Primary structure of prion and CNS distribution
Primate sequences and human mutations
Unstable CAG triplet repeat in spinocerebellar ataxia
Inherited prion disease with 144 base pair gene insertion.
Insertions in the prion protein gene in atypical dementias.
Inherited prion disease with 144 bp insertion -- misdiagnosis
Frequencies of PrP gene variants in cattle
Octapeptides and the scrapie isoform
Polymorphism analysis of the prion gene in cattle.
A two-repeat octapeptide insert mutation in CJD
A prion disease with a novel 96-base pair insertion
Mutations and polymorphisms in prions
Deletions in the prion protein gene are not associated with CJD
British family with a 144 base pair insertion
Prion disease with 144 base pair insertion in Japan
A mutant prion protein displays an aberrant membrane association
Polymorphisms of the prion protein gene in Italian patients
Nine-fold insertion in the prion protein gene
An in-frame insertion in the prion protein gene
Deletion of one octapeptide repeat in fatal familial insomnia
Five, seven, and eight extra octapeptide coding repeats
Octapeptide mutation in the PAX3 gene
American family with an insert mutation in the PRNP amyloid precursor gene.
An insert mutation in the prion gene in a GSS family.

Bovine PrP gene with 5 or 6 repeats
Prion restriction fragment length polymorphism in cattle and sheep.
Sheep prion polymorphisms
hnRNP homologue in C. elegans to hamster prion

Diversity of oligosaccharide linked to prion asparagines.
GPI anchors of the prion proteins contain sialic acid.

Serum amyloid A protein superfamily as constitutive apolipoproteins
Mouse serum amyloid A protein (SAA5) octapeptide inserts
Neuro octapeptide FF-like immunoreactivity in human plasma

hnRNP homologue in C. elegans to hamster prion

Iwasaki M; Okumura K; Kondo Y; Tanaka T; Igarashi H 
Shionogi Institute for Medical Science, Osaka, Japan. 

Nucleic Acids Res 20: 4001-7 (1992) 
The mammalian prion protein (PrPc) is a cellular protein of unknown function, an altered isoform of which (PrPsc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. The evolutionary conservation of the PrP gene has been reported in the genomes of many vertebrates as well as certain invertebrates. In the genome of nematode Caenorhabditis elegans, the sequence capable of hybridizing with the mammalian PrP cDNA probe has been demonstrated, predicting the presence of the PrP gene homologue in C.elegans. In this study, Southern analysis with the hamster PrP cDNA (HaPrP) probe confirmed the previous observation. Moreover, Northern analysis revealed that the sequence is actively transcribed in adult worms. Thus, we screened C.elegans cDNA libraries with the HaPrP probe and isolated a cDNA that hybridizes to the same sequence in C.elegans that hybridized with the HaPrP probe in the Southern and Northern analyses. The deduced amino acid sequence of this cDNA, however, is substantially homologous with heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins rather than mammalian PrPc. The hnRNPs contain the glycine-rich domain in the C-terminal half of the molecule, which also seemed to be in PrPc at the N-terminal half of the molecule. Both of the glycine-rich domains are composed of tracts with high G + C content, indicating that these tracts may due to the hybridizing signals. These results suggest that this cDNA clone is derived from a novel hnRNP gene homologue in C.elegans but not from a predicted PrP gene homologue.
  MTDVEIKAENGSGDASLEPENLRKIFVGGLTSNTTDDLMREFYS
                     QFGEITDIIVMRDPTTKRSRGFGFVTFSGKTEVDAAMKQRPHIIDGKTVDPKRAVPRD
                     DKNRSESNVSTKRLYVSGVREDHTEDMLTEYFTKYGTVTKSEIILDKATQKPRGFGFV
                     TFDDHDSVDQCVLQKSHMVNGHRCDVRKGLSKDEMSKAQMNRDRETRGGRSRDGQRGG
                     YNGGGGGGGGWGGPAQRGGPGAYGGPGGGGQGGYGGDYGGGWGQQGGGGQGGWGGPQQ
                     QQGGGGWGQQGGGGQGGWGGPQQQQQGGWGGPQQGGGGGGWGGQGQQQGGWGGQSGAQ
                     QWAHAQGGNRNY

Diversity of oligosaccharide linked to prion asparagines.

Endo T; Groth D; Prusiner SB; Kobata A 
Department of Biochemistry, University of Tokyo, Japan. 

