_Journal ofNeurology, Neurosurgery, and Psychiatry 1995;58:65-69

Inherited Creutzfeldt-Jakob disease in a Britishfamily associated with a novel 144 base pairinsertion of the prion protein gene

D Nicholl, 0 Windl, R de Silva, S Sawcer, M Dempster, JW Ironside, J P Estibeiro,GM Yuill, R Lathe, R G Will

North ManchesterGeneral Hospital,Crumpsall,Manchester M8 6RB,UKD NichollS SawcerG M YuillAFRC Centre forGenome Research,Kings Buildings,Edinburgh EH9 3JQ,UK0 WindlM DempsterJ P EstibeiroR LatheNational Creutzfeldt-Jakob DiseaseSurveillance Unit,Western GeneralHospital, Crewe Road,Edinburgh EH4 2XU,UKR de SilvaJW IronsideR G WillCorrespondence to:Dr R de Silva.Received 5 April 1994and in revised form13 July 1994Accepted 20 July 1994

AbstractA case of familial Creutzfeldt-Jakobdisease associated with a 144 base pairinsertion in the-open reading frame oftheprion protein gene is described.Sequencing of the mutated allele showedan arrangement of- six octapeptiderepeats, distinct from that of a recentlydescribed British family with an insertionof similar size. Thirteen years previouslythe brother of the proband had died from"Huntington's disease", but re-examina-tion of his neuropathology revealedspongiform encephalopathy and anti-prion protein immunocytochemistrygave a positive result. The independentevolution of at least two distinct patho-logical 144 base pair insertions in Britainis proposed. The importance of main-taming a high index ofsuspicion ofinher-ited Creutzfeldt-Jakob disease in cases offamilial neurodegenerative disease isstressed.

(J Neurol Neurosurg Psychiatry 1995;58:65-69)

Keywords: Creutzfeldt-Jakob disease; prion proteingene; mutations; base pair insertion

Between 6% and 15% of cases of Creutzfeldt-Jakob disease may be familial with an autoso-mal dominant pattern of inheritance.' 2 A

variety of point mutations and base pair inser-tions of the open reading frame of the prionprotein gene have been reported to cosegre-gate with cases of inherited Creutzfeldt-Jakobdisease.36 The pathogenicity of these muta-tions is suggested by their absence in normalsubjects,78 and the apparent development ofspontaneous neurodegeneration in transgenicmice carrying the murine equivalent of theleucine substitution at codon 1029 (character-istically associated with Gerstmann-Strausslersyndrome'0). The region between codons 51and 91 of the open reading frame normallycomprises one nonapeptide and four octapep-tide repeats." Insertions of two and betweenfive and nine extra octapeptide repeats havebeen associated with Creutzfeldt-Jakob dis-ease."' In their paper on a large family fromthe south east of England with a 144 base pairinsertion (six extra repeats), Collinge et aldescribed the remarkable diversity of clinicaland neuropathological features of affectedcases. 12-14 The nucleotide sequence of themutated allele in this family showed that the144 base pair insertion had remained stableover at least six generations.8 In the currentcommunication another British family with abase pair insertion of similar size but novelsequence (unrelated to the original family)is described. There were remarkable similari-ties to the first pedigree with respect to the.diversity of clinical features in affectedmembers.

Case reportThe propositus (III.6, fig 1) presented aged46 with a two month history of progressivevisual disturbance and unsteadiness. He wasunable to operate machinery at work andseemed less concerned about his perfor-mance. One month before admission he couldnot recognise large objects or relatives' faces,and by the time of his admission could onlydistinguish between light and dark. By then hehad difficulty standing independently.Medical history was unremarkable, and hetook no medication. He smoked 20 cigarettesdaily and consumed 4 units of alcoholmonthly. On examination he was euphoric,disoriented in time, and had impaired shortterm memory (with confabulation). He wasmildly dysphasic. He could not perceive handmovements, but pupils were equal with nor-mal responses to light stimulation. There wastremor on maintained posture of his arms.

11

III

IV

Figure I Family tree.

