reaches a plateau. Published kinetic data are all compatible with our mathematical model, so the nucleated polymerisation hypothesis cannot be ruled out on dynamic grounds. [One wonders where these parameters are coming from and how they could possibly warrant such detailed modelling -- webmaster.]
Amirali Hamir Listserve 28 May 1999Amirali Hamir is with the TSE group at Ames, Iowa. He has been a veterinary diagnostic patholgist for the past 20 yrs and has worked in various countries (Tanzania, Kenya, Canada, Australia, Papua New Guinea). His research interests are in comparative neuropathology (mainly infectious etiology). Since arriving in the US, he has been involved with the experimental and field testing of the oral raccoon rabies vaccine (VR-G).
At OSU, he found over 40% of raccoons had neuronal vacuolations in the brainstem. According to Hadlow, these lesions were identical to scrapie in sheep. However, the raccoons were nonclinical and did not show PrP by IHC (done at 2 different labs -- Prusiner's and NVSL). On EM these cases revealed lipid within the vacoules of the neurons.
"This is a good case to illustrate that even in the presence of histopathological lesions, one cannot conclude that it is TSE. Pathologists need to back their diagnosis with IHC or western blots."
Neuronal vacuolation in raccoons (Procyon lotor). Hamir AN, Heidel JR, Picton RA, Rupprecht CE: Veterinary Pathology. 34:250-252, 1997. Neuronal vacuolations in raccoons from Oregon. Hamir AN, Fischer KA: Journal of Veterinary Diagnostic Investigation. (in press), 1999."Spongiform changes affecting the neuropil and neuronal cell bodies have been described in experimental as well as natural cases of rabies in skunks, foxes, and a heifer. Neuronal perikaryonic vacuolation is also seen in scrapie and related spongiform encephalopathes, and in lysosomal and acquired storage diseases of various animals. Naturally occurring transmissible spongiform encephalopathy and storage diseases have not been documented in raccoons (Procyon lotor). Rabies, on the other hand, is commonly encountered in raccoons of the eastern United States.
However, vacuolation of neurons or the neuropil has neither been seen in raccoons with naturally acquired rabies, nor in raccoons experimentally infected with either a dog or a raccoon isolate of the rabies virus. During a 2-year period vacuoles were detected in neurons of 21/50 (42%) raccoons in Oregon. Microscopically the vacuoles were variable in size, were in the perikarya, and were consistently present in pontine nuclei.
Brain tissues were negative for rabies virus antigen by fluorescent antibody test and for protease-resistant protein (PrP) prion by immunohistochemistry. Electron microscopic examination of the brainstem revealed accumulation of electron dense material within neuronal perikarya. Based on light and electron microscopic findings, the accumulated intracellular material appeared to have a high lipid content. These lesions indicate a form of neuronal storage condition. Further research is required to identify the composition of the intracellular lipid material, to elucidate the mechanism of neuronal vacuolation in raccoons, and to understand the basis for the apparent geographic restriction of this lesion."
28 May 1999, new split-off journal Nature Cell Biology, off Medline, webmaster sent pdfComment (webmaster): Basically, their thought here was that if the repeat region of PrP-res binds copper and zinc, chelators of these metals might remove them, de-constraining the structure of that portion of the peptide and opening it up more to protease K attack. In fact, some minor downshifting of gel bands consistent with this scenario is presented for strain-types 1 and 2, which are hypothesized to have differed originally only in this regard. Various chelators are used; a laudable number of controls gels are shown.
Overall, the experiment has rather pushed itself too far forward in the queue. It has not been established that native or reconstituted, sensitive or resistant, prion protein specifically binds copper or zinc nor whether this is limited to the repeat region, nor whether these or other ions such as magnesium are bound elsewhere as well.
EDTA, their best reagent, is not at all specific for transition metals. In a crude brain homogenate at a required minimum level of 20mM, a vast number of irrelevent factors could come into play. Who can recall a PrP-res gel containing the repeat region? -- it seems to be cleaved already either in in vivo fibrils or by PK even without chelators. Since PK is given adequate time to act and the bond around codon 90 is already accessible; cleavage here would then open up the protein to digestion up to the globular domain.
Free cupric ion added at a level of 25 ÁM (Figure 4) to a brain homogenate is physiologically most problematic and would be complexed fully within microseconds to background targets. No one has been able to reconsititute native protein in this way and indeed a copper chaperone and cofactors are generally needed in other proteins. Molecular models of copper binding to the repeat region are not supportive of media equilibration or chelator access.
It was a poor idea to retain the name 'type 2' for the reaction products of both type 1 and type 2, which after all had minor band shifts amounting to 10 and 5 amino acids respectively (though no evidence for changed termini was actually presented). If type 2 is degraded, then it is no longer type 2. Smeared bands slightly downset from 19k are completely inadequate for establishing a common final product from types 1 and 2.
Concluding that type 1 and type 2 sporadic CJD differ by copper binding conformation -- well, a whole lot more work is needed. In prion research, few investigators have the patience for establishing the basic biochemistry -- we are now 9 years into the unknown covalent arginine modifications -- and we see over and over again how difficult it is to interpret effects without a sound underlying foundation.
Nature Cell Biology | Vol 1 | May 1999 pg 55 Jonathan D.F. Wadsworth, Andrew F. Hill, .. Anthony R. Clarke and John Collinge"...Here we show that two different human PrP Sc types, seen in clinically distinct subtypes of classical CJD, can be interconverted in vitro by altering their metal-ion occupancy. The dependence of PrP Sc conformation on the binding of copper and zinc represents a new mechanism for post-translational modification of PrP and for the generation of multiple prion strains, with widespread implications for both the molecular classification and the pathogenesis of prion diseases in humans and animals."