Biochemistry 28: 8380-8 (1989) 
Prion proteins from humans and rodents contain two consensus sites for asparagine-linked glycosylation near their C-termini. The asparagine-linked oligosaccharides of the scrapie isoform of the hamster prion protein (PrP 27-30) were released quantitatively from the purified molecule by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The radioactive oligosaccharides were fractionated into one neutral and three acidic oligosaccharide fractions by anion-exchange column chromatography. All oligosaccharides in the acidic fractions could be converted to neutral oligosaccharides by sialidase digestion. Structural studies on these oligosaccharides including sequential exoglycosidase digestion in combination with methylation analysis revealed that PrP 27-30 contains a mixture of bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6(GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4-(Fuc alpha 1----6)GlcNAc as their core. Variation is produced by the different combination of the oligosaccharides Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, GlcNAc beta 1----, Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Sia alpha 2----6Gal beta 1----4GlcNAc beta 1---- in their outer chain moieties. When both asparagine-linked consensus sites are glycosylated, the diversity of oligosaccharide structures yields over 400 different forms of the scrapie prion protein. Whether these diverse asparagine-linked oligosaccharides participate in scrapie prion infectivity or modify the function of the cellular prion protein remains to be established.

GPI anchors of the prion proteins contain sialic acid.

Stahl N; Baldwin MA; Hecker R; Pan KM; Burlingame AL; Prusiner SB 
Department of Neurology, University of California, San Francisco 94143. 

Biochemistry 31: 5043-53 (1992) 
The only identified component of the scrapie prion is PrPSc, a glycosylinositol phospholipid (GPI)-linked protein that is derived from the cellular isoform (PrPC) by an as yet unknown posttranslational event. Analysis of the PrPSc GPI has revealed six different glycoforms, three of which are unprecedented. Two of the glycoforms contain N-acetylneuraminic acid, which has not been previously reported as a component of any GPI. The largest form of the GPI is proposed to have a glycan core consisting of Man alpha-Man alpha-Man-(NeuAc-Gal-GalNAc-)Man-GlcN-Ino. Identical PrPSc GPI structures were found for two distinct isolates or "strains" of prions which specify different incubation times, neuropathology, and PrPSc distribution in brains of Syrian hamsters. Limited analysis of the PrPC GPI reveals that it also has sialylated glycoforms, arguing that the presence of this monosaccharide does not distinguish PrPC from PrPSc.

GPI at Ser 231, truncation of 23 aa or truncation at Gly 228

Stahl N; Prusiner SB 
Department of Neurology, University of California, San Francisco 94143-0518. 

FASEB J 5: 2799-807 (1991)
Neurodegenerative diseases of animals and humans including scrapie, bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease are caused by unusual infectious pathogens called prions. There is no evidence for a nucleic acid in the prion, but diverse experimental results indicate that a host-derived protein called PrPSc is a component of the infectious particle. Experiments with scrapie-infected cultured cells show that PrPSc is derived from a normal cellular protein called PrPC through an unknown posttranslational process. We have analyzed the amino acid sequence and posttranslational modifications of PrPSc and its proteolytically truncated core PrP 27-30 to identify potential candidate modifications that could distinguish PrPSc from PrPC. The amino acid sequence of PrP 27-30 corresponds to that predicted from the gene and cDNA. Mass spectrometry of peptides derived from PrPSc has revealed numerous modifications including two N-linked carbohydrate moieties, removal of an amino-terminal signal sequence, and alternative COOH termini. Most molecules contain a glycosylinositol phospholipid (GPI) attached at Ser-231 that results in removal of 23 amino acids from the COOH terminus, whereas 15% of the protein molecules are truncated to end at Gly-228. The structure of the GPI from PrPSc has been analyzed and found to be novel, including the presence of sialic acid.

Primary structure of prion and CNS distribution

Kitamoto T; Doh-ura K; Muramoto T; Miyazono M; Tateishi J 
Department of Neuropathology, Kyushu University, Fukuoka, Japan. 

Am J Pathol 141: 271-7 (1992)
We immunohistochemically examined tissue sections from patients with prion protein (PrP) polymorphism using hydrolytic autoclaving enhancement. Abnormal PrP accumulations could be classified into plaque formations (plaque-type) and the diffuse gray matter stainings including synaptic structures (synaptic-type). Insertional polymorphism, a point mutation in codon 102 or 117/129, and a polymorphism in codon 129 (Val129) result in plaque-type PrP accumulations. The patients with codon 102 mutation also have synaptic-type PrP accumulations. However, a point mutation in codon 200 did not show plaque-type accumulations, and only showed synaptic-type PrP accumulations.

