The A117V mutation in the tiso ansmembrane domain
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Safety tips for anatomic studies of possible CJD
Press release: Prion disease mechanism found
Highligts of the Science article
V. Lingappa offers further clarification
Commentary: prions and the endoplasmic reticulum
Why not more genetics of cattle?
Breeding out scrapie -- will it work?
MAFF answers email research queries
Are florid plaques enough?
Protective polymorphisms: codon 219 is better
Anti-prions resurrected
Chromosomal organization of 3 prion genes
Bone marrow: bad news in Blood
S100 Blood check may give simpler CJD diagnosis

Safety tips for anatomic studies of possible CJD

Barbara J. Crain, MD, PhD     [no contact info]
College of American Pathologists
[goes on for 6 pages, the two cites are from 1990; page last modified Jun 12, 1997. Basically bleach or sodium hydroxide, double glove, careful cleanup and disposal. If someone sees problems with recommendations, it might be important to write, as a lot of pathologists might see the CAP web page. -- webmaster]

S100 Blood check may give simpler CJD diagnosis

20 Feb 1998	Times BY NIGEL HAWKES 
BMJ links: free fulltext to everyone!
Maurizio Pocchiari commentary
Main article
PET scan article
BSE and pharmaceuticals
CJD was not diagnosed until eight months after organ donor's death G D Morrice: offline

A SIMPLE test for Creutzfeldt-Jakob disease could come from measuring the levels of a protein in the blood, German scientists have reported. They have found that the protein S100, often found in the bloodstream after brain damage, can be used for diagnosis. The test is simpler and safer than examining spinal fluid for traces of aberrant prion proteins believed responsible for the disease. Rapid diagnosis would be valuable because in the new variant form of CJD, victims often first complain of severe depression or other psychiatric disorders. Only later do more specific symptoms appear. Earlier diagnosis could prevent inappropriate treatment and minimise the risk of victims passing on the disease, such as through blood transfusions.

In the British Medical Journal, a team led by Markus Otto of the Psychiatric Clinic at Georg-August University in G–ttingen examined blood samples from 224 suspected victims. Levels of S100 were about three times as high in the 108 patients ultimately found to have CJD. Originally S100 was thought to be produced when brain cells were damaged by disease or accident, but it is now believed to be a marker for higher levels of activity in cells known to be involved in CJD.

The test does not distinguish between new variant CJD and the classical form, but the new variant occurs in younger people while traditional CJD is found in the elderly, or in patients with relations who have also had it.

A second test is described by a team led by Rajith de Silva of Southern General Hospital, Glasgow. Two cases of new variant CJD were given brain scans in which radioactive materials are injected into the bloodstream and imaged to show the brain. The results showed a decline in blood flow in CJD patients.

Brain-destroying Process Identified

Feb. 5/98 AP Paul Recer
WASHINGTON -- A team from the University of California, San Francisco, was cited as reporting in Science tomorrow that it has discovered a type of prion that attaches to a key structure in neuron cells and triggers a signal that causes the cell to die.

Dr. Vishwanath R. Lingappa, the study's lead author, was cited as saying that the research reveals a process "that is at the heart of at least one prion disease," but he said it is not clear if the same process occurs in all prion diseases.

In the latest study, the California researchers used a form of mutated prion that is different from the mad cow prion to search for an explanation of how the proteins cause disease.

Lingappa was cited as saying they found that when the abnormal prion is made in the cell, it becomes stuck in a structure called the endoplasmic reticulum, a membrane that makes proteins and moves them around within the cell. When the prion is lodged in the membrane, it triggers a signal that causes the cell to die.

A Transmembrane Form of the Prion Protein in Neurodegenerative Disease

5 Feb 98 Science fulltext [subscription req'd]
Ramanujan S. Hegde, James A. Mastrianni, Michael R. Scott, Kathryn A. DeFea, 
Patrick Tremblay, Marilyn Torchia, Stephen J. DeArmond, Stanley B. Prusiner, 
Vishwanath R. Lingappa

Highlights from the article:

At the endoplasmic reticulum membrane, the prion protein (PrP) can be synthesized in several topological forms. The role of these different forms was explored with transgenic mice expressing PrP mutations that alter the relative ratios of the topological forms. Expression of a particular transmembrane form (termed CtmPrP) produced neurodegenerative changes in mice similar to those of some genetic prion diseases. Brains from these mice contained CtmPrP but not PrPSc, the PrP isoform responsible for transmission of prion diseases. Furthermore, in one heritable prion disease of humans, brain tissue contained CtmPrP but not PrPSc. Thus, aberrant regulation of protein biogenesis and topology at the endoplasmic reticulum can result in neurodegeneration.

PrP is a highly conserved, 35-kD brain glycoprotein essential for the transmission and pathogenesis of several neurodegenerative diseases, such as scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease (CJD), and Gerstmann-Straussler-Scheinker (GSS) disease (1). Although the normal function of PrP remains unclear, the pathogenesis of prion diseases requires its expression (2-4) and is often accompanied by the accumulation in the brain of an abnormal isoform of PrP (termed PrPSc). Considerable evidence from biochemical, immunologic, pathologic, and genetic studies argues that PrPSc is the major, if not only, component of the transmissible prion particle [reviewed in (1)]. Furthermore, the data suggest that PrPSc is able to propagate itself in the host by stimulating the conversion of normal cellular PrP (termed PrPC) to PrPSc, leading to its accumulation (5, 6). Thus, although the exact mechanism of PrPC to PrPSc conversion remains unknown, a foundation has been laid for the understanding of prion disease transmission, on which subsequent structural, biochemical, and cell biological studies can be based.

More enigmatic at the present time is our understanding of the biochemical and cell biological events that form the basis for the pathophysiological progression of neurodegeneration in prion diseases. The role of PrPSc in the pathologic process leading to neuronal death is currently unclear. The observation of substantial neurodegeneration in the absence of PrPSc accumulation in some cases of natural and experimental prion disease argues against its accumulation as the sole cause of pathology (7, 8). Conversely, the time course of PrPSc deposition in the brains of mice expressing low levels of PrPC does not correlate with the time course of neurodegeneration (9), raising the possibility that PrPSc is not directly toxic. This possibility is further supported by the demonstration that PrPSc deposition fails to cause disease in brain tissue lacking PrPC (2). Thus, although conversion of PrPC to PrPSc appears to be central to transmission, other aspects of PrP expression, folding, and trafficking may feature in the pathophysiological mechanisms that ultimately cause disease.

Studies of PrP translocation at the endoplasmic reticulum (ER) membrane have revealed unusual features in its biogenesis. Whereas most glycoproteins are synthesized in a single orientation with respect to the membrane of the ER, PrP synthesized in cell-free translation systems can be found in more than one topologic form (10-13). One form appears to be fully translocated into the ER lumen and hence is termed the secretory form (secPrP). This topology is consistent with much of what is known about PrPC, which is on the cell surface, tethered to the membrane by a glycolipid anchor whose cleavage results in release from cells (14). The remainder of the PrP made at the ER spans the membrane, with regions of the molecule exposed to the cytosol. However, the relation between these multiple topological forms of PrP and neurodegenerative disease has not been established, nor have transmembrane forms of PrP been previously detected in the brain.

Here, we used transgenic mice that express various mutations in PrP to examine the possible role of PrP topology in neurodegeneration. Our data indicate that a specific transmembrane form of PrP (termed CtmPrP, see below) can confer severe neurodegeneration in mice with features typical of prion disease. Subsequent analysis of the human neurodegenerative disease caused by the A117V mutation [alanine (A) to valine (V) substitution at position 117] in PrP suggests that its basis lies in increased production of CtmPrP at the ER membrane. These findings identify CtmPrP as a key component in the pathway of neurodegeneration in a specific human disorder (GSS) and may have important implications for a broader range of neurodegenerative diseases.