"So far, PrP Sc types 1 and 4 have been found only in individuals of the MM genotype; type 2 is seen individuals of all genotypes (MM, MV and VV); and type-3 PrP Sc accompanies only the MV or VV genotypes (refs 2, 15, 16 and A.F.H., S.J. and J.C., unpublished observations). "
" When we treated type-1 and type-2 PrP Sc from these patients with 20 mM of the metal-ion chelator EDTA before treatment with proteinase K, the pattern of cleavage was changed. Rather than pro-ducing their distinct patterns, both types gave indistinguishable and common fragment sizes (Fig. 3a).
" We estimated the shift in apparent relative molecular mass from type-1 to type-2 - products and from type-2 MM to type-2 - products to be 1,100 ▒ 300 (mean ▒ s.d.; n = 9) and 650 ▒ 300 (mean ▒ s.d.; n = 9), respectively.
" However, the effectiveness of combined application of triethylenetetramine and dipicolinic acid (Fig. 3f) indicated that chelation of both Cu 2+ and Zn 2+ may be required for generation of type-2 cleavage products from type-1 PrP Sc .
"These findings indicate that the respective conformations of type-1 PrP Sc and type-2 PrP Sc MM may depend upon the presence of metal ions and that metal-ion chelation may induce a conformational change in the protein, exposing a new proteolytic cleavage site that is apparently common to both metal-ion-depleted conformers."
26 May 99 NEJMarticles and press releasesComment (webmaster):I looked at the full text of the NEJM article on "sporadic fatal insomnia" which seeks to carve out a new disease based on 1 individual lacking D178N in a disease with 11 known kindreds worldwide. (The Neurology articles have not yet appeared; this brings the totals to 6 individuals in 24 kindreds -- though we shall see how hard they worked to link seemingly disparate kindreds.)
Most of the article is descriptive pathology. The FFI kindreds was available for comparison were an incomplete set. This does not make for a quantitative basis for concluding that the patient had sporadic FFI, even with depleted fluorodeoxyglucose on the PET. However, they do make the case for a 'pure thalamic dementia' (without saying how many cases per year might not be diagnosed as a CJD). This patient was initially told, 'this is all in your head' and went through an interminable series of expensive and irrelevent tests at an unnamed facility.
Both copies of the gene were carefully sequenced from blood; the patient was met/met. However, the search for somatic mutations was minimally reported (pg 1633) without the primer being disclosed. Only thalamic DNA was considered. People have been ranting for a good decade about somatic CJD; this is the first report I can recall where even minimal effort was expended to find it. (The press releases also cite spontaneous CJD, the promoter and poly A regions were not investigated at all.)
Supposing the adult brain contains 100 billion neurons, the question is how late in its history can a stem cell mutation to D178N give enough daughter cells to later cause sporadic CJD. If familial FFI arises in germ line cells at a 1 per hundred million rate, it would seem that roughly 1,000 somatic FFI events could occur during neural development.
Since any somatic D178N would occur heterozygously and then only in applicable progenitor cell descendants, to find it requires a primer methodology that is completely ineffective at amplifying the normal allele. Negative results should be reported as a lower limit, ie, 'the data show that less than 1 neuron per hundred thousand could have carried a D178N allele.' The actual paper stalls out after conventional microscopy and a few gels.
[Recall that the entire CNS is derived from a neural tube formed in early embryological development. The brain derives from three primary vesicles of the neural tube. By the third week of development, the embryo consists of three cell layers: ectoderm, mesoderm, and endoderm; the neuroectoderm gives rise to the brain, spinal cord, and peripheral nervous system. All neurons come from a common progenitor cell line. The most rostral vesicle is called the prosencephalon. Secondary vesicles give rise to the optic vesicles, which become the retinas and optic nerves, and the telencephalon. What remains of the prosencephalon is now referred to as the diencephalon. The diencephalon differentiates into the thalamus and hypothalamus. The thalamus acts as a relay station between different parts of the brain. Signals from all of the sensory systems, with the exception of smell, pass through here on their way to the cerebral cortex, and the cortex itself uses it to relay signals to other parts of itself.
Subregions of the thalamus include internal medullary lamina, stratum zonale, external medullary lamina, medullary lamina,internal capsule, reticular nucleus, lateral nuclear mass, medial nuclei, anterior nuclei, fork, interlaminar nucle, centromedian nucleus, parafascicular nucleus, central lateral nucleus , reticular nucleus, ventral posterior nucleus,ventral lateral nucleus, metathalamus, medial geniculate nucleus, lateral geniculate nucleus, ventral anterior nucleus, pulvinar, suprageniculate nucleus, nucleus limitans, lateral dorsal nucleus, lateral posterior nucleus, pulvinar, mediodorsal nucleus, medioventral nucleus, nucleus reuniens, paratenial nucleus, etc.] The gustatory thalamus is the functional name for the parvicellular region of the ventroposteromedial nucleus of the thalamus. It is not essential for taste detection, sodium appetite, or conditioned taste aversion, but is critical for food-seeking aspects of taste-guided behavior.
The paper does not address inheritance to any extent; this need not involve the prion gene itself: "No similar neurologic disease was reported in the family" sums up the investigation.
