Molecular Brain Research, 13 (1992) 155-157 © 1992 Elsevier Science Publishers B.V. All fights reserved. 0169-328X/92/$05.00
155
BRESM 80114
A dementing illness associated with a novel insertion in the prion protein gene F. Owen 1, M. Poulter 2, J. Collinge 3, M. Leach 2, R. Lofthouse 2, T.J. A . E . Harding 4
Crow 2
and
1Department of Physiological Sciences, University of Manchester, Manchester (U.K.), 2Division of Psychiatry, Clinical Research Centre, Harrow, Middlesex (U.K.), SSt. Mary's Hospital, London (U.K.) and 4The National Hospital, London (U.K.) (Accepted 5 November 1991) Key words: Prion protein; Creutzfeldt-Jakob disease; Transmissible dementia; Spongiform encephalopathy
Following our previous report of an 144-bp insertion in the open reading frame of the prion protein (PrP) gene, we have now identified a larger, 216-bp, insertion in the gene. The insertion which is in frame encodes 9 extra octapeptide repeat sequences in addition to the 5 repeats normally present and represents the largest insertion so far detected in the PrP gene. Creutzfeldt-Jakob disease (CJD) and Gerstmann Str~ussler syndrome (GSS) are rare neurological diseases that can be transmitted to animals, including primates, through intracerebral innoculation of affected brain material. CJD is usually sporadic but a family history is present in about 15% of cases whereas GSS is almost entirely familial. Both diseases are transmitted vertically in an autosomal dominant manner. CJD and GSS belong to a group of disorders known as the transmissible spongiform encephalopathies which include related animal diseases such as scrapie in sheep and bovine spongiform encephalopathy (BSE) in cattle. The precise nature of the infectious agent remains unknown but the diseases in animals and man are associated with an accumulation in the brain of a host-encoded, abnormal isoform of the priori protein (PrP) which is intimately involved in the disease process 12'13. We have previously reported an insertion in the open reading frame (ORF) of the PrP gene that segregated with the illness in a family with a history of CJD m. The insertion was subsequently shown to encode an extra 6 octapeptide repeat sequences in addition to the 5 repeats normally present in the PrP n. During the course of a recent survey of atypical dementias, we have detected insertions in the O R F of the PrP gene in individuals from an additional 5 families with histories of dementing illness of early onset 9. Of these new cases, individuals from 4 families had insertions whose sequences were identical to that initially described H. Genealogical investigation has since revealed that these four families are
part of a large pedigree with a fatal neurodegenerative disease inherited in an autosomal dominant manner, some cases of which conform to the clinical and neuropathological definition of CAD (in preparation). Several of these cases have been described previously ~'2. The only member so far tested of the remaining family presented at age 55 years with a 2 year progressive history of unsteady gait, self neglect and incontinence of urine. On examination, she was mildly demented with apraxia, occasional myoclonic jerks and gait ataxia. She died several months later. In this patient we report here the finding of a larger insertion in the O R F of the PrP gene encoding an extra 9 octapeptide repeat sequences. D N A was extracted from leukocytes and the ORF of the PrP gene amplified using the polymerase chain reaction (PCR) as described previously 11. The PCR mixture comprised 2/~g DNA, 250 ng of each oligonucleotide primer, 50 mM KC1, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCI2, 0.01% (w/v) gelatin, 200 /~M deoxyribonucleoside triphosphates and 2.5 units of Taq polymerase (Perkin Elmer Cetus) in a total volume of 100 kd. Cycling conditions were 95°C for 1.5 min (denaturing), 50°C for 1.5 min (annealing) and 72°C for 3 min (extending) for 35 cycles in a Techne thermal cycler. Oligonucleotide primer sequences were 5"-AAGAATTCTCTGACATTCTCCTCTTCA-3" (5"-primer) and 5"-AAGGATCCCTCAA G C T G G A A A A A G A - 3 " (3"-primer). The primers included 5"-EcoR1 and B a m H I linkers, respectively, to facilitate cloning. PCR products were ethanol precipitated, digested with EcoRI and B a m H I and size fractionated
Correspondence: E Owen, Department of Physiological Sciences, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
156 on a 1% agarose gel containing 0.5 #g/ml ethidium bromide. The mutant allele was identified under ultraviolet light, excised from the gel and purified using Geneclean (Bio 101), and asymmetrically cloned in pUC8.