PrP topology. The events of secretory and membrane protein biogenesis can be faithfully reconstituted and studied with cell-free translation systems containing ER-derived microsomal membranes (15). Topology of a protein can be assessed by determining whether any regions of the molecule are accessible to proteases added to the outside of the membrane vesicles. Full protection from exogenous protease indicates complete translocation into the ER lumen. Conversely, digestion of certain domains to yield discrete protease-protected fragments indicates a membrane-spanning topology, the exact orientation of which can be clarified by identification of the protected fragments with epitope-specific antibodies. This use of proteases as a probe of topology is distinctly different from the use of proteases as probes of protein conformation (for example, the protease resistance of PrPSc). Because the topology assay is carried out in the absence of detergent, the protection from protease is due to an intact membrane barrier. In contrast, the assay of PrP conformation by use of proteinase K (PK) is carried out in detergent solution, where only particular conformations of PrP, and not a membrane barrier, can protect it from digestion (16).

Previous analyses of PrP topology have suggested that two distinct forms of PrP can be made at the ER: one that is fully translocated (secPrP) and one that is transmembrane (10-12). Digestion of the transmembrane form with proteases added to the outside of the membrane yielded two fragments: One is COOH-terminal derived and glycosylated, and the other is NH2-terminal derived and unglycosylated. These data have been interpreted to indicate that transmembrane PrP chains span the membrane twice, with the NH2- and COOH-termini of the molecule in the ER lumen, protected from proteases added to the cytosolic side (10, 13). However, the findings described below suggest that the NH2- and COOH-terminal fragments reflect the existence of two different transmembrane forms of PrP (Fig. 1A). One form, termed C-trans transmembrane (CtmPrP), has the COOH-terminus in the ER lumen with the NH2-terminus accessible to proteases in the cytosol.

The other form, termed N-trans transmembrane (NtmPrP), has the NH2-terminus in the ER lumen with the COOH-terminus accessible to proteases in the cytosol. Both transmembrane forms appear to span the membrane at the same hydrophobic stretch in PrP (roughly residues 110 to 135, previously termed TM1). For this reason, the proteolytic fragments derived from each transmembrane form share a common domain of PrP approximately from residues 105 to 140 (the residues immediately adjacent to the membrane-spanning domain are not digested by protease, perhaps because of steric hindrance by the membrane itself). Thus, both fragments share the epitope for the 3F4 monoclonal antibody (mAb) (to residues 109 to 112), whereas only the COOH-terminal fragment contains the epitope to the 13A5 mAb (to residues 138 to 141). These differences in antibody reactivity, glycosylation, and size allow the NtmPrP and CtmPrP fragments to be clearly distinguished.

A new paradigm for PrP-induced neurodegeneration. Our studies were motivated by the desire to determine the role in prion diseases played by a transmembrane form of PrP that was first identified through the use of cell-free translation-translocation systems (10). The hypothesis that a specific topologic form of PrP, namely CtmPrP, is involved in the development of neurodegeneration is supported in several ways (summarized in Table 1).

First, two independent mutations of PrP (KHII and AV3), which were initially identified by their ability to favor markedly the synthesis of CtmPrP in cell-free systems, caused accelerated spontaneous neurodegeneration and death in transgenic mice. Second, other PrP mutations in the STE-TM1 domain that abolish expression of CtmPrP in cell-free systems (STE and G123P) did not cause spontaneous disease in transgenic mice.

Third, biochemical examination of PrP topology in the brains of transgenic mice expressing pathogenic mutations of PrP revealed the presence of CtmPrP in vivo, whereas this form was not seen in mice expressing a comparable level of wild-type PrP. Fourth, mice expressing the pathogenic KHII mutation at lower levels did not contain CtmPrP (but did contain secPrP) in brain and, correspondingly, failed to show signs of disease. Fifth, examination of the brains of ill mice failed to reveal the presence or accumulation of protease-resistant PrPSc.

Finally, a naturally occurring human disease-causing mutation in PrP caused increased CtmPrP formation in vitro and in vivo. Taken together, these data strongly suggest that expression of CtmPrP can cause neurodegeneration.

Several lines of evidence currently indicate that the pathologic processes resulting in CtmPrP-associated neurodegeneration are directly relevant to prion diseases. First, the neuropathology observed in the brains of Tg[SHaPrP(KHII)H] and Tg[MH2MPrP- (AV3)] is very similar to that observed in several prion diseases (27, 30). Second, the apparent lack of protease-resistant PrPSc in Tg[SHaPrP(KHII)H] and Tg[MH2MPrP- (AV3)] mice is not unprecedented in prion disease. Rather, in several inherited prion diseases, certain mutations cause neuropathology despite little detectable PrPSc (8, 27, 31).

Finally, it does not appear that the disease observed in this study is due to nonspecific accumulation of misfolded or aggregated PrP molecules. This conclusion is supported by the observation of nearly all of the CtmPrP being located in a post-ER compartment, a lack of excessive accumulation of PrP (that is, no plaques or other localized deposition of PrP), and a lack of neuropathology characteristic of a storage-type disorder (32). Together, these data support the hypothesis that expression of CtmPrP may be involved in the pathologic process occurring in at least a subset of heritable prion diseases.

At present, the data in this study are suggestive of at least three distinct steps in the pathogenesis of CtmPrP-associated neurodegenerative disease (Fig. 7). The first step is preferential synthesis of CtmPrP over other topological forms at the ER membrane. Second, newly synthesized CtmPrP, which may normally be degraded rapidly, exits from the ER. Finally, in a post-ER compartment, CtmPrP is proposed to serve an as yet unknown function to cause, directly or indirectly, neurodegeneration.

There are three lines of evidence for the first step. First, PrP can be made in more than one topological form in cell-free translation systems [(10-12), and this study]. Second, CtmPrP has now been detected in vivo (Fig. 3A). Finally, PrP topology is dependent on both the amino acid sequence [(12), and this study] and trans-acting factors in the cytosol (13) and in the ER membrane (18). Thus, not only is PrP able to be made in multiple topological forms, but this step appears to be regulated in a complex manner. One prediction of this hypothesis is that, just as mutations in cis are able to affect both PrP topology and development of disease, loss or inactivation of specific trans-acting factors might also alter PrP topology in favor of CtmPrP and potentiate development of neurodegeneration.

Once synthesized at the ER, CtmPrP is thought to exit to a post-ER compartment on the basis of the acquisition of resistance to digestion by endo H (Fig. 3B). We postulate that although exit from the ER can occur in some instances, CtmPrP may normally be degraded before exit, a fate observed for some posttranslationally regulated proteins (33). This degradation may explain why the expression of any given PrP construct at the ER in the cell-free system results in a higher percentage of transmembrane PrP chains than were detectable for that construct in brain at steady state. The observation of a lack of CtmPrP accumulation in Tg[SHaPrP(KHII)L] mice, despite readily detectable secPrP, supports this model.

At the present time, we do not know how CtmPrP, upon exit from the ER, is able to cause disease. The correlation of three independent PrP mutants that promote CtmPrP synthesis in cell-free systems, with development of neurodegenerative disease in either transgenic mice or humans, and the detection of CtmPrP in brain suggest a causative role in disease. Whether inappropriately expressed CtmPrP is able to initiate specific signaling events to cause cell death or accomplishes this end by another mechanism remains to be determined. The pathway involving CtmPrP may not be the only pathway of neurodegeneration, although it appears to be the one used in GSS(A117V), as demonstrated here. Recognition of CtmPrP and its involvement in some cases of GSS should facilitate the biochemical identification of other steps in a putative signaling cascade. Proteins undergoing topological regulation like CtmPrP may be involved in neurodegenerative diseases besides those currently attributed to prions.