Protein conformation was not studied. As noted, the PrP-Sc fails to strain-type out as FFI because it is partly glycosylated unlike D178N which has excess glycan at 180. "About 19k" does not establish a great deal. The glycoform ratio is that of sporadic CJD, not FFI. Strong clinical and histopathological parallels do exist to FFI but the lack of deeper molecular support trumps these findings.
What is going on? Quite possibly nothing. Here was an insomnia that proved to have protease K resistant prion accumulation and infectivity to mice. It was probably initially misdiagnosed as FFI (ie, make lemonade out of a lemon). Phenotypes have not proven interesting nor instructive so far in TSEs, though much ink has been spilled over splitting them as MDs try to stake claim to "new" diseases. They are too variable, even within the same family with a familial form with identical haplotype. This paper only cites one of several papers reporting FFI variability.
However, the phenomenon itself is novel: a sporadic CJD that presents more or less like FFI. Perhaps there is an interesting explanation, for example, over-production of a particular partial proteolytic fragment as is seen in AD. While they have not gotten to the bottom of the matter, the descriptive aspect has been well done. Let's hope there is follow-up (sequencing of a larger portion of the gene, determination of termini, MS of glycans, somatic sequences seriously sought, heritability looked at harder, and so on).
J A. Mastrianni, R Nixon, R Layzer, G C. Telling, Dong Han, S J. DeArmond, S B. Prusiner N Engl J Med 1999;340:1630-8. full text available for $10 online next day fax.
Neurology (in press). Parchi P, Capellari S, Chin S, et al.
Ann Neurol (in press). Parchi P, Giese A, Capellari S, et al.
press release by researchers. 26 May 99 Associated Press By KATHARINE WEBSTERResearchers have discovered a brain-wasting disease that begins with severe insomnia and ends in hallucinations and death. The disease, sporadic fatal insomnia, is caused by the same type of deformed proteins, known as prions, that cause mad cow disease and its human variant, Creutzfeldt-Jakob disease, according to two studies.
There is no known treatment or cure. It is not clear what causes the disease, but scientists know it is not inherited. And unlike one type of Creutzfeldt-Jakob disease, it is not believed to come from infected meat.
Instead, scientists suspect it is caused by a spontaneous mutation in a single brain or nerve cell. [People have speculated about somatic CJD for decades -- is there support in these articles? -- webmaster] So far, they have identified only six cases of the disease, but there could be others that were misdiagnosed as other mind-destroying illnesses such as Alzheimer's disease.
Prions are proteins with Jekyll-and-Hyde personalities that cluster in the brain. When their molecules are folded into the correct shape, prions are benign, though no one knows their function. Folded the wrong way, prions induce other proteins to mimic them. The misfolded prions then accumulate in parts of the brain, causing the tissue to break down and become full of holes, like a sponge.
Different prion diseases attack different parts of the brain, causing characteristic types of dementia and death. Some are infectious, like mad cow disease in cattle and scrapie in sheep and goats. In humans, prion diseases are either inherited, caught from eating contaminated meat, or spontaneous.
Researchers have previously identified an inherited prion disorder whose main symptom is sleeplessness, called fatal familial insomnia. In a case outlined in Thursday's New England Journal of Medicine, a 44-year-old patient had all the symptoms of the inherited disease, including prions with the same ``signature,'' but did not have the inherited mutation. The case was reported by Dr. James Mastrianni, a neurologist at the University of Chicago. [Again, it seems most unlikely that a quantitative Bruce-type signature was established for anyl of these cases. -- webmaster]
The spontaneous, or sporadic, versions of the prion diseases are rare, but more common than their inherited counterparts. Prions are still controversial. Some researchers believe it is impossible for proteins to replicate and cause disease, since they do not contain genetic material. These researchers believe the diseases are instead caused by as-yet-undiscovered viruses.
However, the new studies add to evidence the infection is caused by the abnormal shape of the prions, not another agent, Drs. Pierluigi Gambetti and Piero Parchi of Case Western Reserve University said in an accompanying editorial. Gambetti and Parchi have described five similar cases of non-inherited fatal insomnia in a study scheduled to appear in the journal Neurology.
New England Journal of Medicine -- May 27, 1999 -- Vol. 340, No. 21 editorial Pierluigi Gambetti, M.D. Piero Parchi, M.D. Case Western Reserve University Cleveland, OH 44106In 1920 and 1921, Creutzfeldt [sic] and Jakob described a rapidly progressive form of dementia that was apparently sporadic; the description of the familial form appeared shortly thereafter. (1) Between 1928 and 1936, Gerstmann, Straussler, and Scheinker described a slowly progressive familial disease characterized by cerebellar signs. The discovery in 1957 of a third such disease, kuru, a subacute condition affecting a New Guinean tribe that practiced cannibalism, suggested that Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, and kuru were related. Since all three diseases were shown to be transmissible to animals, they were grouped under the name "transmissible spongiform encephalopathies" and later came to be known as prion diseases as well. In 1974, the first case of iatrogenic Creutzfeldt-Jakob disease was reported (Table 1).