and the one reported here to R1, R2, R2, R3, R2, R3/, R2/, R 2, R2, R2, R3/, R2, R 3, R 4. Apart from R1 the repeats have identical amino acid sequences but can be distinguished from each other by changes in the nucle-
The insertion was sequenced on both strands of 5 recombinant clones using the strategy and primers previously described 11 and was found to comprise an extra 9
otide sequences. R 2 / a n d R3/differ from R 2 and R 3 by a single base and have not been observed in the wild-type sequence. Both the 6 extra repeat and 9 extra repeat insertions occur on alleles encoding the methionine form of the amino acid polymorphism at codon 129 in h u m a n prion protein 8 and cannot have resulted from one recom-
in-frame repeat sequences in addition to the 5 repeats present in the wild-type allele. The consensus sequences of the wild-type 7 and mutant alleles are presented in Table I in the format previously published with invariant bases in uppercase and variant bases in lower case letters respectively 11. The nucleotide sequence flanking the
bination event. All the pedigrees in question reside in a relatively small area of Southern England, raising the possibility of c o m m o n ancestry with a degree of genetic instability in this form of mutation. However, the gene-
insertion was identical to that of the wild type allele 7. The sequence of the wild-type repeats can be abbreviated to R1, R 2, R e, R3, R 4, the insertion with 6 extra
alogical studies have revealed that the 6 extra repeat insertion appears to have remained stable over at least 6
repeats to R~, R2, R2, R2, R3, R2, R3/, R2, R2, R3, R4
generations so the solution to this question may require
TABLE I Nucleotide and amino acid sequences of wild-type and mutant alleles a. Wild.type allele
CCT Pro CCT Pro CCT Pro CCc Pro CCT Pro
CAg Gin CAT His CAT His CAT His CAT His
GGc Gly GGT Gly GGT Gly GGT Gly GGT G|y
GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly
GGt Gly GGC Gly GGC Gly GGC Gly GGC Gly
ggc Gly
GGc Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly
GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly
GGt Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly
ggc Gly -
TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp
GGG Gly GGG Gly GGG Gly GGa Gly GGt Gly
CAG Gin CAG Gin CAG Gin CAG Gin CAa Gin
TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp
GGG Gly GGG Gly GGG Gly GGa Gly GGG Gly GGG Gly GGa Gly GGG Gly GGG Gly GGG Gly GGG Gly GGG Gly GGa Gly GGt Gly
CAG Gin CAG Gin CAG Gin CAG Gin CAG Gin CAG Gin CAG Gin CAG Gin CAG Gln CAG Gln CAG Gln CAG Gin CAG Gin CAa Gin
R1 Re Re R3 R4
b. Mutant allele
CCT Pro CCT Pro CCT Pro CCc Pro CCT Pro CCc Pro CCT Pro CCT Pro CCT Pro CCq" Pro CCc Pro CCT Pro CCc Pro CCT Pro
CAg Gln CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His
-
R1 R2 R2 R3
R2 R3/ R2/ R2 R2 R2 Ra/ R2
R3 R4
157 further characterisation of the relevant PrP genes to produce the full haplotypes. In these families insertions have only b e e n found in individuals who have died from, are suffering with or at risk to the dementing illness. H o w e v e r , G o l d f a r b et al. 4 have r e p o r t e d an insertion in a transmitted case of CJD (in an individual of English descent; Dr. P. Brown, personal communication) with 'a bizarre family history of ill-defined neurological disease'. No information regarding the latter case has been published so it is not possible to evaluate its relevance to the role of insertions in the PrP gene in prion-related disease. H o w e v e r , the
presence of mutations in the PrP gene, other than insertions, in patients with CJD or GSS seems to be well established 3-5 and transgenic mice expressing mutant prion protein spontaneously develop neurological disease with spongiform degeneration and gliosis6. It would be rem a r k a b l e therefore if these rare insertions in the PrP gene, in families with histories of dementing illnesses, were not intimately involved in the disease process. M o r e o v e r , it is already clear that the pedigree with the 6 extra r e p e a t insertion, which has been assembled through recent genealogical studies, will yield a lod score in excess of 9 with no obligate recombinants.