Some questins answered

10 Feb 1998 Vishwanath R. Lingappa comments

On the eukaryotic ER translocon

1. It is important to remember that Sec 61p, SRP receptor and TRAM are the MINIMUM components of the translocation channel (termed the translocon). Our working hypothesis is that there are translocation accessory factors (termed TrAFs) that facilitate or regulate biogenesis of various subsets of specialized proteins. PrP is the first proteins shown to use such accessory factors (we have just submitted a manuscript describing a "protective factor" in normal brain that prevents transmembrane PrP expression). The Science paper makes the argument through a genetic mutation in the translocation substrate (i.e. PrP), that the pathway defined by TrAF activity is pathophysiologically relevant.

Why is 106-126 not a known protein motif?

2. Regarding evolutionary conservation. It is hard to know, at this point, if conservation is for TrAF-related purposes or for some other reason. The problem here is that the machinery for other translocation-related events, e.g. signal sequence recognition, stop transfer recognition, is highly conserved, but the sequences recognized are remarkably degenerate. Thus sequence homology has not proved to be an effective way to recognize all signal sequences or stop transfer sequences. Much more reliable is FUNCTIONAL screening for translocation activity. I expect this to be the case for STE and TrAF recognition. Put another way, the machinery is a lot smarter than we are, at present, and is able to recognize and decode information (e.g. based on secondary structure) where we would not recognize primary amino acid sequence homology.

Keep in mind that there are some special features about translocation that make it unlike simple hormone-receptor type interactions: It takes place while the chain is nascent and hence changing and not at equilibrium; It takes place in a restricted space (the aqueous translocation channel) that is not accessible to the cytosol -- except underspecial circumstances that are themselves regulated, see Cell 85, 217 1996), etc. Thus, relatively low affinity interactions, which facilitate rapid "off" rates for receptor binding, but whose "on" rates are maintained by the high local concentration of the nascent chain may be the rule, and we really have not had a lot of precedents to study these previously.

Will A117V be transmissible, ie is there a new toxic mechanism?

Note in DR Brown et al. Nature 1996 Mar 28;380(6572):345-347, Exp Neurol 1997 Jul;146(1):104-112, Histol Histopathol 1997 Jul;12(3):883-892, and Glia 1997 Oct;21(2):253-257 have found evidence for 106-126 causing oxidative damage to mciroglia. They also argued that the mechanism is differant from that with beta-amyloid 25-35 in Eur J Neurosci 1997 Jun;9(6):1162-1169. Is your mechanism different from this?

3. Regarding GSS infectivity, I want to mainly emphasize the point that, regardless of the mechanism of transmission, the issue of why cells actually die remains. The findings on transmembrane PrP allow the question of why prion diseases result in neurodegeneration to be conceptualized in terms of signal transduction and activation of apoptotic or other pathways.

What was the rationale for the variants used, eg, KH to II and AV3?

4. Historically, we started with observations in cell-free translation systems. In those systems, the simple human mutations (e.g. see A117V presented as Figure 4B in the Science paper) had relatively subtle effects. So instead, we asked which mutations have dramatic effects in cell-free systems, and found some (e.g. KH to II and AV3). From there we went into mice -- and found that the mutations with the most extreme behavior in cell-free analysis, caused very rapid disease, e.g. within 60 days of birth. The key point is that human mutations such as A117V could never be as extreme as KH>II because otherwise it would never be passed along in the germline. Our approach was to make the extreme point with the KH>II and AV3 in mice -- and then go back and show that the same process, albeit in a more subtle manifestation that allowed the patient to survive past reproductive age, was at work in the human A117V GSS.

Does the chain span once or twice, as in J Biol Chem 1994 269(24):16810.

5. Simply put, since the protein has an amino terminal cleaved signal sequence, and had both amino and carboxy terminal protected domains, it must have spanned twice. In the meantime, the 3F4 monoclonal antibody became available -- which showed that the two protected domains could NOT be generated from the same population of nascent chains, because BOTH fragments contain the 3f4 epitope. Hence it must have been two populations of chains, one of which went in N-trans, the other of which went in C-trans.

If this is confusing, welcome to the club. One explanation is that the nascent chain is folding WITHIN the translocation channel (see our review in Cell last November about the translocation channel) and from that complex, nascent folded state, it can fall either of three ways: with N-trans, with C-trans, or fully translocated.

My personal view is that the double transmembrane orientation may prove not to be wrong, but rather to be TRANSIENT, with the chain "slipping" one or the other domain (N vs C) back into the cytoplasm, some of the time. But this is just speculation at present.

Regardless, none of this changes the fundamental conclusion we came to ten years ago, namely, that PrP could be expressed as both a secretory form and as an integral membrane form, dependent on accessory translocation machinery. What the science paper fundamentally shows is that this observation from cell-free translation systems is fundamentally involved in the pathogenesis of neurodegenerative disease.

I think the most exciting point is that, if the ER can make these kinds of decisions with these kinds of signalling consequences in this case, what else is it doing of this sort in other systems, organs -- and diseases.

Are there two very different mechanisms giving rather similar end results?

The way I like to think of all of this is that prion disease involves two unusual features: transmission and neurodegeration. BSE, CJD, scrapie, are examples of prion disease that show both features, while GSS shows only the latter. Most of the work in the field has focused on transmission. But regardless of the mechanism of transmission, little has been known as to why the neurons actually die.

By looking at GSS in which protease-resistant PrP Sc does NOT accumulate, we are able to "look for the needle without the complication of the haystack". Formally, we do not know whether the pathway of neurodegeneration in CJD, BSE and scrapie will involve ctmPrP or not. I agree with you that it seems a bit of a coincidence me to say that two very different mechanisms give similar end results. My hunch is that, since the histopathology is the same, ctmPrP may well be involved in all of these diseases, but that remains to be demonstrated. The problem is that the accumulation of PrP Sc makes it technically difficult to see a small amount of transmembrane form above the "noise".

At the moment, we are no closer to a pathogenic mechanism with ctm PrP -- except that it allows for transmembrane signalling (e.g. perhaps turning on an apoptosis program), and provides a first "fish hook" with which to tease out the later steps in such a putative pathway. To my mind, the most exciting implication has to do with the basic biology of the ER and its ability to do extraordinary things.

Our paper has important implications for three areas of biology. First, for prion diseases, we have shown:

a) that a prion disease not easily explained by the existing paradigm (i.e. in which PrPSc, the abnormal form associated with BSE, CJD and scrapie is not found, and in which prion disease is not infectious), has a novel form of prion protein, termed ctmPrP, and hence can now might be explained as being different from infectious prion diseases in this respect.

b) that separate from the question of transmission (which has been the focus of most prion research up until now) is the question of why neurons die in prion disease. Now we have insight into a form of neuronal death that looks just like prion disease (i.e. same histology and clinical signs), but without transmission, hence perhaps revealing the pathway of cell death, in some -- or perhaps all -- prion disease, and maybe even other neurodegenerative disorders, or even diseases of other organs (in other work, we show that the molecular machinery in the ER that specifically suppresses ctmPrP expression in the normal brain, is present in other tissues that do not express PrP. Hence, PrP is just one of a family of gene products whose biogenesis is likely to be regulated in this fashion).

Second, our results have some profound implications for basic cell biology:

a) that the endoplasmic reticulum (ER), a part of the cell that most scientists thought they understood, is doing something truly extraordinary and unanticipated: it is making decisions as to whether proteins are to be secretory or to span the membrane, from which they can signal other parts of the cell -- in the case studied here, with fatal consequences.

b) Furthermore, we know from other experiments that in the normal brain the ER contains a protective factor that prevents the expression of ctmPrP. Thus, unravelling the details of this pathway may suggest a simple treatment for these disorders: boosting the activity of the protective pathway by which the brain normally eliminates ctmPrP.

Finally, there is a profound implication for basic biology. If the ER can take one gene, that for the prion protein, and make it be expressed in such completely different ways, in effect, making it behave like two different genes in one, then the potential information content of the genome is far greater than might have been imagined.