For many years, Gerstmann-Straussler-Scheinker disease, kuru, and Creutzfeldt-Jakob disease, in its sporadic, inherited, and iatrogenic forms, were the only prion diseases known to occur in humans. (1) This changed with the discovery of two new diseases: fatal familial insomnia and new-variant Creutzfeldt-Jakob disease, a distinct type observed in the United Kingdom that is believed to have been transmitted from cattle to humans through the consumption of contaminated meat. (2,3) In this issue of the Journal, Mastrianni et al. describe a patient with yet another prion disease: the sporadic form of fatal familial insomnia. (4)
With its distinctive features, fatal familial insomnia has widened the clinical and pathologic spectrum of prion diseases. (1) Fatal familial insomnia is a genetically determined condition, associated with the presence of both a mutated codon at position 178 (D178N) and the normal codon for methionine at position 129 on the mutant allele of the prion protein gene (PRNP). The presence of the codon for methionine at position 129, which is the site of a methionine-valine polymorphism, is required for the expression of fatal familial insomnia, since the presence of the codon for valine at the same position on PRNP results in the expression of a subtype of familial Creutzfeldt-Jakob disease.
To date, 24 kindreds affected by fatal familial insomnia have been described, all of which carry the mutant codon 178 plus the normal methionine codon at position 129. Fatal familial insomnia first appears between the ages of 40 and 60 years, and persons who are affected survive for a period of 7 to 33 months, depending on the genotype at codon 129; patients who are homozygous for methionine at this codon have a shorter survival than those who are heterozygous. The disease is characterized by marked reduction of total sleep time along with distinct polysomnographic changes and restlessness, alteration of autonomic functions, and motor signs such as ataxia, dysarthria, and myoclonus.
The histopathological signature of fatal familial insomnia is atrophy of the anteroventral and mediodorsal thalamic nuclei and olives, with mild or no spongiform degeneration of the cerebral cortex. Like most prion diseases, fatal familial insomnia is characterized by the presence of an aberrant isoform of the normal prion protein, called scrapie prion protein (PrPSc). (5) PrPSc contains a fragment that is resistant to treatment with protease and is thought to be the infectious agent. (5)
Two major types of the PrPSc isofo
rm associated with human prion diseases have been identified on the basis of the relative molecular mass of their core fragment (the unglycosylated PrPSc fragment that is resistant to proteases once the sugar chains have been removed). (6) The core fragment of type 1 PrPSc has a molecular mass of 21 kd; the type 2 fragment has a molecular mass of 19 kd. A further subdivision of these two types has been proposed on the basis of the ratio of their glycoforms (see below). (7)
The classifications based on the molecular mass and the glycoform ratio help to characterize the prion diseases with which the different types of PrPSc are associated. Indeed, the PrPSc associated with fatal familial insomnia has a combination of features that distinguish it from the PrPSc isoforms associated with the other human prion diseases. The PrPSc of fatal familial insomnia is present in the brain at low concentrations and with a characteristic regional distribution; the molecular mass of the core fragment is approximately 19 kd (the same as that of type 2), and the unglycosylated form is markedly underrepresented. (8,9)
Mastrianni et al. (4) describe a 44-year-old man with insomnia, dysautonomia, and ataxia, followed toward the end of the fatal 16-month course by hallucinations and myoclonus. Histopathological examination showed lesions that were indistinguishable in type and regional distribution from those of fatal familial insomnia; the amount, the distribution, and the molecular mass of PrPSc in the brain were also similar to those in fatal familial insomnia. However, a rigorous search of PRNP failed to identify the mutation at codon 178 that is associated with fatal familial insomnia. Since the clinical, histopathological, and major PrPSc features were strikingly similar to those of fatal familial insomnia but PRNP mutations were absent, Mastrianni et al. argue that their patient had a sporadic form of fatal insomnia. They term this condition "sporadic fatal insomnia." One might question the establishment of a novel disease on the basis of a single case, but this is not a concern since we have also recently described five patients with similar features. (10) Therefore, sporadic fatal insomnia is unquestionably here to stay as a rare new member of the group of prion diseases (Table 1).
The study by Mastrianni et al. not only describes sporadic fatal insomnia but also provides insight into the mechanisms of prion diseases. Two aspects of their study are especially important. The first is their experimental transmission of sporadic fatal insomnia to mice. Approximately six months after mice that were genetically engineered to express a chimeric mouse-human prion protein were intracerebrally inoculated with brain homogenate from the affected patient, a rapidly fatal prion disease developed. Mastrianni et al. then compared the brains of these mice with brains obtained in a previous study from genetically identical mice that had been inoculated with brain homogenate from patients with fatal familial insomnia or sporadic or familial Creutzfeldt-Jakob disease. All the mice inoculated with brain homogenates from subjects with fatal familial insomnia or sporadic fatal insomnia had lesions of similar types and distributions and a similar distribution of newly formed PrPSc in their brains. Moreover, the molecular mass of the PrPSc core fragment was 19 kd in these mice. In contrast, these characteristics were different in the mice inoculated with homogenate from patients with typical sporadic or familial Creutzfeldt-Jakob disease, and the molecular mass of their PrPSc was 21 kd.
Therefore, sporadic fatal insomnia and fatal familial insomnia are phenotypically similar when they occur naturally in humans, and they also cause a similar prion disease and maintain the characteristics of their protease-resistant PrPSc fragment when they are transmitted to genetically engineered mice. Infectious preparations that contain PrPSc are considered to belong to the same prion "strain" if, on inoculation in genetically compatible animals, they cause a similar disease and maintain the molecular mass of the PrPSc fragment.
The first prion strains to be identified were isolated from sheep with scrapie, and scientists long believed that the strain type was specified by the nucleic acid of the infectious agent. Increasing evidence, some of which comes from the study by Mastrianni et al., indicates that the conformation of PrPSc is what differentiates individual prion strains. (8,11,12) This conclusion led Mastrianni et al. to propose that PrPSc isoforms associated with sporadic fatal insomnia and fatal familial insomnia have the same conformation, which in turn is the primary determinant of the disease phenotype.