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Nicholl, Windl, de Silva, Sawcer, Dempster, Ironside, Estibeiro, Yuill, Lathe, Will66

Tone and reflexes were physiological andsymmetric, and plantar responses were down-going. Gait was unsteady.The following investigations were normal

or negative: routine biochemistry and haema-tology, liver function tests, autoantibody pro-file, syphilis serology, TSH, copper studies,and HIV serology. Examination of CSFshowed 4 lymphocytes per mm' and a proteincontent of 0 16 g/l. Computerised tomogra-phy showed enlarged ventricles, and cerebraland cerebellar cortical atrophy. Electro-encephalography showed bilaterally synchro-nous, repetitive, triphasic discharges at 1 persecond on a background of slow rhythms.Pattern reversal visual evoked potentials couldnot be performed, and in response to a flashstimulus the late components were dispersed(P3 at 178 ms bilaterally).

Figure 2 (A) Occipital cortex in case III. 6 showith severe reactive astrocytosis (haematoxylin-e(B) Immunocytochemistry for prion protein shoeproduct around areas of confluent spongiform ch

reaction after hydrolytic autoclaving and haema

..~~~~~~~~~~~~.

A~~~~~~~AP~

Figure 3 (A) Putamen in case III. 4 showingloss in an irregular distribution (haematoxylin-(B) Immunocytochemistry for prion protein shcproduct around areas ofconfluent spongiform c

technique after hydrolytic autoclaving with hae

Myoclonic jerks were noted soon afteradmission, and he developed dressing apraxia.Frontal release signs recurred, and paratonicrigidity of his limbs emerged. Gait ataxiaworsened and he became bedbound.Terminally he had rest and startle myoclonus,and became akinetic and mute. He died onemonth after admission.The patient's brother III.4 (fig 1) was

thought to have died from Huntington'sdisease at the age of 39, 13 years previously.His terminal illness had been of five years'duration, and had been characterised by chor-eiform movements, gait disturbance, anddementia. Several other members of thepedigree had also succumbed to dementingillnesses, variously described as "shellshock",Huntington's disease, general paralysis of theinsane, and "organic dementia" (fig 1). Afterhis brother's presentation, III.4's neuro-pathology was reviewed.

Materials, methods, and resultsNEUROPATHOLOGYAfter necropsy, the brain from case III.6 wasfixed in 10% formalin and submitted tothe National Creutzfeldt-Jakob disease Sur-veillance Unit for investigation. The fixedbrain weighed 1234 g and showed a moderatedegree of cortical atrophy in the cerebralhemispheres. No other relevant externalabnormalities were noted. Tissue blocks from

g 5t ^ all cortical areas, the basal ganglia, thalamus,hypothalamus, brainstem, and cerebellumwere decontaminated in 96% formic acid forone hour before routine processing into paraf-fin wax. Five ,um sections were cut andstained by conventional histological tech-

B niques and by immunocytochemistry for

neuronal loss prion protein using a polyclonal antibody?wing spongiformchaxng and raised against scrapie associated fibrils (cour-

wing a positive reaction with a dark stain tesy of Dr J Hope, MRC/AFRC Neuro-tange in the occipital cortex (avidin-biotin pathogenesis Unit, Edinburgh).ctoxylin counterstain, originally x 240). From case III.4, unstained paraffin sections

were obtained from the cerebellum, frontal,parietal, temporal, and occipital lobes, basalganglia, and thalamus. These were stained byconventional histological techniques and byimmunocytochemistry for prion protein as

described earlier.In case III.6, the sections of the cerebral

hemispheres showed a spongiform encepha-lopathy involving the cerebral cortex in awidespread distribution with particularlysevere occipital and parietal involvement.

A > t y ; B:Reactive gliosis in these regions was conspicu-.). ! :i,.,'...ousas well as spongiform change and neu-

ronal loss. The visual cortex showed status

spongiosus. Spongiform change was noted inthe basal ganglia, particularly involving theputamen, and was also noted in a patchy dis-tribution throughout the molecular layer inthe cerebellar cortex. Prion protein immuno-

E!- B cytochemistry showed a positive reaction inthe cerebral cortex within and around areas of

Iconfluent spongiform change with neuronal spongiform change (fig 2). In the cerebellum,-eosin, originally x 200). focal deposition of prion protein wasowing dense deposition of the dark reaction accompanied by a more diffuse staining in the;hange in the putamen (avidin-biotinmatoxylin counterstain, originally x 240). granular layer. No evidence of Huntington's

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Inherited Creutzfeldt-Jakob disease in a British family associated with a novel 144 base pair insertion of the prion protein gene