Commentary: prions and the endoplasmic reticulum

20 Feb 98 webmaster commentary
This is a very interesting and extraordinarily thorough article in the 5 Feb 98 Science, which is part of a substantial series of endoplasmic reticulum articles going back 8-10 years by VR Lingappa et al. of the UCSF group. The article is 26 screen-scrolls, so long and detailed.

The two basic ideas here are (1) an explanation for A117V familial CJD (2) a new pathogenicity mechanism other than Prp-Sc and overproduction, involving nascent chain stuck trans-membrane in everted topology (ctm: the carboxy terminus is inside the lumen, the N-terminus in the cytosol). A GPI-anchored protein normally ends up completely in the lumen of the ER.

Now it is true that A117V is obscure -- 3-4 families max worldwide. It is thrown in with 'GSS' though the phenotype varies even within the members of the same family, ataxic or telencephalic, and within a given codon 129. It is classed with P102L, P105L, A117V, Y145stop as difficult to transmit or nontransmissible. [The nearest CpG hotspot, G127S, might fall within this group.] One family had a double mutant: an silent GCA 117 GCG on the normal allele accompaning GCA 117 GTA [which should have a coupled origin].

A117V occurs in a critical conserved region of the protein, 99-130 WNKPSKPKTNM KHMAGAAAAGAVVGGLGGYMLG, that does not come out too well in nmr. This region is divided here into STE (stop transfer effector) 104-112 and TM-1 (transmembrane) 113-135. Rougly, some nascent chain get stuck trans-membrane at the strongly hydrophobic stretch/positive lysine cap (ntm or ctm topology ). The STE region has always reminded me of KKRPKPGGWNT, immediately distal to the signal peptide.

In the paper, they find very striking correlations in various engineered alleles [del-STE, G123P, KH 110 111 II, amd A113 115 118V] between pathology and the amount of ctm. These alleles oddly don't include A117V itself (or P102L, P105L) though a frozen A117V brain was examined somewhat unsatisfactorily.

Next, they looked but did not find Prp-Sc or overproduction. While their methods were sensitive, transmission was unfortunately not studied. While it is hard to build a case around something not detected, they do suggest from the correlations that accummulated ctm is the origin of pathogenicity in the alleles studied. There are no ideas on whether or why ctm per se is toxic, what subsequent toxic events its accumulation might trigger, whether the mechanism is only applicable to 'proximal' CJD, nor any explanation that I could see for how there could be a new type of pathogenicity with an outcome seemingly so similar to the old conformational conversion type.

While nobody "needed" another mechanism or complication with this disease, here is such a proposal. The one unifying thought is that now it seems that essentially all familial CJD can be attributed, one way or another, to problems in export and accumulation, as opposed to loss of structure or function in mature protein. It is unclear whether STE and TM function in the ER by design or by accident. Neither is a recognized protein motif.

This article is best understood by considering the primary interests of the main authors, RS Hegde and Vishu Lingappa, which have to do with the larger issues of the endoplasmic reticulum translocon, as articulated in the 28 November 97 issue of Cell mini-review [see also preceding articles].

The prion protein, because of its quixotic 106-126 domain, provides them a handle on active topics such as distinct functions for proteins with multiple membrane topologies and regulation and properties of the ER translocon in health and disease. The CJD mutation A117V is conveniently located in this domain, but for their purposes the ER effects they want to study are better exhibited in engineered mutants such as KH-->II.

I think they have a valid research program here and the prion protein is a good model system for them. If you don't give a hoot about the ER and its woes, this could still provide valuable insights on a crucial prion protein domain and minor mutants in it, but overall it has very little to do with most topics in CJD and the ultimate pathogenetic mechanism.

In my view, there has to be a universal core mechanism of toxicity across the set of 20 amyloid cross-beta congophilic disorders (Occam's razor, Glenner paradigm) and the ER translocon cannot play more than an early role in certain specific instances. I don't agree that the ER translocon is a promising therapeutic target -- this would be affecting thousands of proteins at a time.

After reviewing the full literature on A11V, I conclude below that it will contain rogue conformer and prove transmissible by the usual mechanism, contrary to this paper. It might get there by a novel pathway, but I don't agree that the final outcome is anything new. Why rogue conformer should lead to toxicity remain completely unknown; I don't agree that studying early processes in the ER will illuminate this. It may well illuminate early steps in how particular mutants get to rogue conformer.

There may well be diseases solely manifested by direct sequelae of a dysfunctional interaction and resultant topologies of a mutant protein with the endoplasmic reticulum. Engineered mutants such as KH-->II are valid objects of study here. But this is not to study CJD.

Another very interesting idea put forward by the authors is whether two N/C orientations relative to the membrane could be associated with distinct prion functions. That is, prion protein speculatively has a double life. The precedent they provide is ductin, said to be a subunit of vacuolar H+ ATPase in one transmembrane configuration and a component of a connexin gap junction in the other. D Levy reviews such cases in Essays Biochm 1996 31 49-60. Maybe -- but if double function then knockouts would give double trouble. Yet these have mild effects.

The 106-126 domain is central to understanding the prion protein and CJD. It bridges the conventional globular domain and repeat region; its extreme evolutionary conservation speaks to strong selective pressure on its bafflingly bland primary sequence. A117V would seem harmless enough but it isn't.

From theory and cell-free work early on, it was identified as potentially trans-membrane (TM domain, lipid bilayer spanning), which would make prion protein an integral membrane protein. However, no support is found for this in normal brain: the prion protein is a conventional extra-cytoplasmic protein anchored by its GPI lipids to the membrane, not by any peptide domain. The free peptide reportedly forms membrane pores, slanted hairpins, chimeric helix and sheet, amyloid fibrils, and is the toxicity counterpart of beta-amyloid 1-43.

The ER needs to 'decide' as nascent chains come in, how to orient successive domains. The signal domain, the 106-126/stop-transfer effector, and the pre-GPI domain are the main known mileposts for prion protein. Surprisingly [maybe shouldn't be], the signal and pre-GPI domains are very similar structurally, and in fact sometimes functionally interchangeable. [J. Mol. Biol. (1998) 275, 25±33].

According to the Science paper reviewed here, the prion domain 106-126, perhaps unavoidably because of its final destination and role, seems to be sending mixed messages to the endoplasmic reticulum. The A117V mutation tilts the balance of achieved orientations, perhaps to that appropriate to an integral transmembrane segment, valine being much more hydropobic than alanine. Overall, a nice result with substantial documentation, more work needed as always, and I look forward to learning what happens next.

A very recent article in Nature on a mutant presenilin showed that it too exerted its effect in the endoplasmic reticulum -- but by affecting proteolytic clipping that produces beta amyloid 1-42. The same mechanism could be operative with A117V: the abnormal topology simply leads to a peptide giving the usual amyloid fiber.

Literature on A117V

      Tranchant C, et al.    
       Neurofibrillary tangles in Gerstmann-Straussler-Scheinker syndrome with the A117V prion gene mutation. 
       J Neurol Neurosurg Psychiatry. 1997 Aug; 63(2): 240-246. 
       PMID: 9285466; UI: 97429828.

     Tateishi J, et al. 
       Experimental transmission of Creutzfeldt-Jakob disease and related diseases to rodents. 
       Neurology. 1996 Feb; 46(2): 532-537. 
       PMID: 8614527; UI: 96181989.

     Kaneko K, et al.     
       Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform. 
       Proc Natl Acad Sci U S A. 1995 Nov 21; 92(24): 11160-11164. 
       PMID: 7479957; UI: 96074667.

     Mastrianni JA, et al. 
       Prion disease (PrP-A117V) presenting with ataxia instead of dementia. 
       Neurology. 1995 Nov; 45(11): 2042-2050. 
       PMID: 7501157; UI: 96063532.

     Tateishi J, et al.  
       Inherited prion diseases and transmission to rodents. 
       Brain Pathol. 1995 Jan; 5(1): 53-59. Review. 
       PMID: 7767491; UI: 95284998.