Primate sequences and human mutations

Cervenakova L; Brown P; Goldfarb LG; Nagle J; Gajdusek DC 
Laboratory of Central Nervous System 

Proc Natl Acad Sci U S A 91: 12159-62 (1994) 
Based on the analysis of genomic DNA from single healthy animals of each of five primate species, nucleotide and predicted amino acid sequences of the infectious amyloid precursor gene of higher apes (Gorilla and Pan) and Old World (Macaca) and New World (Ateles, Saimiri) monkeys showed 95-99% homology to the human sequences, corresponding to their phylogenetic distance from humans. Two of 18 amino acids that differed from humans resulted from nucleotide changes at sites of mutations in humans with familial forms of spongiform encephalopathy (a deleted codon within the codon 51-91 region of 24 bp repeats and a substitution at codon 198). In each of the five animals, codon 129 specified methionine, the more common of the two polymorphic genotypes in humans. Because genotypic homology did not correlate with experimental transmission rates of human spongiform encephalopathy, primary structural similarity of the infectious amyloid precursor protein in humans and experimental primates may not be an important factor in disease transmissibility.

Unstable triplet repeat in spinocerebellar ataxia

Goldfarb LG; Vasconcelos O; Platonov FA; Lunkes A; Gajdusek DC 
Laboratory of Central Nervous System

Ann Neurol 39: 500-6 (1996) 
A Siberian kindred with spinocerebellar ataxia genetically linked to the SCA1 locus on chromosome 6p has been screened for the CAG triplet expansion within the coding region of the SCA1 gene. The kindred includes 1,484 individuals, 225 affected and 656 at risk, making this collection the largest spinocerebellar ataxia type 1 (SCA1) pedigree known. Each of the studied 78 SCA1 patients carried an expanded allele containing a stretch of 39 to 72 uninterrupted CAG repeats. Normal alleles had 25 to 37 trinucleotide repeats. Expanded alleles containing 40 to 55 repeats were found in 26 at-risk relatives. The number of CAG repeats in the mutated allele was inversely correlated with age at disease onset. Cerebellar deficiency was present in each patient and its severity was moderately affected by the number of CAG repeats. In contrast, the associated signs, dysphagia, diffuse skeletal muscle atrophy with fasciculations, and tongue atrophy were absent or mild in patients with low CAG repeat numbers, but severely complicated the course of illness in patients with a larger number of repeat units.

Inherited prion disease with 144 base pair gene insertion.

Poulter M; Baker HF; Frith CD; Leach M; Brown J; et al 
Division of Psychiatry, Clinical Research Centre, Harrow, UK. 

Brain 115 ( Pt 3): 675-85 (1992)
Genealogical and molecular studies were carried out in four families in which early onset dementia is inherited as an autosomal dominant. These studies indicated that the four families derive from four siblings whose parents were born in the late 18th century in South-East England. The disease was found to be closely linked to a 144 bp insertion within the open reading frame of the prion protein (PrP) gene with a maximum LOD score of 11.02 at zero recombination. Within the general population the PrP gene is polymorphic at codon 129 (allele frequency approximately 30% valine, 70% methionine). The insertion in this family is always within a methionine-129 allele. The age at death of affected individuals whose normal allele encoded methionine at codon 129 was significantly lower than those whose normal allele encoded valine. The clinical features which were very variable and the neuropathological findings, which sometimes included spongiform encephalopathy, but which often did not, are described fully in the accompanying article (Collinge et al., 1992).

Insertions in the prion protein gene in atypical dementias.

Owen F; Poulter M; Collinge J;  Harding AE; Hardy J; et al 
Division of Psychiatry, Clinical Research Centre, Harrow, Middlesex, United Kingdom. 

Exp Neurol 112: 240-2 (1991)
A number of mutations have been demonstrated in the open reading frame (ORF) of the prion protein (PrP) gene in patients with familial Creutzfeldt-Jakob disease or Gerstmann-Straussler syndrome. On the basis of detecting an insertion in the ORF of the PrP gene in a patient originally suspected to be suffering from familial Alzheimer-type dementia, we screened 101 individuals with atypical dementias for the known PrP gene mutations. Insertions were found in five individuals, whereas none of the other reported mutations in the PrP gene was detected in the present study. One of the five insertions was larger than that described previously, suggesting that the individuals with these mutations are unlikely to be all lineally related and that insertions in the PrP gene may not be uncommon in prion diseases.