1 Collinge, J., Harding, A.E., Owen, F., Poulter, M., Lofthouse, R., Boughey, A.M., Shah, T. and Crow, T.J., Diagnosis of Gerstmann Str~ussler syndrome in familial dementia with prion protein gene analysis, Lancet, ii (1989) 15-17. 2 Collinge, J., Owen, E, Poulter, M., Leach, M., Crow, T.J., Rossor, M.N., Hardy, J., Mullan, M.J., Janota, I. and Lantos, EL., Prion dementia without characteristic pathology, Lancet, 336 (1990) 7-9. 3 Doh-Ura, K., Tateishi, J., Sakaki, H., Kitamoto, T. and Sataki, Y., Pro-leu change at position 102 of prion protein is the most common but not the sole mutation related to Gerstmann-Str~iussler syndrome, Biochim. Biophys. Acta, 163 (1989) 974-979. 4 Goldfarb, L.G., Brown, P., Goldgaber, D., Garruto, R.M., Yanagihara, R., Asher, D.M. and Gajdusek, D.C., Identical mutations in unrelated patients with Creutzfeldt-Jakob disease, Lancet, 336 (1990) 174-175. 5 Hsiao, K.K., Baker, H.E, Crow, T.J., Poulter, M., Owen, E, Terwilliger, J.D., Westaway, D., Ott, J. and Prusiner, S.B., Linkage of a prion protein missense variant to GerstmannStr~iussler syndrome, Nature, 388 (1989) 342-345. 6 Hsiao, K.K., Scott, M., Foster, D., Groth, D.F., De Armond, S.J. and Prusiner, S.B., Spontaneous neurodegeneration in transgenic mice with mutant prion protein, Science, 250 (1990) 1587-1590.
7 Kretzchmar, H.A., Stowring, L.E., Westaway, D., Stubblebine, W.H., Prusiner, S.B. and De Armond, S.J., Molecular cloning of human prion protein cDNA, DNA, 5 (1986) 315-324. 8 0 w e n , E, Poulter, M., Collinge, J. and Crow, T.J., Codon 129 changes in the prion protein gene in caucasians, Am. J. Hum. Genet., 46 (1990) 1215-1216. 9 0 w e n , E and Poulter, M., CoUinge, J., Leach, M., Shah, T., Lofthouse, R., Chen, Y., Crow, T.J., Harding, A.E., Hardy, J. and Rossor, M.N., Insertions in the prion protein gene in atypical dementias, Exp. NeuroL, 112 (1991) 240-242. 10 Owen, E, Poulter, M., Lofthouse, R., Collinge, J., Crow, T.J., Risby, D., Baker, H.E and Ridley, R.M., Insertion in prion protein gene in familial Creutzfeldt-Jakob disease, Lancet, i (1989) 51-52. 11 Owen, E, Poulter, M., Shah, T., Collinge, J., Lofthouse, R., Baker, H., Ridley, R., McVey, J. and Crow, T.J., An in-frame insertion in the prion protein gene in familial Creutzfeldt-Jakob disease, Mol. Brain Res., 7 (1990) 273-276. 12 Prusiner, S.B., Prions and neurodegenerative diseases, N. Engl., J. Med., 317 (1987) 1571-1581. 13 Westaway, D., Carlson, G.A. and Prusiner, S.B., Unravelling prion diseases through molecular genetics, Trends Neurosci., 12 (1989) 221-227.