Sporadic forms of prion diseases are thought to be caused by a random change in the conformation of the normal, or cellular, prion protein (PrPC) that results in its conversion into PrPSc. (5) The phenotypic similarity of the sporadic and familial diseases suggests that the PrPSc conformation associated with fatal familial insomnia can also be generated in the absence of the D178N mutation. Therefore, the repertoire of conformational changes of PrPSc may be relatively limited -- a factor that may facilitate the discovery of treatments. This is good news because it suggests that inherited forms, which can be more easily reproduced experimentally, are valid models for study of the sporadic forms.
The second important aspect of the study by Mastrianni et al. relates to their finding that in sporadic fatal insomnia, the ratio of the PrPSc glycoforms differs from that in fatal familial insomnia. Under normal conditions, one of the steps in the posttranslational modification of PrPC is the nonobligatory acquisition of sugar chains at its two glycosylation sites. (1,5) This results in the presence of PrPC forms with two sugar chains, one sugar chain, or none at all. The unglycosylated form is markedly underrepresented in PrPSc preparations from patients with fatal familial insomnia, whereas it is well represented in preparations from those with sporadic Creutzfeldt-Jakob disease. (6,8) Mastrianni et al. found that in sporadic fatal insomnia, in contrast to fatal familial insomnia, the unglycosylated form of PrPSc is well represented and the ratio of the glycoforms is similar to that in sporadic Creutzfeldt-Jakob disease. We also found this to be true in each of our five patients with sporadic fatal insomnia. (10)
Mastrianni et al. explain that the scarcity of the unglycosylated form in fatal familial insomnia results from the proximity of one of the PrPC glycosylation sites to the D178N mutation, which leads to excessive glycosylation. An alternative explanation supported by experimental evidence is that D178N, like other PrnP mutations, preferentially destabilizes the unglycosylated form of PrP because this form lacks the stabilizing effect of the sugars. (1,13) Whatever the mechanism, the low proportion of the unglycosylated form of PrPSc in fatal familial insomnia appears to be a direct effect of the mutation, and therefore this characteristic would be absent in sporadic fatal insomnia. Furthermore, since sporadic fatal insomnia and fatal familial insomnia are phenotypically indistinguishable, it appears that the glycoform ratio is not a primary determinant of the prion strain or of the disease phenotype. (7)
Our findings (10) and those of Mastrianni et al. establish the existence of the sporadic form of fatal insomnia. Other variants of sporadic Creutzfeldt-Jakob disease have also been recently characterized. (14) These findings emphasize the need to monitor prion diseases and to assess their potential risks to public health. Surveillance is required not only to detect novel variants of prion diseases but also to monitor their prevalence and to determine whether they are sporadic, inherited, or acquired by infection from animals or humans. Many European countries already have surveillance systems. In the United States, the Centers for Disease Control and Prevention are expanding existing surveillance mechanisms by establishing a center with expertise in molecular pathology.
Neurology 1998 Dec;51(6):1715-7 Johnson MD, Vnencak-Jones CL, McLean MJWe describe clinical and pathologic features of a patient with fatal familial insomnia (FFI) whose prion (PrP) genotype is D178N coupled with methionine at codon 129 on his mutant allele and valine at codon 129 on his normal allele. A cousin (genetic half brother) with identical PrP genotypes exhibited strikingly different clinical and pathologic changes. Comparison of these cousins shows the phenotypic heterogeneity of FFI and suggests that the phenotypic expression of D178N is influenced by multiple factors.
Neurology 1998 Nov;51(5):1398-405 Zerr I, Giese A, Windl O, ... Skworc K, Bodemer M, Kretzschmar HA, Poser SClinical and pathologic features of patients with the D178N-129M mutation living in Germany: Patients with clinically suspected Creutzfeldt-Jakob disease (CJD) were seen in an ongoing, prospective epidemiologic study from June 1993 to August 1997 throughout Germany. Suspect patients were referred to the CJD unit by the participating hospitals or physicians. Patients were seen by a physician, and each patient underwent a detailed neurologic examination. Prion protein gene (PRNP) analysis was performed to distinguish patients with familial forms of CJD.
The constellation D178N-129M was identified in eight individuals; in one patient, the diagnosis was made by neuropathologic examination. Four affected men and five women belong to eight unrelated families. A family history of a neurodegenerative disorder was recalled in only five patients. In contrast to the first reported fatal familial insomnia (FFI) patient, none of our patients complained of severe, untreatable insomnia in the early stages. Dysautonomia was observed in varying degrees in most patients. The clinical course of these patients resembled sporadic CJD. In six patients, brain tissue was available for neuropathologic study. In one patient, the neuropathologic examination showed changes that were more reminiscent of forms of sporadic CJD; in the remaining five, the histopathology was typical of FFI. The clinical presentation in patients with FFI may vary to a great extent. Genotyping of the patients was crucial in providing laboratory confirmation of the diagnosis of FFI, even when there was no family history of a prion disease.
Neurology 1998 Mar;50(3):688-92 Rossi G, Macchi G, Porro M,...Molini GE, Sasanelli F, Bugiani O, Tagliavini FFatal familial insomnia (FFI) is an inherited prion disease linked to a mutation at codon 178 of the PRNP gene that results in aspartic acid to asparagine substitution, in coupling phase with methionine at position 129. The disease is characterized clinically by insomnia with disturbances of the autonomic, endocrine, and motor systems and neuropathologically by selective degeneration of the thalamus. Phenotypic variability is well known and has been linked to homozygosity or heterozygosity at PRNP codon 129. We report the clinical, neuropathologic, and biochemical findings and genomic analysis of a patient with FFI from a new Italian kindred. Although homozygous for methionine at codon 129, this patient showed some clinical and pathologic features most commonly found in heterozygotes.