1 2 3 4 5 6 7

2036 --

1636

1018

506/17 --

396

1018

--- 874

546

---- 472

1-- 402

Figure 4 Analysis ofprion protein gene PCR products by gel electrophoresis. 10 ofeach PCR reaction was loaded directly (lanes 2-4) or after P3vuII digest (lanes S and 6).PCR was performed on genomic DNA ofpatient III. 6 (lanes 2 and 5) and an unrelatedpatient without any mutation of the open readingframe of the prion protein gene (lanes 3and 6). A PCR reaction with no template DNA was added as a contamination control(lane 4). The sizes of the PCR products were estimated by comparison with markerDNA(lanes 1 and 7, 1 kb ladder, GicoBRL; sizes on the left in bp). Calculated values of thepublished human prion protein gene sequence fragments are given in bp on the right.

disease or any other primary neuro-

degenerative disorder was noted. Occasionalblood vessels in the deep grey matter andcentral white matter regions showed arterio-sclerosis with patchy calcification but therewas no evidence of appreciable ischaemicdamage.

In case III.4, spongiform change was notedin a patchy distribution in the cerebral hemi-

spheres, most conspicuously in the temporaland frontal lobes. Cortical atrophy in theseregions was accompanied by neuronal lossand reactive gliosis. Spongiform change wasalso present in a patchy distribution in thecerebellar cortex and the basal ganglia. Therewas no evidence of Huntington's disease orany other primary neurodegenerative disor-der. Immunocytochemistry for prion proteinshowed a patchy positive reaction in the cere-bral cortex in relation to areas of spongiformchange (fig 3). Occasional astrocytes alsoshowed punctate prion protein immunoreac-tivity within the cytoplasm.

MOLECULAR BIOLOGYGenomic DNA was extracted from the bloodof patient III.6 and control subjects by stan-dard techniques.'5 The open reading frame ofthe prion protein gene was amplified by thepolymerase chain reaction (PCR) in a 50 ulreaction containing 1 jug of genomic DNA, 50mM KC1, 10 mM Tris HC1 (pH 9-0), 0-1%Triton X-100, 1-5 mM MgCl,, 0-2 mM ofeach deoxynucleotide triphosphate, 1 pM ofeach oligonucleotide primer, and 2-5 unitsAmplitaq DNA polymerase (Perkin ElmerCetus). The sequence of the oligonucleotideprimers were 5'-CGCAAGCTTGAACTCT-GAACATTCTCCTCTTC-3' and 5'-TTC-GAATTCCTCCCTCAAGCTGGAAAAAG-3', which are situated 5' or 3' to the humanopen reading frame of the prion proteingene. 16 The mixture was overlayered withmineral oil and subjected to 35 temperaturecycles (05' 95°C, 1' 55°C, 2' 72°C) in an

Omnigene thermal cycler. To analyse theproduct, 10 jul of the reaction was loaded onto a 1-5% agarose gel either directly or aftercleavage with the restriction endonucleasePvuII, electrophoretically separated, andstained with ethidium bromide. To clone andsequence the PCR products, they were cut

Sequence comparison of the normal and mutated tandem repeat regions of the prion protein gene

Mutant allele Mutant allele Mutant allele(patient III. 6) (Owen, et al'7) (Owen et al'9)144bp insert 144 bp insert 216 bp insert

Normal alleleCCT CAG GGCPro Gln GlyCCT CAT GGTPro His GlyCCT CAT GGTPro His GlyCCC CAT GGTPro His GlyCCT CAT GGTPro His Gly