155
BRESM 80114
A dementing illness associated with a novel insertion in the prion protein gene F. Owen 1, M. Poulter 2, J. Collinge 3, M. Leach 2, R. Lofthouse 2, T.J. A . E . Harding 4
Crow 2
and
1Department of Physiological Sciences, University of Manchester, Manchester (U.K.), 2Division of Psychiatry, Clinical Research Centre, Harrow, Middlesex (U.K.), SSt. Mary's Hospital, London (U.K.) and 4The National Hospital, London (U.K.) (Accepted 5 November 1991) Key words: Prion protein; Creutzfeldt-Jakob disease; Transmissible dementia; Spongiform encephalopathy
Following our previous report of an 144-bp insertion in the open reading frame of the prion protein (PrP) gene, we have now identified a larger, 216-bp, insertion in the gene. The insertion which is in frame encodes 9 extra octapeptide repeat sequences in addition to the 5 repeats normally present and represents the largest insertion so far detected in the PrP gene. Creutzfeldt-Jakob disease (CJD) and Gerstmann Str~ussler syndrome (GSS) are rare neurological diseases that can be transmitted to animals, including primates, through intracerebral innoculation of affected brain material. CJD is usually sporadic but a family history is present in about 15% of cases whereas GSS is almost entirely familial. Both diseases are transmitted vertically in an autosomal dominant manner. CJD and GSS belong to a group of disorders known as the transmissible spongiform encephalopathies which include related animal diseases such as scrapie in sheep and bovine spongiform encephalopathy (BSE) in cattle. The precise nature of the infectious agent remains unknown but the diseases in animals and man are associated with an accumulation in the brain of a host-encoded, abnormal isoform of the priori protein (PrP) which is intimately involved in the disease process 12'13. We have previously reported an insertion in the open reading frame (ORF) of the PrP gene that segregated with the illness in a family with a history of CJD m. The insertion was subsequently shown to encode an extra 6 octapeptide repeat sequences in addition to the 5 repeats normally present in the PrP n. During the course of a recent survey of atypical dementias, we have detected insertions in the O R F of the PrP gene in individuals from an additional 5 families with histories of dementing illness of early onset 9. Of these new cases, individuals from 4 families had insertions whose sequences were identical to that initially described H. Genealogical investigation has since revealed that these four families are
part of a large pedigree with a fatal neurodegenerative disease inherited in an autosomal dominant manner, some cases of which conform to the clinical and neuropathological definition of CAD (in preparation). Several of these cases have been described previously ~'2. The only member so far tested of the remaining family presented at age 55 years with a 2 year progressive history of unsteady gait, self neglect and incontinence of urine. On examination, she was mildly demented with apraxia, occasional myoclonic jerks and gait ataxia. She died several months later. In this patient we report here the finding of a larger insertion in the O R F of the PrP gene encoding an extra 9 octapeptide repeat sequences. D N A was extracted from leukocytes and the ORF of the PrP gene amplified using the polymerase chain reaction (PCR) as described previously 11. The PCR mixture comprised 2/~g DNA, 250 ng of each oligonucleotide primer, 50 mM KC1, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCI2, 0.01% (w/v) gelatin, 200 /~M deoxyribonucleoside triphosphates and 2.5 units of Taq polymerase (Perkin Elmer Cetus) in a total volume of 100 kd. Cycling conditions were 95°C for 1.5 min (denaturing), 50°C for 1.5 min (annealing) and 72°C for 3 min (extending) for 35 cycles in a Techne thermal cycler. Oligonucleotide primer sequences were 5"-AAGAATTCTCTGACATTCTCCTCTTCA-3" (5"-primer) and 5"-AAGGATCCCTCAA G C T G G A A A A A G A - 3 " (3"-primer). The primers included 5"-EcoR1 and B a m H I linkers, respectively, to facilitate cloning. PCR products were ethanol precipitated, digested with EcoRI and B a m H I and size fractionated
Correspondence: E Owen, Department of Physiological Sciences, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
156 on a 1% agarose gel containing 0.5 #g/ml ethidium bromide. The mutant allele was identified under ultraviolet light, excised from the gel and purified using Geneclean (Bio 101), and asymmetrically cloned in pUC8.