Neurology 1997 Aug;49(2):552-8 McLean CA, Storey E, Gardner RJ, Tannenberg AE, Cervenakova L, Brown PFatal familial insomnia (FFI) is an inherited prion disease characterized by progressive insomnia and dysautonomia with only modest cognitive impairment early in the disease, associated with atrophy and gliosis in the medial thalamus, but without spongiform change. FFI is associated with an aspartic acid to asparagine mutation at codon 178 of the PrP gene (D178N) in conjunction with methionine at the codon 129 polymorphic site on the mutant allele (cis-129M). We report a pedigree with this genotype in which marked clinicopathologic phenotypic heterogeneity occurred including typical Creutzfeldt-Jakob disease, FFI, and what was thought to be an autosomal dominant cerebellar ataxia (ADCA)-like-illness, suggesting that the genotype-phenotype correlation is not as tight for this mutation as is frequently supposed.
Neurology 1997 Jul;49(1):126-33 Cortelli P, Perani D, Parchi P, Grassi F, Montagna P, De Martin M, Castellani R, Tinuper P, Gambetti P, Lugaresi E, Fazio FWe used [18F]-2-fluoro-2-deoxy-D-glucose (FDG) and PET to study regional cerebral glucose utilization in seven patients with fatal familial insomnia (FFI), an inherited prion disease with a mutation at codon 178 of the prion protein gene. Four patients were methionine/methionine homozygotes at codon 129 (symptom duration, 8.5 +/- 1 months) and three were methionine/valine (MET/VAL129) heterozygotes (symptom duration, 35 +/- 11 months).
A severely reduced glucose utilization of the thalamus and a mild hypometabolism of the cingulate cortex were found in all FFI patients. In six subjects the brain hypometabolism also affected the basal and lateral frontal cortex, the caudate nucleus, and the middle and inferior temporal cortex. Comparison between homozygous or heterozygous patients at codon 129 showed that the hypometabolism was more widespread in the MET/VAL129 group, which had a significantly longer symptom duration at the time of [18F] FDG PET study.
Comparison between neuropathologic and [18F] FDG PET findings in six patients showed that areas with neuronal loss were also hypometabolic. However, cerebral hypometabolism was more widespread than the histopathologic changes and significantly correlated with the presence of protease-resistant prion protein (PrPres). Our findings indicate that hypometabolism of the thalamus and cingulate cortex is the hallmark of FFI, while the involvement of other brain regions depends on the duration of symptoms and some unknown factors specific to each patient. The present data also support the notion that PrPres formation is the cause of neuronal dysfunction in prion diseases.
Am Heart J 1999 Jun;137(6):1173-1178 Fagih B, Eisenberg MJIn 1996, the Quebec Ministry of Health and Social Services, concerned that reuse of devices contaminated with blood or blood products could cause the transmission of Creutzfeldt-Jakob disease (CJD), discontinued its practice of reusing angioplasty catheters despite the significant cost savings reuse had afforded the health care system for several years. The objective of this study was to establish whether the medical literature provides documentation of any cases in which CJD was transmitted by reused percutaneous transluminal coronary angioplasty (PTCA) equipment.
A Medline search was performed to identify previous studies that examined this issue. Key words for the search included PTCA, CJD, and material and equipment reuse. A substantial amount of effort has been spent on the study of PTCA catheter reuse in relation to the risk of infection, toxicity, and catheter breakage as well as cost. In Quebec, studies by the Conseil d'Evaluation des Technologies de la Sante investigated the effectiveness of cleaning and sterilizing PTCA equipment and considered the possibility that reuse of single-use catheters, hemodialyzers, and cardiac pacemakers could spread CJD. A number of other studies found evidence that iatrogenic transmission was responsible for several cases of CJD by direct implantation in or adjacent to the central nervous system during neurosurgery. CJD was also transmitted to human beings by injection of pituitary growth hormone and to mice through cerebral inoculation of contaminated blood and urine.
However, there were no documented cases of CJD occurring as a complication of PTCA equipment reuse. The current literature indicates that there are no known cases of CJD attributable to the reuse of PTCA devices contaminated by blood or to the transfusion of blood or blood products. This practice is associated with a very low risk of CJD transmission. With the considerable cost savings made possible by reuse of PTCA equipment, reimplementation of this practice should be considered by health delivery systems, provided that stringent methods of cleaning and sterilization are observed.
Comment (webmaster): Simply performing a Medline search does not constitute publishable research. Anyone in the field could have told the authors there were no known cases and no known studies. Much more ominous in this regard is the new Australian study on overall surgical risk. The issue is, do the authors want to keep re-using this equipment in the context of nvCJD, which is too new to allow angioplasmy cases to have emerged?
Archives of Virology 144 Issue 4 (1999) pp 829-834 A. Bossers*, F. L. Harders, M. A. Smits * 145 page thesis just released in the form of a book.Comment (webmaster): Is scrapie a genetic disease? Now no one doubts that 8 extra prion repeats in a genetically engineered sheep would cause scrapie in and of itself. However, such mutations have not been seen in actual scrapie sheep so far. Instead, various alternatives alleles to wildtype are found, with A136V homozygotes being the most suspicious of these. Because of pre-existing infection and facility contamination issues, it is not an easy matter to simply to raise a flock of these and see if they contract scrapie.