GOTGlyGOTGlyGOTGly

GGOTGly

GGOTGly

GOTGlyGOCGlyGOCGly

GGOCGly

GGOCGly

GOC TGO

Gly Trp

-G-- TG

Trp

-G-- TG

Trp

-G-- TG

TrP

-G-- TG

Trp

GOGGlyGOGGlyGGOGly

GGOAGly

GGOTGly

CAG

GlnCAG

Gln

CAGGlnCAGGlnCAAGln

Only in mutant alleles

CCC CATPro HisCCT CATPro His

GGT GGT GGC ---

Gly Gly Gly -

GGT GGT GGC ---

Gly Gly Gly -

TGG GGG CAGTrp Gly GlnTGG GGA CAGTrp Gly Gln

RI RI

R2

RIRI

R2

R2

R3

R4

R2

R2 R2

R3

R2

R3gR2

R3gR2

R3g R3

R2a R4

R2

R2

R3

R2

R3gR2

R3gR2

R3

R4

R2

R3R2R3gR2R2

R3

R4

R2a<R2

R2

Repeats are named according to the proposed nomenclature of Goldfarb et al 4 and the top to bottom order reflects the 5' to 3'direction. The nucleotide and amino acid sequences of the repeats in the normal allele and of the repeats R2a and R3g, unique tomutated alleles, are presented. Silent nucleotide changes in R2a, R3, R3g and R4 compared with R2 are underlined. Thediffering regions between the mutated alleles with 144 bp are printed in bold, and the position of the three additional repeats inthe allele with a 216 bp insert is indicated.

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Nicholl, Windl, de Silva, Sawcer, Dempster, Ironside, Estibeiro, Yuill, Lathe, WiU

PvuII

874 bp

-,

144 bpFigure 5 Schematic representation of the human openreadingframe of the prion protein gene. The open readingframe is shown as a dotted bar and the five tandem repeatsare indicated. The size of the PCR product of a normalallele (874 bp) exceeds the open readingframe at the 5'and 3 ends, and the approximate position and scale of thesix repeat inserts (144 bp) are indicated. The restrictionenzyme PvuII cleaves the prion protein geneasymmetrically.

with the restriction endonucleases MspI andSau3A to create a large fragment includingmost of the open reading fraction of theprion protein gene. The digestion products ofthe alleles were isolated from an agarose gelseparately. They were cloned into the vectorpBluescriptIIKS(-) (Stratagene), that wasitself cut with the enzymes Cla I and BamH Ito create compatible ends. The inserted openreading frame was sequenced withSequenaseII (United States Biochemicals) asdouble stranded material according to themanufacturer's recommendations. Thesequencing primers used were either the T3and T7 primers (Stratagene) or the syntheticoligonucleotides 5'-TGGCACCCACAGTC-AGT-3' and 5'-TTCTCCCCCTTGGTGG-T-3', which correspond to internal sequencesof the human open reading frame of the prionprotein gene.'6

Agarose gel electrophoresis of the PCRproducts from the genomic DNA of III.6showed two major products of about 850 bpand 1000 bp in size (fig 4, lane 2). This indi-cated that one prion protein allele in III.6 hadan insertion of about 150 bp. In normal sub-jects (and patients with Creutzfeldt-Jakob dis-ease without extra octapeptide repeats in theirprion protein gene) both prion protein allelesare represented by a single 850 bp fragment(fig 4, lane 3). A digest of the PCR amplifiedmaterial with the restriction endonucleasePvuII showed in a normal subject two frag-ments of 400 bp and 470 bp, correspondingto the 5' and 3' parts of the gene respectively(fig 4, lane 6; fig 5). The same treatment ofthe PCR product of III.6 showed a thirdband at about 550 bp (fig 4, lane 5) indi-cating the presence of an insertion of about150 bp in the 5' part of the gene. This locali-sation matched to the only known insertionsite within the prion protein gene that isassociated with familial Creutzfeldt-Jakob dis-ease, the octapeptide repeat region, and thesize estimation of the mutated PCR productwas compatible with a 144 bp insertion (fig5).

Archival CNS tissue from III.4 was avail-able, but several attempts at DNA extraction

and amplification of the open reading frameof the prion protein gene were unsuccessful.This was probably due to the old age and pro-longed fixation time of the tissue.The open reading frame of the prion pro-

tein gene of III.6 was further analysed aftersubcloning and sequencing. To excludeamplification errors caused by the Taq DNApolymerase, the products of five PCR reac-tions were analysed independently. Theresults were consistent and confirmed a 144bp insertion in the octapeptide region of themutated allele. The arrangement of therepeats was different from the sequencedescribed in the previous British pedigreewith an insertion of the same size.'7 Whereasthe first (Rl, R2, R3) and the last (R2, R3,R4) three repeats were identical, the order ofthe five repeats in the middle of the mutatedallele was R3, R2, R3g, R2, R3g comparedwith R2, R3, R2, R3g, R2 in the previouslydescribed pedigree (table). Although thesechanges have not influenced the amino acidsequence of the octapeptides, the nucleic acidsequence clearly establishes this as a case offamilial Creutzfeldt-Jakob disease with a novel144 bp insertion in the octapeptide repeatregion of the prion protein gene.