Inherited prion disease with 144 bp insertion -- misdiagnosis

Collinge J; Brown J; Hardy J; Ridley R; et al 
Department of Biochemistry and Molecular Genetics, St Mary's Hospital Medical School,

Brain 115 ( Pt 3): 687-710 (1992)
A large family with autosomal dominant segregation of presenile dementia, and other neurological and behavioural features is described. At various times, family members have carried diagnoses of Alzheimer's disease, Huntington's disease, Parkinson's disease, myoclonic epilepsy, atypical dementia, Pick's disease, Creutzfeldt-Jakob disease and Gerstmann-Straussler syndrome. Molecular genetic studies have enabled classification of this disease at the molecular level as one of the group of inherited prion diseases. Here we describe the phenotype of inherited prion disease (PrP 144 bp insertion).
GPI at Ser 231, truncation of 23 aa or truncation at Gly 228

Bovine PrP gene with 5 or 6 repeats

Goldmann W; Hunter N; Martin T; Dawson M; Hope J 
AFRC Institute for Animal Health, Edinburgh, U.K. 

J Gen Virol 72 ( Pt 1): 201-4 (1991)
Current models of the virus-like agents of scrapie and bovine spongiform encephalopathy (BSE) have to take into account that structural changes in a host-encoded protein (PrP protein) exhibit an effect on the time course of these diseases and the survival time of any man or animal exposed to these pathogens. We report here the sequence of different forms of the bovine PrP gene which contain either five or six copies of a short, G-C-rich element which encodes the octapeptide Pro-His-Gly-Gly-Gly-Trp-Gly-Gln or its longer variants Pro-Gln/His-Gly-Gly-Gly-Gly-Trp-Gly-Gln. Out of 12 cattle, we found eight animals homozygous for genes with six copies of the Gly-rich peptide (6:6), while four were heterozygous (6:5). Two confirmed cases of BSE occurred in (6:6) homozygous animals..

Prion restriction fragment length polymorphism in cattle and sheep.

Ryan AM; Womack JE 
Department of Veterinary Pathobiology, Texas A&M 

Anim Genet 24: 23-6 (1993) 
Brains affected by the progressive neurological disease bovine spongiform encephalopathy (BSE) contain scrapie-associated fibrils and the protease-resistant isoform of prion protein. The gene encoding the normal host prion protein (PRNP) has been mapped to human chromosome 20 and mouse chromosome 2 with the hamster cDNA probe pEA974. Using this probe and a panel of bovine-rodent hybrid somatic cells, we have mapped PRNP to bovine syntenic group U11 (100% concordancy). PRNP restriction fragment length polymorphisms (RFLPs) were detected with five of six enzymes (BglII, EcoRI, HindIII, MspI and TaqI) in sheep, in contrast to one of 16 enzymes (HincII) in cattle. Codominant segregation of the bovine HincII RFLP was demonstrated in six backcross pedigrees. While PRNP RFLPs are tightly linked to scrapie incubation period, and consequently susceptibility or resistance to disease in rodents and sheep, the relationship between the PRNP RFLPs and BSE incubation period has not been determined.

Sheep polymorphisms

Goldmann W; Hunter N; Smith G; Foster J; Hope J 
Institute for Animal Health, AFRC and MRC Neuropathogenesis Unit, Edinburgh, U.K. 

J Gen Virol 75 ( Pt 5): 989-95 (1994) 
Genotype analysis showed that dimorphisms in codons 136 and 171 of the ovine PrP gene correlated with control of disease incidence and modulation of incubation time. Crucially, the functional effects of these domains of PrP were shown to alternate depending on the isolate of infecting agent.

Frequencies of PrP gene variants in cattle

Hunter N; Goldmann W; Smith G; Hope J 
Institute for Animal Health, Neuropathogenesis Unit, Edinburgh. 

Vet Rec 135: 400-3 (1994) 
Bovine spongiform encephalopathy (BSE) is one of a family of scrapie-like diseases which affect various mammals. Polymorphisms and mutations of the PrP gene have been associated with the incidence of experimental and natural scrapie in other animals and this study of the bovine PrP gene was undertaken to discover whether there was a similar association with PrP genotype in cattle with BSE. There are two known polymorphisms of the coding region of the bovine PrP gene, a silent HindII restriction site polymorphism and a difference in the number of an octapeptide repeated sequence (either five or six copies). An analysis of 370 cattle in Scotland revealed no difference between the frequencies of these PrP genotypes in healthy cattle and cattle with BSE.

Octapeptides and the scrapie isoform

Rogers M; Yehiely F; Scott M; Prusiner SB 
Department of Neurology, University of California, San Francisco 94143. 