In one animal husbandry nightmare scenario, the genetically modified food scare of the 1700's, familial scrapie was inbred into sheep centuries ago in the form of A136V. This would be like a royal family harboring P102L instead of hemophilia. This unfortunate sheep line not only developed scrapie to some extent if allowed to live to old age but also was capable of propagating it horizonally and maternally, jump-starting the infection in sheep with similar and dissimilar genetics.
If one takes New Zealand's claims to be scrapie-free at face value (no outside group has ever validated this), and if a flock of VRQ/VRQ sheep could be identified and raised in isolation to advanced age and compared to an ARQ/ARQ control group, the question of A136V causality might be settled. (Alternatively, a super-sensitive test for scrapie might allow young sheep to be screened for incipient scrapie.)
The first issue has been settled by the paper below. That is, the highest risk allele does exist in New Zealand at about 1 in 1,666 sheep, or 27,611 sheep in the population of 43 million at any given time. However, tens of thousands of sheep need to be genotyped in order to collect an adequate study population of VRQ/VRQ. While this seems like an extremely excessive effort for a simple test of a point mutation, there are significant implications to breeding out scrapie. No sheep were actually tested for scrapie here or in the papers from Hunter's group.
I would like to see New Zealand adopt an explicit policy of allowing foreign researchers to do what they need to do, ie, IHC on a sample of these 27,611 sheep. Otherwise, spare us the scrapie-free anecdotes. If New Zealand truly are scrapie-free, what is there to be afraid of? The fact is,like everyone else, they really don't know if A136V scrapie can be a purely genetic disease.
In sheep, codon A136V, Q171R, and to a minor extent 154H contribute to the susceptibility of sheep for scrapie . The polymorphisms at codons 112, 137, 141, 171 (Q to H), and 211 are rare and have not been associated yet with a scrapie phenotype. In sheep breeds that carry PrP VRQ at codons 136, 154, and 171, there is a high susceptibility to scrapie. The PrP ARR allelic variant is associated with resistance in all breeds investigated so far. In breeds in which the PrP VRQ allele is rare or absent, for instance the Suffolk breed, the wildtype PrP ARQ allele is associated with susceptibility to scrapie. In breeds that contain the PrP VRQ allele, the PrP VRQ /PrP VRQ sheep are at greatest risk from scrapie followed by the PrP VRQ /PrP ARQ sheep, at least in Britain and Europe. In breeds in which the PrP VRQ allele is rare or absent, the PrP ARQ /PrP ARQ sheep are at greatest risk. In sheep with the latter homozygous PrP genotypes survival times of less than 14 months have been recorded (L. van Keulen, pers. comm.).
Is scrapie is a genetic disease arising spontaneously from sheep with certain PrP alleles in the absence of any infectious agent? Observations on the transmission of scrapie in embryo transfer experiments however indicated that scrapie is not purely a genetic disease. In addition, sheep with PrP genotypes at greatest risk from scrapie have been found in Cheviot and Suffolk sheep from Australia and/or New Zealand. Since these countries are free from scrapie for decades and scrapie has become a notifiable disease in New Zealand since 1955, it is likely that scrapie is not a spontaneous genetic disease linked to the PrP gene alone, although development of the disease is strongly influenced by PrP.
Here we determined the PrP allele- and genotype frequencies of the most dominant sheep breed in NewZealand since 1900, the Romney Marsh. This breed was first imported from Britain into New Zealand in 1852. It was used to develop two new breeds, the Perendale and the Coopworth. The Romney Marsh and these two new breeds form the bases of the entire New Zealand sheep flock, which consists of about 46 million sheep, 58% Romney Marsh, 17% Perendale and Coopworth, and 25% other breeds. No new Romney Marsh sheep have been imported into New Zealand since the beginning of this century.
The authors collected blood samples of 140 random animals less than two years of age from 14 different flocks, scattered all over the country. These farms normally breed their own replacement animals and only purchase rams. The sampled flocks are likely representative for the entire Romney Marsh flock.
PCR amplified products were analysed by DGGE) which not only identifies known PrP alleles but also new ones. However no new ones were found, though AF141RQ was found in 7 sheep. The analysis revealed the presence of five different PrP alleles in the New Zealand Romney Marsh breed, including alleles linked to the highest scrapie susceptibilities (PrP VRQ allele, linked to the highest susceptibility, was present at a frequency of 2.5%, but never in homozygotes). Flock VIII had an unusually high PrP VRQ frequency of 15%, so VRQ / VRQ of2.25%, one per 44 animals expected; flock X had a relatively high frequency of PrP AHQ, and flocks XII and XIV were high in PrP AF141RQ .
PrP genotype frequencies: homozygotes in blue ARQ/ARQ 21 wildtype ARR/ARR 32 resistance AHQ/AHQ 1 weak susceptibility VRQ/ARQ 4 first allele is high susceptibility VRQ/ARR 3 double difference ARQ/ARR 54 ARQ/AF141RQ 5 ARQ/AHQ 9 ARR/AHQ 9 ARR/AF141RQ 2 Total 140
No VRQ homozygotes were found in 140 sheep; however they should be present in the population at a frequency of about one per 1666 animals. Three of the 140 tested animals (2,9%) contain the PrP VRQ /PrP ARQ genotype. In an environment where scrapie is endemic, such animals would also succumb to scrapie.