DiscussionThe clinical course of the propositus was typi-cal of Heidenhain's variant of Creutzfeldt-Jakob disease. 18 In this form occipitalblindness is an early and prominent feature,and seems to correlate with severe spongiformchange of the visual cortex (as here). Thefinding of an insertion of 144 extra base pairsin the N-terminal octapeptide repeat region ofthe open reading frame of his prion proteingene was consistent with familial Creutzfeldt-Jakob disease. Re-evaluation of his brother'sneuropathology confirmed the presence ofspongiform encephalopathy, although due totechnical reasons an identical insertion in hisprion protein gene could not be demon-strated. Other members of this pedigree hadbeen diagnosed as having a variety of neuro-degenerative illnesses, and the variety of labelsattached to them in previous generations isremarkably similar to the previously reportedpedigree from the south east of England.'2Although the size of the insertion was identi-cal in these two families, there was no knownrelationship and, as demonstrated, thesequence of the extra repeats was different.Given the similar size of the insertion and theidentical amino acids encoded, however, it ishardly surprising that a similar spectrum ofclinical phenotypes was noted.

Various sizes of insertions of the open read-ing frame of the prion protein gene havebeen associated with familial Creutzfeldt-Jakob disease.3'5 6 Recently Duchen et alhave described a family with nine extrarepeats,5 19 and although larger by three extrarepeats the similarity in sequences betweenthis and the insertion in the present report isnoteworthy (table). The complete octapeptiderepeat sequence of the current family can be

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Inherited Creutzfeldt-Jakob disease in a British family associated with a novel 144 base pair insertion of the prion protein gene

represented by the first six and the last fiverepeats of the larger sequence. There is insuffi-cient knowledge at present regarding themechanisms leading to the generation of extrarepeats to judge whether the similarities in thesequences of the extra repeats in these twopedigrees arose by chance or whether theyevolved from a common ancestor.The clinical illness of III.4 was compatible

with Huntington's disease with choreiformmovements, progressive intellectual decline,and a family history of neurodegenerative dis-ease. Neuropathological findings in this caseand the clinical, neuropathological, and mole-cular biological findings in his brother, how-ever, make this diagnosis untenable. The genemutation responsible for Huntington's diseasehas been identified recently,20 but cases ofhereditary chorea without this mutation (anexpansion of a trinucleotide repeat sequence)are described.2' Familial Creutzfeldt-Jakobdisease is clearly an important diagnosis tobear in mind in these cases. Expansions oftrinucleotide repeat sequences are increas-ingly recognised in inherited neurological dis-ease, and seem to correlate with the clinicalphenomenon of anticipation.22 This is not aclinical feature of inherited Creutzfeldt-Jakobdisease, and as noted from the molecular bio-logical perspective, there is stability inoctapeptide repeat sequences in successivegenerations.The present report emphasises the impor-

tance of neuropathological examination incases of undiagnosed dementia, althoughCreutzfeldt-Jakob disease as a cause seems tobe rare.2' Nevertheless, the importance ofestablishing this diagnosis, especially where ithas implications for succeeding generations, isreadily apparent. The feasibility of offeringpresymptomatic diagnosis to families withinherited Creutzfeldt-Jakob disease hasalready been investigated.24 This was compli-cated, however, not only by ethical dilemmas,but also by methodological problems (the lessefficient amplification of alleles carrying inser-tions in PCR). These are likely to be espe-cially acute when considering antenataldiagnosis.

Necropsies were performed by Dr De Kretser and Dr Brownat the North Manchester General Hospital. Archival materialfrom case III.4 was made available by the Huntington's diseasebrain bank (Dr Bruton). The Centre for Genome Researchreceives funding from the AFRC, and this work was supportedby its BSE programme (grant No LRG 15/344). The NationalCreutzfeldt-Jakob disease Surveillance Unit is funded by theDepartment of Health. Immunocytochemistry was performedby Ms C Barrie (supported by the AFRC, grant No AG15/610). Mrs H Southworth and Miss J MacKenzie providedsecretarial assistance.

1 Will RG, Matthews WB, Smith PG, Hudson C. A retro-spective study of Creutzfeldt-Jakob disease in Englandand Wales 1970-1979 II: epidemiology. J NeurolNeurosurg Psychiatry 1986;49:749-55.