Proc Natl Acad Sci U S A 90: 3182-6 (1993)
We examined the role of the N and C termini in the formation of PrPSc in persistently infected, mouse neuroblastoma (ScN2a) cells. Neither deletion of amino acids 23-88, which are also removed by proteinase K in the formation of PrP 27-30, nor deletion of the five octapeptide repeats within this region altered synthesis of PrPSc. Elongation of PrP with one, two, four, or six octapeptide repeats in addition to the five found in wild-type PrP did not alter the synthesis of PrPSc. Truncation of the C terminus was accomplished by substituting a translation stop codon for the predicted glycosylinositol phospholipid (GPI) anchor-attachment signal corresponding to amino acids 231-254. Expression of this C-terminal PrP mutant in ScN2a cells produced PrPSc that appeared to lack a GPI anchor. We conclude that neither the GPI anchor nor the N-terminal 66 amino acids are required for the synthesis of PrPSc as measured by the acquisition of limited resistance to proteinase K digestion. Whether these truncated or elongated PrP molecules are competent to participate in the formation of infectious prions remains to be established. Insertion of six additional octapeptide repeats between codons 51 and 90 did cause CJD in mouse cell culture.

Polymorphism analysis of the prion gene in cattle.

Neibergs HL; Ryan AM; Womack JE; Spooner RL; Williams JL Anim Genet 25: 313-7 (1994) Polymerase chain reaction (PCR) primers designed to amplify the octapeptide repeat region of the bovine prion gene were used to test the association of genotypes with bovine spongiform encephalitis (BSE) in 56 BSE-affected and 177 unaffected animals. Three alleles (A,B,C) were detected as single-strand conformation polymorphisms (SSCPs) and two alleles (1,2--representing six or five copies of the octapeptide repeat respectively) were detected as amplified double-strand fragment length polymorphisms (AMFLPs). Observed genotypes of SSCPs and AMFLPs were analysed by chi-square. The SSCP genotypes of nuclear family members of animals with BSE and BSE-affected animals were different (P < 0.001, P < 0.01) from unrelated animals of the same breed without BSE. No genotypic differences were found between the BSE-affected animals and their relatives (P > 0.469). No AMFLP genotypic differences were detected between BSE-affected animals, their relatives, unrelated animals of the same breed or animals of different breeds (P > 0.05). These data suggest that BSE-affected animals and their relatives are more likely to have the AA SSCP genotype than unrelated animals of the same breed or animals of different breeds.

A two-repeat octapeptide insert mutation in CJD

Goldfarb LG; Brown P; Gajdusek DC 
Laboratory of CNS Studies, National Institute of Neurological Disorders and Stroke

Neurology 43: 2392-4 (1993)
We report a family in which the proband died of clinically typical, neuropathologically verified Creutzfeldt-Jakob disease; her still-living mother suffers from a progressive dementia of many years' duration, and her maternal grandfather died after a similar illness. The proband, her mother, and two of three young first-degree relatives all have an identical insert mutation in the PRNP gene consisting of a twice-repeated 24-nucleotide sequence in the region between codons 51 and 91.

A prion disease with a novel 96-base pair insertion

Campbell TA; Palmer MS; Will RG; Gibb WR; Luthert PJ; Collinge J There are coding mutations in the prion protein gene in familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease, and other phenotypes that make up the inherited prion diseases. Insertional mutations consisting of two, five, six, seven, eight, and nine additional octapeptide repeat elements are seen in the inherited prion diseases and usually present as atypical dementias with considerable intrafamilial phenotypic variability. A four-octarepeat insertion was reported previously in an individual without neurodegenerative disease who died of hepatic cirrhosis. Here we report a novel four-octarepeat insertional mutation in a case with classical clinical, electroencephalographic and histopathologic features of CJD with the unusual finding of pronounced prion protein immunoreactivity of the molecular layer of the cerebellum.

Mutations and polymorphisms in prions

Palmer MS; Collinge J 
Hum Mutat 2: 168-73 (1993) 
Inherited forms of prion diseases are associated with mutations in the prion protein gene. A common polymorphism at codon 129 is also implicated in the predisposition of individuals to sporadic or iatrogenic forms of the disease. This update lists all the currently published mutations and polymorphisms together with their clinical phenotypes, and discusses the significance of the codon 129 genotype in inherited, sporadic, and iatrogenic cases. There are two categories of mutation. Insertions of additional numbers of an octapeptide lying within an octapeptide repeat region now account for six variations and there are also six point mutations.

Deletions in the prion protein gene are not associated with CJD.