Genetics of BSE

Listserve 9 Feb 98
Question: is there a pattern of homozygocity/heterozygocity at specific codons in cattle equivalent to the traits in humans (e.g. 129) and mice. I realize that in most cases, detailed sequencing may have been done on only a small subset of the 171,000 case-animals, but what were the results? Can they be used to predict or explain the geographic clustering, perhaps through in-breeding? Response: No. very little sequencing has been done. Or should I say, very little sequencing has been reported. There have been no transgenetic studies of high repeat alleles of the bovine gene. I should say the reason for this was:

So far, cattle are not like sheep with various common point alleles. There is a common extra repeat insertion which, for various reasons, is a weak cause per se for concern. There was a rumor circulating among researchers of a double extra repeat, which is quite a bit more problematic, at least in humans, for familial CJD. If a few hundred BSE cows were sequenced, and a double repeat shows up, that makes it in all liklihood an allele.

However, because of the mechanism of extra repeat generation, 6- and 7- repeat populations may be at much higher risk of generating further longer repeat alleles: one more round and things are definitely in familial BSE territory and with much higher frequency than human familial repeat CJD (which is roughly half of all familial CJD) which lacks this generator population.

This scenario is sort of an enhanced Gibbs Principle: cattle genetics are one step removed from being direct susceptibility types per se like sheep alleles, they are susceptibility types for throwing out self-sufficient familial BSE. Somewhat similar spino-cerebellar ataxia intermediate polyglutamine repeat carriers except that 'anticipation' or earlier onset in subsequent generations is predicted only in a much weaker statistical sense.

A recent paper addressed to what extent BSE was being passed on maternally versus via genetics.

Genetic interpretation of heightened risk of BSE in offspring of affected dams

Ferguson,NM, Donnelly,CA, Woolhouse,MEJ, Anderson,RM
Proceedings Of The Royal Society Of London Series B-Biological  Sciences, 1997, Vol.264, No.1387, pp.1445-1455
An analysis is presented of the results of a cohort study designed to test whether or not the aetiological agent of bovine spongiform encephalopathy (BSE) in cattle can be transmitted maternally (vertically) from dam to offspring.

Various genetic models are fitted to the data under the assumption that the results could be explained entirely by genetic predisposition to disease (as opposed to maternal transmission) given exposure of offspring of diseased and unaffected dams to contaminated cattle feed. The analyses suggest that the results could be explainedby the hypothesis of genetic predisposition , provided a large difference exists in the susceptibility of resistant and susceptible hosts, and explore the ranges of genotypic parameters and frequencies consistent with the limited currently available data.

The results presented are broadly robust, even under the scenario that a portion of the observed maternally enhanced risk of BSE is due to a low level of maternal transmission in late incubation stage dams.

Anti-prions resurrected

12 Feb 98 webmaster opinion
A new article appeared on an old subject, the lack of stop codons in the -1 reading frame of prion genes, Biol Chem [not JBC] 1997 Dec;378(12):1521-1530.

The article is mostly about Hsp 70, which has a more dramatic stretch of 2000 stopless nucleotides, with an extension of the effect to a larger range of species for the prion gene, and a lame scheme to connect the two phenomena. The authors do not trouble themselves with the context provided by analysing all ORFs in complete genomes such as yeast or much by way of other human genes. Like so many others before them, they make no effort to track down the source of the complementary anti-prion RNA found in knockout mice.

The 834 stopless stretch in human prion drops down to 600 as more species, overwhemingly just old world primates, are considered. Stopless stretches in the -1 reading frame are expected statistically if there is high GC content in third position (as in prion genes) or low use of TTA, CTA (leu), and TCA (ser) codons and indeed the authors cite another recent paper of theirs which apparently invalidates the current paper.

Now 5 scientists can't spend a whole afternoon doing research without having some good ideas. The first was to look for potential splice sites (the -1 frame has no start codon in sight) such as AG/GTGGGT using Grail and Netgene Interestingly enough, a site was reported right at the YPP at the beginning of the repeat region. Unbeknownst to the authors, this is the previously reported conserved Helic C region in mRNA secondary structure. Curious.

Secondly, they considered the different effect of CJD mutations on sense and anti-sense strands and the overall conservation of translated anti-sense protein. P105L generates additionally an anti-sense stop codon. A117A is coding-silent but cys to arg anti-sense, be more interesting if A117A led to CJD. M129V changes sense susceptibility but is silent anti-sense. D178N is silent anti-sense. ( I am having to supply these for the paper.)

The authors buy in to the implausibly borderline idea of Brentani for a natural protein-protein interaction of the products from the two strands though dsRNA regulation is also considered.

There are now about twice as many known prion sequences as considered in this paper. I haven't checked yet to see if the open reading frame on the complementary strand holds up. It would be much more interesting to experimentally pursue the complementary RNA in knockout mice. It doesn't show up on Blast searches but I haven't checked ESTs or human genome project lately.

Chromosomal organization of 3 prion genes

22 Feb 98 webmaster alert
There is a set of 3 new prion sequences at GenBank, U67922 sheep, U29185 human, and U29186 short incubation mouse . These are gigantic: 31,412 bp for sheep, 35,522 for humans, and 38,814 for mouse. The protein part is only 771 or 2% of these. The sheep sequence starts thousands of bases upstream of exon 1 and ends thousands of bases downstream of exon 3.

Then Lee et al. seem to have used powerful software for analysing untranscribed and untranslated material using results from the big mammalian genome projects. I know the meaning of only a very few of the identified repeats. Then there is the issue of comparing the three species and what similarities and differences mean.

I guess the idea is to separate wheat from chaff. One application would be to nvCJD [or sporadic], whether the early victims had over-producing regulatory alleles. Another is to whether these will have 2-3 exon transcripts like hamster in different parts of the brain.

Let us hope the paper will be out soon.

"RepeatMasker is a program that screens DNA sequences for interspersed repeats known to exist in mammalian genomes as well as for low complexity DNA sequences. The output of the program is a detailed annotation of the repeats that are present in the query sequence as well as a modified version of the query sequence in which all the annotated repeats have been masked (replaced by Ns). On average, over 40% of a human genomic DNA sequence is masked by the program."
"Sputnik is a denovo microsatelite identifying program. It looks for SSRs, by just looking for repeated sequence motifs (e.g. ACACACAC, etc.). We wrote the software here in the lab. I apologize that the link is no longer active. The programmer who did the work for us initially is no longer with us. It is a very simple program written in C.

Due to the fact that Lee Hood's Lab is very large and separated over miles, I wouldn't count on an immediate genbank update. The site at Serac is coming down and will be replaced by a new site, where we will have links to all of these programs. It will be a couple of months before it is up and it will be at

Chris Abajian, who wrote sputnik, has moved to a company. Go to their web page. Then go to link to sputnik. Or you can get Chris's email there.

LOCUS       OAPRP       31412 bp    DNA             MAM 19-FEB-1998
DEFINITION  Ovis aries prion protein gene, complete cds.
NID         g1778172
SOURCE      sheep.