2 Masters CL, Gajdusek DC, Gibbs CJ. The familial occur-rence of Creutzfeldt-Jakob disease and Alzheimer's dis-ease. Brain 1981;104:535-58.

3 Goldfarb LG, Brown P, Gajdusek DC. The moleculargenetics of human transmissible spongiformencephalopathy. In: Prusiner SB, Collinge J, Powell J,Anderton B, eds. Prion diseases of humans and animals.London: Ellis Horwood, 1992:139-53.

4 Kitamoto T, Ohta M, Doh-ura K, Hitoshi S, Terao Y,Tateishi J. Novel missense variants of prion protein inCreutzfeldt-Jakob disease or Gerstmann-Straussler syn-drome. Biochem Biophys Research Commun 1993;191:709-14.

5 Duchen LW, Poulter M, Harding AE. Dementia associ-ated with a 216 base pair insertion in the prion proteingene. Brain 1993;116:555-67.

6 Goldfarb LG, Brown P, Little BW, Cervenakova L,Kenney K, Gibbs CJ, Gajdusek DC. A new (two-repeat)octapeptide coding insert mutation in Creutzfeldt-Jakobdisease. Neurology 1993;43:2392-4.

7 Doh-ura K, Tateishi J, Sasaki H, Kitamoto T, Sakaki Y.Pro to Leu change at position 102 of prion protein is themost common but not the sole mutation related toGerstmann-Straussler syndrome. Biochem BiophysResearch Commun 1989;163:974-9.

8 Poulter M, Baker HF, Frith CD, Leach M, Lofthouse R,Ridley RM, et al. Inherited prion disease with 144 basepair gene insertion 1. Genealogical and molecular studies.Brain 1992;115:675-85.

9 Hsiao K, Scott M, Foster D, Groth DF, DeArmond SJ,Prusiner SB. Spontaneous neurodegeneration in trans-genic mice with mutant prion protein. Science 1990;250:1587-90.

10 Hsiao K, Baker HF, Crow TJ, Poulter M, Owen F,Terwilliger JD, et al. Linkage of prion protein missensevariant to Gerstmann-Straussler syndrome. Nature1989;338:342-5.

11 Owen F, Poulter M, Collinge J, Leach M, Shah T,Lofthouse R, et al. Insertions in the prion protein gene inatypical dementias. Exp Neurol 1991;112:240-2.

12 Collinge J, Brown J, Hardy J, Mullan M, Rossor MN,Baker H, et al. Inherited prion disease with 144 base pairgene insertion 2. Clinical and pathological features.Brain 1992;115:687-710.

13 Coilinge J, Owen F, Poulter M, Leach M, Crow TJ,Rossor MN, et al. Prion dementia without characteristicpathology. Lancet 1990;336:7-9.

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15 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: alaboratoty manual, 2nd ed. New York: Cold SpringHarbor Laboratory Press, 1989.

16 Kretzschmar HA, Stowring LE, Westaway D, StubblebineWH, Prusiner SB, DeArmond SJ. Molecular cloning ofa human prion protein cDNA. DNA 1986;5:315-24.

17 Owen F, Poulter M, Shah T, Collinge J, Lofthouse R,Baker H, et al. An in-frame insertion in the prion proteingene in familial Creutzfeldt-Jakob disease. MolecularBrain Research 1990;7:273-6.

18 Meyer A, Leigh D, Bagg CE. A rare presenile dementiaassociated with cortical blindness (Heidenhain's syn-drome). J Neurol Neurosurg Psychiatry 1954;17: 129-33.

19 Owen F, Poulter M, Collinge J, Leach M, Lofthouse R,Crow TX, Harding AE. A dementing illness associatedwith a novel insertion in the prion protein gene.Molecular Brain Research 1992;13:155-7.

20 The Huntington's Disease Collaborative Research Group.A novel gene containing a trinucleotide repeat that isexpanded and unstable on Huntington's disease chro-mosomes. Cell 1993;72:971-83.

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doi: 10.1136/jnnp.58.1.65 1995 58: 65-69J Neurol Neurosurg Psychiatry

 D Nicholl, O Windl, R de Silva, et al. base pair insertion of the prion protein gene.British family associated with a novel 144 Inherited Creutzfeldt-Jakob disease in a

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