Palmer MS; Mahal SP; Campbell TA; Hill AF; Sidle KC; Laplanche JL; Collinge J 
Hum Mol Genet 2: 541-4 (1993) 
The human prion diseases (spongiform encephalopathies) Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler syndrome (GSS), are neurodegenerative disorders characterised by the accumulation of an abnormal isoform of the prion protein. The normal prion protein is a phosphatidyl inositol anchored, membrane bound sialoglycoprotein of widespread tissue distribution but expressed predominantly in the brain. 15% of prion diseases are autosomal dominant genetic disorders associated with mutations in the gene encoding the prion protein. To date six pathogenic amino acid substitutions have been identified in affected family members, in addition to five distinct insertional events which occur within a region of the protein comprising four tandem octapeptide repeats. We have investigated deletions within this region and have identified three specific deletions. We report here that these deletions are not associated with CJD and represent a new class of polymorphism within the prion protein gene.

British family with a 144 base pair insertion

Nicholl D; Windl O; de Silva R; Sawcer S; Will RG 
J Neurol Neurosurg Psychiatry 58: 65-9 (1995) 
A case of familial Creutzfeldt-Jakob disease associated with a 144 base pair insertion in the open reading frame of the prion protein gene is described. Sequencing of the mutated allele showed an arrangement of six octapeptide repeats, distinct from that of a recently described British family with an insertion of similar size. Thirteen years previously the brother of the proband had died from "Huntington's disease", but re-examination of his neuropathology revealed spongiform encephalopathy and anti-prion protein immunocytochemistry gave a positive result. The independent evolution of at least two distinct pathological 144 base pair insertions in Britain is proposed.

Prion disease with 144 base pair insertion in Japan

Oda T; Kitamoto T; Tateishi J; Mitsuhashi T; Yanai K; et al 
Department of Neuropsychiatry, National Shimofusa Sanatorium, Chiba, Japan. 

Acta Neuropathol (Berl) 90: 80-6 (1995) 
We describe an insert mutation in the prion protein (PrP) gene in a Japanese family line that encodes six octapeptide repeats. This is the second report to date of an inherited prion disease with a 144-base pair insertion, although the order of the repeat sequences differ from that reported for the disease in an English family line.

A mutant prion protein displays an aberrant membrane association

Lehmann S; Harris DA 
Department of Cell Biology and Physiology, Washington University School of Medicine

J Biol Chem 270: 24589-97 (1995)
Inherited forms of prion disease have been linked to mutations in the gene encoding PrP, a neuronal and glial protein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. One familial form of Creutzfeldt-Jakob disease is associated with a mutant PrP containing six additional octapeptide repeats. We report here our analysis of cultured Chinese hamster ovary cells expressing a murine homologue of this mutant PrP. We find that, like wild-type PrP, the mutant protein is glycosylated, GPI-anchored, and expressed on the cell surface. Surprisingly, however, cleavage of the GPI anchor using phosphatidylinositol-specific phospholipase C fails to release the mutant PrP from the surface of intact cells, suggesting that it has an additional mode of membrane attachment. The phospholipase-treated protein is hydrophobic, since it partitions into the detergent phase of Triton X-114 lysates; and it is tightly membrane-associated, since it is not extractable in carbonate buffer at pH 11.5.

Polymorphisms of the prion protein gene in Italian patients

Salvatore M; Genuardi M; Pocchiari M 
Laboratory of Virology, Istituto Superiore di Sanita, Rome, Italy. 

Hum Genet 94: 375-9 (1994) 
Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disorder characterized by the accumulation of the amyloid protein PrP in the CNS. Two coding polymorphisms of the PrP gene (PRNP) are a methionine (Met) to valine (Val) change at codon 129, and a deletion in the octapeptide coding region. In the United Kingdom, homozygosity at codon 129 appears to be associated with a predisposition to develop CJD. However, in Japan, where allelic frequencies and genotype distribution are significantly different, such an association has not been demonstrated. To determine whether such deletion(s) or codon 129 polymorphisms of PRNP predispose to the development of CJD in Italian patients, 31 sporadic CJD patients with no known PRNP mutations, and 186 unrelated control subjects were studied. Genotypic frequencies at codon 129 in these Italian CJD patients revealed a significant excess of methionine alleles, and a different genotype distribution in comparison with the normal Italian population. Deletions of a 24-bp segment located in the PrP octapeptide coding region were found in two control subjects, but in none of the sporadic CJD patients.

Nine-fold insertion in the prion gene

Owen F; Poulter M; Collinge J; Leach M; Lofthouse R;
Department of Physiological Sciences, University of Manchester, U.K. 