  AUTHORS   Lee,I.Y., Westaway,D., Smit,A.F., Wang,K., Cooper,C., Yao,H.,
            Prusiner,S.B. and Hood,L.
  TITLE     Structure and Organization of Chromosomal Regions Carrying the
            Mammalian Prion Gene from Three Species
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 31412)
  AUTHORS   Lee,I.Y.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-AUG-1996) Department of Molecular Biotechnology,
            University of Washington, Box 352145, Seattle, WA 98195-2145, USA
COMMENT     Interspersed Repeats were identified with RepeatMasker (available
            from Simple
            sequence repeats were identified with sputnik (available from  [dead]

     repeat_region   1..316
     repeat_region   complement(321..540)
     repeat_region   588..613
     repeat_region   complement(1035..1159)
     repeat_region   1745..2082
     repeat_region   2092..2203
     repeat_region   2194..2492
     repeat_region   2482..2561
     repeat_region   2590..2683
     repeat_region   complement(2684..2842)
     repeat_region   3011..3174
     repeat_region   3650..3942
     repeat_region   3756..4215
     mRNA            join(5666..5717,8139..8236,22268..26295)
                     /product="prion protein"
     exon            5666..5717
     exon            8139..8236
     repeat_region   complement(12806..13148)
     repeat_region   complement(13146..13321)
     repeat_region   complement(13227..13648)
     repeat_region   complement(16433..16524)
     repeat_region   16566..16654
     repeat_region   complement(17457..17737)
     repeat_region   17745..18115
     repeat_region   complement(18418..18594)
     repeat_region   complement(18594..18713)
     repeat_region   18974..19174
     repeat_region   complement(19211..19358)
     repeat_region   complement(19372..19741)
     repeat_region   complement(19741..19839)
     repeat_region   complement(21583..21689)
     exon            22268..26295
     CDS             22278..23048
                     /product="prion protein"




     repeat_region   23801..24187
     repeat_region   24709..24867
     repeat_region   24889..26108
     repeat_region   complement(27249..27348)
     repeat_region   27347..27574
     repeat_region   complement(27569..28141)
     repeat_region   28142..28334
     repeat_region   complement(28335..29738)
     repeat_region   complement(30285..30335)
     repeat_region   complement(30369..30546)
     repeat_region   complement(30807..30950)
     repeat_region   complement(31255..31404)
     repeat_region   complement(31287..31412)
BASE COUNT     8889 a   6517 c   6464 g   9542 t
        1 gatccaccca gtccatccta aaggagatca gtcctgggtg ttcattggaa ggactgatgt
       61 tgaagctgaa actccaatac tttggccacc tgatgcgaag agctgactca ttggaaaaga
      121 ccctgatgct gggagggatt gagggcagga ggagaagggg acgacagagg atgagatggt

Protective polymorphisms: codon 219 is better

7 Feb 98 Lancet S.Shibya et al.
E219K is a common polymorphism in Japan. 85 Japanese cases of sporadic CJD ware anaylysed. There were no heterozyotes glu/lys at 219, in strong chi-squared conflict , 0.0027, with incidence in the Japanese population of about 12%. I do not see any data on the status of codon 129 of these patients but met/met is the rule in Japan whereas E219K has never been seen in Europeans. Heterozygosity is known to modulate in cis (one family, 4 cases) but not in trans (2 families) P102L non-congophilic familial GSS. Mol Brain Res 1995 30: 385-88 and J Neurol Neurosurg Psychiatry 1997 May;62(5):454-457

The authors observe that UK sporadic CJD with met/met at codon 129 is higher than expected [37% population but 83% of cases] but met/val has this turned around [51% population, 9% of sporadic]. So glu/lys is giving complete protection so far at codon 219 while met/val is only giving partial protection. These data do not shed any direct light on nvCJD in met/val.

Prion Protein Expression in Human Leukocyte Differentiation.

Blood 1998 Mar 1;91(5):1556-1561 full text + free. 
Dodelet VC, Cashman NR
The cellular isoform of the prion protein (PrPC) is a small glycoprotein attached to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol anchor. This molecule is involved in the pathogenesis of prion diseases in both humans and animals. We have characterized the expression patterns of PrPC during human leukocyte maturation by flow cytometry with monoclonal antibodies to PrPC, the glycan moiety CD15, and the stem cell marker CD34.

We observe that prion protein is present on CD34(+) bone marrow (BM) stem cells. Although lymphocytes and monocytes maintain PrPC expression throughout their differentiation, PrPC is downregulated upon differentiation along the granulocyte lineage. In vitro retinoic acid-induced differentiation of the premyeloid line HL-60 into granulocyte-like cells mimics the suppression of PrPC in granulocyte differentiation, as both PrPC mRNA and protein are downregulated.

These data suggest that selected BM cells and peripheral mononuclear cells may support prion agent replication, because this process is dependent on availability of PrPC. Additionally, retinoic acid-induced extinction of PrPC expression in HL-60 cells provides a potential model to study PrP gene regulation and protein function. Finally, these data suggest the existence of cell-specific glycoforms of PrPC that may determine cellular susceptibility to infection by the prion agent.


On the basis of our current report, BM cells, including CD34+ stem cells, might be expected to harbor the prion agent, despite current guidelines which do not recognize BM as high-risk tissue.28,29 In the periphery, monocytes and lymphocytes, which express PrPC, would be expected to support prion replication, whereas PrPC negative erythrocytes and granulocytes would not.

This contention is supported by a limited number of transmission experiments30,31 and by recent data >from Blattler et al,32 who show that prion agent infectivity detected in spleen by bioassay is dependent on the expression of PrPC by spleen cells and does not accumulate by nonspecific "carry-over" from the original innoculum.

An additional implication of our study is that PrPC must be differentially glycosylated in the nervous system as opposed to the periphery.

PrPSc, the abnormal disease-associated isoform of PrPC, has been found to possess a high proportion of N-linked glycan chains terminating in the glycosyl moiety stage-specific embryonic antigen 1 (SSEA-1), also known as CD15.33

This carbohydrate moiety has been found to participate in cell-cell adhesion and tissue differentiation early in development, and probably plays an important role in immunocyte adhesion in inflammatory processes.34,35

In the peripheral blood, granulocytes, which express no PrPC, display prominent immunoreactivity for CD15. Lymphocytes, which display no surface CD15, do express PrPC. Only peripheral monocytes possess surface immunoreactivity for both PrPC and CD15, allowing the possibility that monocyte PrPC may be modified with the SSEA-1 moiety.

Because glycosylation may participate in the brain-region-specific replication of the scrapie agent,36 it is possible that certain PrPC on different peripheral cells might be more likely to participate in scrapie infection.

Of interest, some data indicate that the monocyte-macrophage cell lineage is critical to the propagation of scrapie infectivity in the periphery. Scrapie infection in severe combined immunodeficient mice is apparently dependent on dendritic cells,37 which are tissue macrophages specialized for immune presentation. Moreover, peripheral blood cells containing scrapie infectivity are relatively radiation insensitive,38 which is consistent with the end-mitotic nature of peripheral monocytes.

Finally, monocytes may be unique among peripheral blood cells for their capability of processing PrPC to PrPSc.39 Because other peripheral blood cells also express PrPC, the capability of monocytoid cells to support scrapie agent replication may relate to a posttranslational modification, such as SSEA-1 modification of the terminal PrPC glycans.

Breeding out scrapie -- will it work?

Alex Bossers Netherlands 20 Feb 98
Maybe I can give a short explanation of sheep DNA tests as carried out by us in the Netherlands as well as by the MAFF.

Blood samples are collected from i.e. breeding rams or from all sheep residing in a single flock. Total DNA is extracted by various methods. Both alleles of the PrP-ORF are PCR amplified and further analyzed by:

Sequencing (MAFF?!)
DGGE (NL ref Bossers et al. JGV 1996 77:2669-2673)
ASA+RFLP (UK+?+NL Belt et al. ref JGV 1995 76:509-517)
New fast and cheap techniques are in development like Taqman (combining PCR and ASA in a single tube measuring fluorescense).

The advantage of sequencing is that new polymorphisms can be discovered and that all known polymorphisms can be determined. Disadvantage: time-consuming, expensive,.....

The advantage of DGGE is that new polymorphisms can also be discovered, all known polymorphic positions can be determined by a semi-rapid and not so expensive test. One more important fact is that using DGGE polymorphisms occuring within a single allele can be determined (i.e. 136V171R can in DGGE be scoored as alle1 136V... + allele 2 171R. Not as 136V171R as allele 1 and wt as allele 2). This may become a problem as soon as more than a single polymorphisms is found within a single sheep PrP allele (like the FFI/CJD 129M178N->FFI while 129V178N->CJD).