and the one reported here to R1, R2, R2, R3, R2, R3/, R2/, R 2, R2, R2, R3/, R2, R 3, R 4. Apart from R1 the repeats have identical amino acid sequences but can be distinguished from each other by changes in the nucle-
The insertion was sequenced on both strands of 5 recombinant clones using the strategy and primers previously described 11 and was found to comprise an extra 9
otide sequences. R 2 / a n d R3/differ from R 2 and R 3 by a single base and have not been observed in the wild-type sequence. Both the 6 extra repeat and 9 extra repeat insertions occur on alleles encoding the methionine form of the amino acid polymorphism at codon 129 in h u m a n prion protein 8 and cannot have resulted from one recom-
in-frame repeat sequences in addition to the 5 repeats present in the wild-type allele. The consensus sequences of the wild-type 7 and mutant alleles are presented in Table I in the format previously published with invariant bases in uppercase and variant bases in lower case letters respectively 11. The nucleotide sequence flanking the
bination event. All the pedigrees in question reside in a relatively small area of Southern England, raising the possibility of c o m m o n ancestry with a degree of genetic instability in this form of mutation. However, the gene-
insertion was identical to that of the wild type allele 7. The sequence of the wild-type repeats can be abbreviated to R1, R 2, R e, R3, R 4, the insertion with 6 extra
alogical studies have revealed that the 6 extra repeat insertion appears to have remained stable over at least 6
repeats to R~, R2, R2, R2, R3, R2, R3/, R2, R2, R3, R4
generations so the solution to this question may require
TABLE I Nucleotide and amino acid sequences of wild-type and mutant alleles a. Wild.type allele
CCT Pro CCT Pro CCT Pro CCc Pro CCT Pro
CAg Gin CAT His CAT His CAT His CAT His
GGc Gly GGT Gly GGT Gly GGT Gly GGT G|y
GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly
GGt Gly GGC Gly GGC Gly GGC Gly GGC Gly
ggc Gly
GGc Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly
GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly GGT Gly
GGt Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly GGC Gly
ggc Gly -
TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp
GGG Gly GGG Gly GGG Gly GGa Gly GGt Gly
CAG Gin CAG Gin CAG Gin CAG Gin CAa Gin
TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp TGG Trp
GGG Gly GGG Gly GGG Gly GGa Gly GGG Gly GGG Gly GGa Gly GGG Gly GGG Gly GGG Gly GGG Gly GGG Gly GGa Gly GGt Gly
CAG Gin CAG Gin CAG Gin CAG Gin CAG Gin CAG Gin CAG Gin CAG Gin CAG Gln CAG Gln CAG Gln CAG Gin CAG Gin CAa Gin
R1 Re Re R3 R4
b. Mutant allele
CCT Pro CCT Pro CCT Pro CCc Pro CCT Pro CCc Pro CCT Pro CCT Pro CCT Pro CCq" Pro CCc Pro CCT Pro CCc Pro CCT Pro
CAg Gln CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His CAT His
-
R1 R2 R2 R3
R2 R3/ R2/ R2 R2 R2 Ra/ R2
R3 R4
157 further characterisation of the relevant PrP genes to produce the full haplotypes. In these families insertions have only b e e n found in individuals who have died from, are suffering with or at risk to the dementing illness. H o w e v e r , G o l d f a r b et al. 4 have r e p o r t e d an insertion in a transmitted case of CJD (in an individual of English descent; Dr. P. Brown, personal communication) with 'a bizarre family history of ill-defined neurological disease'. No information regarding the latter case has been published so it is not possible to evaluate its relevance to the role of insertions in the PrP gene in prion-related disease. H o w e v e r , the
presence of mutations in the PrP gene, other than insertions, in patients with CJD or GSS seems to be well established 3-5 and transgenic mice expressing mutant prion protein spontaneously develop neurological disease with spongiform degeneration and gliosis6. It would be rem a r k a b l e therefore if these rare insertions in the PrP gene, in families with histories of dementing illnesses, were not intimately involved in the disease process. M o r e o v e r , it is already clear that the pedigree with the 6 extra r e p e a t insertion, which has been assembled through recent genealogical studies, will yield a lod score in excess of 9 with no obligate recombinants.