Brain Res Mol Brain Res 13: 155-7 (1992)
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.

An in-frame insertion in the prion protein gene

Owen F; Poulter M; Shah T; Collinge J; Lofthouse R;
Division of Psychiatry, Clinical Research Centre, Harrow, Middlesex, U.K. 

Brain Res Mol Brain Res 7: 273-6 (1990)
In a pedigree with Creutzfeldt-Jakob disease we identified a 144-bp insertion in the open reading frame of the prion protein (PrP) gene. The insertion is in-frame and codes for 6 extra uninterrupted octapeptide repeats in addition to the 5 that are normally present in the N-terminal region of the protein. The possibility that this mutation may prove relevant to elucidating the mechanism of horizontal transmission of the spongiform encephalopathies is discussed.

Deletion of one octapeptide repeat in fatal familial insomnia.

Reder AT; Mednick AS; Brown P; Spire JP;  Ovsiew F; et al 
Department of Neurology, University of Chicago School of Medicine, IL, USA. 

Neurology 45: 1068-75 (1995) 
We report a 42-year-old man who, for 8 months, had intermittent motor abnormalities and mild difficulty falling asleep. A diagnosis of fatal familial insomnia (FFI) became evident over the next 6 months when he developed progressive insomnia, myoclonus, sympathetic hyperactivity, and dementia. The amyloid or prion protein (PrP) genotype showed features typically seen in FFI, with a 178Asn mutation and a 129Met polymorphism. There was also a deletion of one octapeptide repeat, suggesting that the association of 178Asn mutation with the 129Met polymorphism is not due to "founder effect." Western immunoblot showed a trace of protease-resistant PrP in the thalamus--which had the most significant neuronal loss and gliosis--a moderate amount of PrP in the fronto-temporal area, and no detectable protein elsewhere in the brain. Endocrine studies showed that a circadian modulation of hormonal levels could be maintained despite a near-total absence of sleep. Administration of gamma-hydroxybutyrate induced a remarkable increase in slow-wave sleep.

Five, seven, and eight extra octapeptide coding repeats

Goldfarb LG; Brown P;  Gajdusek DC 
Laboratory of Central Nervous System Studies
Proc Natl Acad Sci U S A 88: 10926-30 (1991) 
The PRNP gene, encoding the amyloid precursor protein that is centrally involved in Creutzfeldt-Jakob disease (CJD), has an unstable region of five variant tandem octapeptide coding repeats between codons 51 and 91. We screened a total of 535 individuals for the presence of extra repeats in this region, including patients with sporadic and familial forms of spongiform encephalopathy, members of their families, other neurological and non-neurological patients, and normal controls. We identified three CJD families (in each of which the proband's disease was neuropathologically confirmed and experimentally transmitted to primates) that were heterozygous for alleles with 10, 12, or 13 repeats, some of which had "wobble" nucleotide substitutions. We also found one individual with 9 repeats and no nucleotide substitutions who had no evidence of neurological disease. These observations, together with data on published British patients with 11 and 14 repeats, strongly suggest that the occurrence of 10 or more octapeptide repeats in the encoded amyloid precursor protein predisposes to CJD.

Octapeptide mutation in the PAX3 gene causing Waardenburg syndrome

Tassabehji M; Read AP; Newton VE; Patton M; Gruss P; Harris R; Strachan T 
University Department of Medical Genetics, St Mary's Hospital, Manchester, UK. 

Nat Genet 3: 26-30 (1993) 
Waardenburg syndrome (WS) is a combination of deafness and pigmentary disturbances, normally inherited as an autosomal dominant trait. The pathology involves neural crest derivatives, but WS is heterogeneous clinically and genetically. Some type I WS families show linkage with markers on distal 2q and in three cases the disease has been attributed to mutations in the PAX3 gene. PAX3 encodes a paired domain, a highly conserved octapeptide and probably also a paired-type homeodomain. Here we describe a further three PAX3 mutations which cause WS; one alters the octapeptide motif plus the presumed homeodomain; a second alters all three elements and the third alters the paired box alone. The latter occurs in a family with probable type 2 WS, a clinical variant usually considered not to be allelic with type 1 WS.

Serum amyloid A protein superfamily as constitutive apolipoproteins

Whitehead AS; de Beer MC; Steel DM; Rits M; Lelias JM;
Department of Immunology, Children's Hospital, Boston, Massachusetts. 