The advantage of ASA+RFLP is that it is quick and cheap, however no new polymorphisms will be discovered as well as nothing extra will be known about linking polymorphisms to a single allele. The latest method still in development: 'Taqman' has the great advantage that it is quick, cheap and the total procedure from blood sample to final result can be automated. Sample flow can (and will) be very high. Especially interesting for eradication programs.


	Sequencing (MAFF UK)  about 30 pounds per sample
	DGGE (NL) about 85 dutch guilders per sample
	Future Taqman about 50 dutch guilders [US $25]or less per sample

All the prices are dependant on no of samples, time of testing (breeding season) etc. Sample flow of DGGE is low at the moment. Sample flow of the Taqman test will be 60,000 samples minimum per year (only breeding seasons!).

Contacting persons are again:

	UK/MAFF: Michael Dawson
	NL/DGGE?...: Mari Smits

Question on scrapie eradication:

In the UK the "resistant" genotype in predominantly blackface sheep is most susceptable to the strain of scrapie usually found in whiteface breeds (esp:Cheviot) in the UK; vice versa applies. Thus selection for the "resistance" genotype only applies to specific scrapie strains and may be creating animals which are "susceptable" to other strains of scrapie. Any comments?


The resistant genotype or allele is ARR and confers resistance to EVERY breed described thus far. Don't mix up the experiments with the so-called CH1641 and SSBP/1 'strains' which are actually ISOLATES. See the extensive discussion in our latest PNAS paper (Bossers et al 1997 94:4931-4936) for more details. I attached part of this discussion below:

Not only the primary PrPC sequence was found to determine the conversion characteristics but also the primary amino acid sequence of PrPSc. PrPSc(VQ/VQ) converted PrPCVQ, PrPCAQ, and PrPCAR with decreasing efficiencies. In contrast, PrPSc(AQ/AQ) converted PrPCVQ almost as efficiently as the PrPCAQ variant. The PrPCAR variant was poorly converted by both PrPSc isolates.

This suggests that scrapie susceptibility is not only determined by the PrP genotype of the acceptor animal but also by the PrP genotype of the animal that produced the infectious PrPSc. This is consistent with the finding that the SSBP/1 scrapie isolate obtained from PrPVQ NPU-Cheviot sheep is best transmitted to PrPVQ sheep (12, 17).

It is also consistent with the striking behaviour of the CH1641 scrapie isolate, which was primarily isolated from a positive line (mainly PrPVQ carrying) NPU Cheviot sheep, when passaged in 'positive-line' or 'negative-line' (non-PrPVQ) Cheviot sheep. The first (primary) intracerebral passage of this >positive-line= material to positive-line Cheviot sheep resulted in short incubation times.

Passage of the primary CH1641 isolate into 'negative-line' Cheviot sheep resulted in longer incubation times (33) probably due to polymorphism barriers. If the negative-line passaged isolates were subsequently passaged in 'negative-line' Cheviot sheep the incubation times in this line of sheep decreased (17, 33). A subsequent passage from these 'negative-line' to 'positive-line' Cheviot sheep increased the incubation times dramatically (17, 33) again probably due to the 'polymorphism barrier'.

Question about sequencing methods:

Why not use the new method just described in PNAS?
Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: 
Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing 
PNAS Vol. 95, Issue 4, 1681-1685, February 17, 1998
Jarmo K–rkk–, Susanna  Annunen, Tero Pihlajamaa, Darwin J. Prockop,, and Leena Ala-Kokko
Previously, an assay called conformation sensitive gel electrophoresis (CSGE) was developed for scanning PCR products for the presence of single-base and larger base mismatches in DNA.

The assay was based on the assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by single-base mismatches in double-stranded DNA and thereby increase the differential migration in electrophoretic gels of heteroduplexes and homoduplexes. Here the sensitivity of assays by CSGE was improved by limiting the maximal size of the PCR products to 450 bp and making several changes in the conditions for PAGE.

With the improved conditions, CSGE detected all 76 previously identified single-base changes in a large series of PCR products from collagen genes that contain multiple exons with highly repetitive and GC-rich sequences. In a survey of 736 alleles of collagen genes, CSGE detected 223 unique single-base mismatches that were confirmed by nucleotide sequencing.

CSGE has the advantage over other methods for scanning PCR products in that it is simple, requires no special preparation of PCR products, has a large capacity, and does not use radioactivity.


I didn't see the PNAS paper yet you are referring to (PNAS 17 Feb 1998 p1681-1685) but the abstract tells me enough (?).

The CSGE (Conformation Sensitive Gel Electrophoresis) assay they describe indeed has the advantages as outlined before; detection of new mutations, detecting polymorphisms present on a single alle. However, these assays, even if automated, take very long times to perform. For instance the DGGE technique which is more or less similar to the CSGE, you still have to analyze your PCR product on PAGE which takes for a DGGE in our case about 24h running time. Sample flow is generally low: 1 gel approx 36 lanes minus 10 controls leaves about 26 samples to be analyzed in a single run.

Sometimes bands in such gels comigrate at the same speed (especially the case for samples homozygous for a particular gene) which have to be reanalyzed by making it an artificially heterozygote (mizing with a known cloned variant). So if you have bad luck all your 26 samples have to be reanalyzed. Then the final part of analyzing the specific banding patterns (like bar codes) is a job which is very hard to automate and in general has to be done by hand. More or less: DGGE as well as CSGE are GREAT methods however sample flow is low and thereby costs per sample are in general high(er).

The new single tube technique, Taqman, in contrast has the disadvantage of detecting no new polymorphisms, in general more polymorphisms in a single allele cannot be discriminated from a heterozygous one. The GREAT advantage of the technique is its high sample flow. It can be fully automated from blood to final result. The PCR amplification step is also the detection step, so results of the test will be available in less then half a day! Sure, you (we) need to invest 1.500.000-2.000.000 Dutch guilders for the complete system but the sample flow compensates it all leaving a final price of about 50 guilders per sample or less.


what advice are you giving to sheep breeders?


At the moment we give breeding advice depending on breed: In general we advice NOT to use the PrP-VRQ variant (136 valine is in many breeds associated with susceptibility) and if possible to introduce the PrP-ARR variant (171 Arginine is in all breeds associated with resistance (even in experimental scrapie)). In breeds where VRQ is common the above breeding advice is almost no problem. In breeds like Suffolk where VRQ is rare and the wildtype ARQ (136A/154R/171Q) is aasociated with susceptibility although with an incomplete penetrance. In these breeds we try to advice to introduce more ARR and ofcourse no VRQ. So in principle all VRQ carriers get negative breeding advice (even if the genotype is VRQ/ARR!).


Can the ANetherland eradilcate scrapie by avoiding certain alleles? I am trying to understand the big picture about the Netherlands' strategy for breeding out scrapie. The rams, which are few in number, get fully sequenced so ARR and no new polymorphisms. VRQ (and ARQ) Suffolk lambs are separated out by Taqman in a timely manner from ARR breeder replacements. The population rapidly tilts to ARR, at least on participating farms.

Glenn Fahnestock and Reed Holyoak expressed a concern that "it is equally plausible that, by selecting for supposed resistance, we are likely to be creating a carrier state (delayed onset of symptoms) and disseminate scrapie further."

ARR is resistant and seldom found with clinical scrapie, but not a bulletproof vest as I recall. This could still work because resistance could mean harder transmission because of lower titre, declining titre over time, and perhaps zero titre in the end. Provided feed was TSE-free and movement was restricted to ARR herds, this might work, especially if supplemented by antibody testing and so on. Is this more or less the plan?


Indeed we do see the potential carrier state as a significant 'problem' for the future. That's for instance why I'm working hard to collect enough data (+in vitro) to make an overall susceptibility list of all the nine different sheep PrP alleles. If for instance the AHQ allele (136A154H171Q) also turns out to be more or less restant, this allele would be interesing for breeding programs also.