Similar data were reported for Merino sheep of Australia and Poll Dorsetts of New Zealand [Hunter N, Cairns D (1998) J Gen Virol 79: 2 079-2 082]. See also: Hunter N, Cairns D, Foster JD, Smith G, Goldmann W, Donnelly K (1997) Is scrapie solely a genetic disease? Nature 386: 137.
2 June 99 ListserveThe following two reports have just appeared on the EU DG24 Web site:
- Scientific Steering Committee: Surveillance of TSEs in sheep and goat in relation to the risk of infection with bovine spongiform encephalopathy agent and related actions to be taken at EU level (Adopted by the SSC at its meeting on 27-28 May 1999).
- A sheep experimentally infected orally with 0.5g of infected bovine brain homogenate developed clinical disease at 734 days and was subsequently shown to have BSE infectivity in its brain and spleen, demonstrates that even small amounts of BSE-infected MBM fed to sheep are likely to have presented a risk of BSE infectivity;
- With the evidence for the inclusion of ruminant derived meat-and-bone meal in sheep feeds, it should be assumed that certain sheep flocks in these circumstances were exposed to BSE. The infection, if it replicated in a pregnant female, may well be sustained in the sheep flocks by vertical and horizontal transmission. One of the main problems, however, is obtaining data on historical feeding practices in different countries, regions and flocks. The crucial feature is whether or not concentrates have been fed rather than MBM due to the problems of cross contamination of supposedly MBM free diets;
- Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate scrapie CH1641 (J.Hope et al, 1999). After inoculation of this scrapie strain to AA HH or HR QQ "negative" sheep, a C type PrP was reisolated, a similar PrP glycoform type C as after inoculation of the same type of sheep with BSE. This suggests that molecular strain-typing could distinguish BSE from group A or B types of scrapie in sheep but, as the frequency of CH1641 or other C-type scrapie isolates is unknown, it may have limited usefulness for positively identifying BSE in the national flocks. Furthermore, since CH1641 pre-dates the outbreak of BSE by 15 years - it was isolated from a natural case of scrapie culled in June 1970 - its biochemical similarity to BSE provides some circumstantial support that BSE could have originated from a C-type sheep scrapie strain.
- In two "positive" line Cheviot sheep orally challenged with BSE, an atypical TSE strain was isolated. One of the explanations could be that indicating that the phenotype of the BSE agent may not be stable on serial passage within sheep (see SSC opinion on BSE in sheep). If this is confirmed, a major problem will arise when strain typing TSE isolates from sheep using the present methodology (which currently involves mice inoculation and western blot analysis);
"..Attention was drawn by the Scientific Veterinary Committee to the high incidence of familial CJD in the Orava region of Slovakia at a level of about 30 times the normal level of 1 per million adults per year. This form of familial CJD is attributed to mutations in the PrP gene at codon 200. This particular mutation is not fully penetrant and some of the workers in Slovakia have suggested that exposure to sheep scrapie may also be necessary for disease to result [ie, jump-start the disease in people with a genetic predisposition -- webmaster]. This is partly because scrapie had been identified in sheep in the region following a period since the late 19th century when scrapie was thought to be absent from the country."
- The clinical signs of experimental BSE in sheep could differ from the typical clinical signs of scrapie. This could depend on the effect of the different strain of agent and also possibly depending on the genotype of sheep. This is because after experimental inoculation of BSE an atypical wasting syndrome with slight neurological signs was observed in one ewe.
This could cause a major problem in the frame of an epidemiosurveillance network based only upon a clinical suspicion after typical scrapie signs. Moreover, it cannot be excluded that a surveillance targeted on scrapie susceptible genotypes of sheep might miss BSE cases in scrapie resistant flocks. Discrimination between natural BSE (if it occurs) and scrapie, not only at the clinical but also the pathological or aetiological level, by a rapid, reliable and cost-effective test is currently not possible and is likely to be a dot on the horizon for some time to come. But it should be underlined that the development of a practical test to distinguish between scrapie and BSE is an absolute priority. This would enable the small ruminant scrapie/BSE situation in the different countries to be assessed and measures to be taken if necessary to avoid economic barriers based only upon hypotheses.
- Scrapie is a concealed or partially concealed disease in most countries in which it occurs and even financial incentives to report may not reveal the extent of the disease. Anonymous questionnaires could be a more effective means of estimating the true incidence....."
c. Creating an efficacious information network between research groups to keep all the concerned groups continuously informed on the progress made in the different TSE in sheep experiments.
The present report of the TSE/BSE ad hoc group is substantially based on the work of a Working group chaired by Dr E. Vanopdenbosch who was also the rapporteur. The other members of the group were: Dr N. Hunter, Dr B. Schreuder, Dr L. Detwiler, Prof M. Woolhouse, Dr Elsen and Prof.M.Ulvund. Dr R. Bradley also prepared various contributions.
- Scientific Steering Committee: Opinion on Monitoring some important aspects of the evolution of the Epidemic of BSE in Great-Britain (Status, April 1999) adopted by the Scientific Steering Committee at its meeting of 27-28 May 1999
"The current and expected evolution of number of BSE cases in the UK (1999-2004) are in line with all models, but the tail of the epidemic will not necessarily present a constant decline, certainly not when small numbers are involved; a reassessment can be needed if by applying better diagnostic measures and an improved quality of surveillance a much higher than expected number of cases is revealed (potential problem of underreporting). In the latter context it is mentioned that the relative importance of underreporting may increase as the incidence decreases at the tail-end of an epidemic."