J Biol Chem 267: 3862-7 (1992) 
A novel serum amyloid A protein (SAA) has been identified as a normal apolipoprotein component of non-acute phase high density lipoprotein. This novel SAA has been designated "constitutive" SAA (C-SAA) to distinguish it from "acute phase" SAA (A-SAA). C-SAA was partially sequenced, and immunochemical analyses indicated that it constitutes a distinct subclass of apolipoproteins within the SAA superfamily. A C-SAA cDNA clone was isolated from a human liver library and sequenced. The clone predicts a pre-C-SAA molecule of 130 residues from which an 18-residue leader peptide is cleaved. The 112-residue mature molecule is 8 residues longer than human A-SAA; the size difference is due to the presence of an octapeptide between positions 70 and 77 that is not found in the corresponding region of human A-SAA. Paradoxically, octapeptides of similar composition are found at similar positions in the A-SAAs of a number of other species. The C-SAA octapeptide specifies the first two residues of a NSS tripeptide, the only potential N-linked glycosylation site in the molecule. Studies indicate that approximately 50% of these sites are glycosylated.

Neuro octapeptide FF-like immunoreactivity in human plasma.

Sundblom DM; Panula P; Fyhrquist F 
Unit of Clinical Physiology, Minerva Institute for Medical Research, Helsinki, Finland. 

Peptides 16: 347-50 (1995)
In order to examine whether neuropeptide FF (NPFF), an octapeptide with pain-modulating and blood pressure-raising properties in the rat, is present in circulating human blood, a radioimmunoassay (RIA) was established. Thus, plasma NPFF may represent leakage of the peptide from nervous tissue.

Mouse serum amyloid A protein (SAA5) octapeptide inserts

de Beer MC; Kindy MS; Lane WS; de Beer FC 
Department of Biochemistry, University of Kentucky Medical Center, Lexington 40536. 

J Biol Chem 269: 4661-7 (1994) 
A novel member of the mouse serum amyloid A protein family, SAA5, has been identified as a normal apolipoprotein component of non-acute-phase high density lipoprotein (HDL). The structure of SAA5 was derived from a clone isolated from a normal Balb/c liver cDNA library. The clone predicts a pre-SAA5 molecule of 130 residues from which an 18-residue leader peptide is cleaved. The mature molecule has an octapeptide insert spanning from position 70 to 77. Similar inserts are found in human C-SAA and, paradoxically, in acute-phase SAA molecules of a number of other species. There is 48% amino acid identity between apo-SAA5 and the other mouse SAA proteins and 57% identity between the human C-SAA and apo-SAA5.

American family with an insert mutation in the PRNP amyloid precursor gene.

Brown P; Goldfarb LG;  Gajdusek DC 
Laboratory of CNS Studies, NINDS, NIH, Bethesda, MD 20892. 

Neurology 42: 422-7 (1992) 
An American family of English origin with an unusually early onset and long-duration form of Creutzfeldt-Jakob disease (CJD) had a heterozygous insert mutation in the region of repeating octapeptide coding sequences between codons 51 and 91 of the PRNP gene on chromosome 20. Affected members were 23 to 35 years old at the onset of illnesses that lasted from 4 to 13 years, yet experimental transmission of disease from the proband (11-year duration) produced a typically brief incubation period and duration of illness in each of three inoculated primates. Also, the PrP amyloid protein that accumulates in CJD brain was only barely detectable in extracted brain tissue from one case with massive spongiform change and was undetectable in another case with no spongiform change, perhaps because of epitope shielding by a configurational change in the protein induced by the mutation. Analysis of this and other families with similar inserts suggests that such mutations in the PRNP gene not only predispose to CJD, but also modify its phenotypic expression.

An insert mutation in the prion gene in a GSS family.

Goldfarb LG; Brown P; Vrbovska A; Baron H; McCombie
Laboratory of CNS Studies, NINDS, NIH, Bethesda, MD 20892. 

J Neurol Sci 111: 189-94 (1992) 
We report the finding of an insert mutation in the chromosome 20 amyloid precursor gene in a family with neuropathologically-verified, experimentally-transmitted Gerstmann-Straussler-Scheinker syndrome (GSS). The insert consisted of 8 extra copies of a repeating octapeptide coding sequence in the region between codons 51 and 91; it was identified in the proband and a presently unaffected at-risk niece by full sequencing of the open reading frame, and was visualized electrophoretically in the proband and 6 of 12 at-risk relatives. Although affected members in this French-Breton family have shown a variety of clinical profiles, including durations of illness that ranged from 3 months to 13 years, all autopsied cases (including the patient with the shortest illness) have had the distinctive multicentric amyloid plaques that define GSS as a nosologic entity.