1 Collinge, J., Harding, A.E., Owen, F., Poulter, M., Lofthouse, R., Boughey, A.M., Shah, T. and Crow, T.J., Diagnosis of Gerstmann Str~ussler syndrome in familial dementia with prion protein gene analysis, Lancet, ii (1989) 15-17. 2 Collinge, J., Owen, E, Poulter, M., Leach, M., Crow, T.J., Rossor, M.N., Hardy, J., Mullan, M.J., Janota, I. and Lantos, EL., Prion dementia without characteristic pathology, Lancet, 336 (1990) 7-9. 3 Doh-Ura, K., Tateishi, J., Sakaki, H., Kitamoto, T. and Sataki, Y., Pro-leu change at position 102 of prion protein is the most common but not the sole mutation related to Gerstmann-Str~iussler syndrome, Biochim. Biophys. Acta, 163 (1989) 974-979. 4 Goldfarb, L.G., Brown, P., Goldgaber, D., Garruto, R.M., Yanagihara, R., Asher, D.M. and Gajdusek, D.C., Identical mutations in unrelated patients with Creutzfeldt-Jakob disease, Lancet, 336 (1990) 174-175. 5 Hsiao, K.K., Baker, H.E, Crow, T.J., Poulter, M., Owen, E, Terwilliger, J.D., Westaway, D., Ott, J. and Prusiner, S.B., Linkage of a prion protein missense variant to GerstmannStr~iussler syndrome, Nature, 388 (1989) 342-345. 6 Hsiao, K.K., Scott, M., Foster, D., Groth, D.F., De Armond, S.J. and Prusiner, S.B., Spontaneous neurodegeneration in transgenic mice with mutant prion protein, Science, 250 (1990) 1587-1590.
7 Kretzchmar, H.A., Stowring, L.E., Westaway, D., Stubblebine, W.H., Prusiner, S.B. and De Armond, S.J., Molecular cloning of human prion protein cDNA, DNA, 5 (1986) 315-324. 8 0 w e n , E, Poulter, M., Collinge, J. and Crow, T.J., Codon 129 changes in the prion protein gene in caucasians, Am. J. Hum. Genet., 46 (1990) 1215-1216. 9 0 w e n , E and Poulter, M., CoUinge, J., Leach, M., Shah, T., Lofthouse, R., Chen, Y., Crow, T.J., Harding, A.E., Hardy, J. and Rossor, M.N., Insertions in the prion protein gene in atypical dementias, Exp. NeuroL, 112 (1991) 240-242. 10 Owen, E, Poulter, M., Lofthouse, R., Collinge, J., Crow, T.J., Risby, D., Baker, H.E and Ridley, R.M., Insertion in prion protein gene in familial Creutzfeldt-Jakob disease, Lancet, i (1989) 51-52. 11 Owen, E, Poulter, M., Shah, T., Collinge, J., Lofthouse, R., Baker, H., Ridley, R., McVey, J. and Crow, T.J., An in-frame insertion in the prion protein gene in familial Creutzfeldt-Jakob disease, Mol. Brain Res., 7 (1990) 273-276. 12 Prusiner, S.B., Prions and neurodegenerative diseases, N. Engl., J. Med., 317 (1987) 1571-1581. 13 Westaway, D., Carlson, G.A. and Prusiner, S.B., Unravelling prion diseases through molecular genetics, Trends Neurosci., 12 (1989) 221-227.