The ARR allele in scrapie sheep is only found in VRQ/ARR sheep. ARR/ARR sheep are resistant to sheep scrapie (natural and experimental) as well as to BSE (experimental). These experimental inoculations (i.c. + s.c. ; Goldmann et al. JGV 1994 75:989-995) show the good resistance of ARR/ARR sheep to various scrapie ISOLATES (SSBP/1 and CH1641) and BSE. Indeed we do have experimental data which could point to a potential carrier state: the in vitro conversion (Bossers et al. 1997 PNAS 94:4931-4936).

In the latter we we showed that ARR PrP-C is converted with a VERY LOW efficiency into a protease resistant state. Our current thoughts about that are that the ARR PrP-C molecule caps and thereby blocks the growing PrP-Sc polymer. However, in vivo this could turn out so that the PrP-Sc molecules (aggregates) bind to PrP-C and get internalized in the cell where it could stay for years.

Many sheep from scrapie free as well as from scrapie infected flocks (all aged 5-6 years or older) with the ARR/ARR or ARR/XXX genotype have been examined by immunohistochemistry (CNS+lymphoid) and none showed neuropathological effects neither they showed staining of accumulated PrP-Sc molecules. This indicates that if the ARR/ARR is a carrier it will be at a very low efficiency.

The best thing to do next is to get ARR/ARR sheep from scrapie infected flocks and bring them under well defined conditions in a scrapie free flock and see whether i.e. VRQ or ARQ sheep in that flock get scrapie (exporting ARR/ARR sheep from scrapie infected flocks to NZ would be ........). This procedure is currently under investigation but these experiments take very long and are hard to control (what is scrapie free?).

Good help from MAFF

Thu, 26 Feb 1998 webmaster
I wrote to MAFF some time back asking for more information on completed experiments:
SE1803 Transmissibility of BSE to pigs by oral exposure to brain homogenate [became SE1817] and
SE 1804 Transmissibility of BSE to cattle by oronasal exposure to placentae of affected cattle [became SE1818].

I received a helpful response within their policy timeframe, though how one obtains a copy of the consultation cited is still unclear. Most interestingly, the scientists there had the good sense to further passage brains from asymptomatic animals at the end of their lifespan. In other words, they used a two stage amplification process, not counting on detection methods on first passage, especially across a species difference. This should be done more widely, for example in human A117V in short-lived mice, or in any situation with apparent negative transmission.

Sounds like we should be taking greater advantage of this service MAFF is providing. They could make it easier on themselves by putting more of it on their Web site so as not to have to respond to individual requests and it sounds like they may be moving in this direction.

MAFF policy: 'Chief Scientist's Group's approach to all requests for information on research is always to assume that the information should be released, except where disclosure would not be in the public interest... Wherever possible, CSG will do its best to respond to requests within 15 working days.There will be some instances when we cannot release information on research: policy options, intellectual property, proposed publication, commercial confidence - Whenever it is deemed necessary to withhold information, CSG staff will provide a full explanation of the reasons and confirm the section of the Code under which the disclosure exemption is claimed. '
MAFF response:
"The work for these projects was started in 1990 and to date the animals challenged, both pigs and cattle have remained negative for BSE. The interim results of this work have been published in the following reference:

M Dawson, G A H Wells, B N J Parker, M E Francis, A C Scott, S A C Hawkins, T C Martin, M M Simmons and A R Austin. Transmission studies of BSE in cattle, pigs and domestic fowl. In: Transmissible Spongiform Encephalopathies. Proceedings of a consultation on BSE with the Scientific Veterinary Committee of the Commission of the European Communities held in Brussels, 14-15 September 1993. Edited by R Bradley and B Marchant. Pages 161-167.

In both projects the challenged animals reached what would be their negative end point - 5 years for chickens, 7 for pigs. Tissues from these animals will be assayed further to look for evidence of infectivity despite the absence of clinical disease or pathological lesions. These subpassages will now run for a further 2-4 years before completion. Any significant findings will be published and placed on our web site."

Are florid plaques enough?

Herbert Budka, M.D.
Professor of Clinical Neuropathology
Institute of Neurology, University of Vienna


Florid plaques are seen in Kuru, and in some of the rarer forms of CJD. Are florid plaques are correlated with relative youth at onset? Two of the three were only 28 and 38 years old, but one was 52.

Because florid plaques are called the most striking criterium for nvCJD, are florid plaques in kuru brains too and if they differ from the nvCJD plaques.

Florid plaques have also been reported in 3 dura mater recipients:
Lane,K.L.; Brown,P.; Howell,D.N.; Crain,B.J.; Hulette,C.M.; Burger,P.C.; DeArmond,S.J. - Creutzfeldt-Jakob Disease in a Pregnant Woman with an Implanted Dura Mater Graft - Neurosurgery 1994 Apr; 34(4): 737-40

Kopp,N.; Streichenberger,N.; Deslys,J.P.; Laplanche,J.L.; Chazot,G. - Creutzfeldt-Jakob-disease in a 52-year-old woman with florid plaques - The Lancet 1996; 348(N9036): 1239-40

Takashima,S.; Tateishi,J.; Taguchi,Y.; Inoue,H. - Creutzfeldt-Jakob disease with florid plaques after cadaveric dural graft in a Japanese woman - The Lancet 1997 Saturday 20 September; 350(9081)


Our report in January 1997 was based on the study of one Kuru brain only which we considered then as one of the last available in neuropathological archives. Although very rare florid plaques were seen, neuropathology was dissimilar from nv-CJD and more akin to sporadic CJD with plaques or iatrogenic CJD. Apparently this report prompted others to dive into their archives and do similar studies on more Kuru cases. Our early results have recently been confirmed in a joint study from Melbourne and Edinburgh, based on 11 Kuru brains and presented last September at the International Neuropathology Congress in Perth:

McLean A et al: Comparative neuropathology in kuru and new variant CJD. Brain Pathol 1997;7:1247 (abstract). They write "Florid plaques were much more numerous in nv-CJD than in Kuru. The neuropathology of these two diseases, although superficially similar, differs markedly in the character and quantity of PrP deposits."

A July 1997 letter to Lancet (Lantos P et al: Is the neuropathology of new variant Creutzfeldt-Jakob disease and kuru similar? Lancet 1997;350:187-188) reports on 2 Kuru brains and, in contrast to our and McLean's data, concludes that "This preliminary study shows that there are similarities between nv-CJD and kuru, particularly in the pattern of PrP deposition." However, they saw florid plaques "only occasionally in kuru".

The mere occasional presence of florid plaques is of no certain diagnostic significance at present. We found them, at meticulous search, in other CJD brains as a rare feature (J A Hainfellner, H Budka: Immunomorphology of human prion diseases. In: Transmissible Spongiform Encephalopathies: Prion Diseases, L Court, B Dodet (eds), pp 75-80. Paris: Elsevier 1996). The Kopp et al case with florid cases, cited above, later turned out NOT to be nv-CJD based on PrP glycotyping by Western blotting (Deslys, J. P., Lasmezas, C. I., Streichenberger, N., Hill, A., Collinge, J., Dormont, D., Kopp, N: New variant Creutzfeldt-Jakob disease in France. Lancet 1997;349:30-31).

This is why, at the WHO consultation earlier this month, we proposed as neuropathological diagnostic criteria for nv-CJD, "Spongiform encephalopathy with abundant PrP deposition, in particular multiple fibrillary PrP plaques surrounded by a halo of spongiform vacuoles ("florid" plaques, "daisy-like" plaques) and other PrP plaques, AND amorphous pericellular and perivascular PrP deposits especially prominent in the cerebellar molecular layer". Thus it is the PROMINENCE of florid plaques IN ASSOCIATION WITH PrP deposition criteria which is diagnostic, at least at present according to the 24 neuropathologically identified cases.

What I wanted to transmit is that interpretation of the neuropathology of various TSE types needs experience and cannot be reduced to a "yes or no" question on florid plaques. Indeed, it requires consideration of several criteria which are both qualitative and quantitative; if this is done, differences are not so subtle.

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