九州大学学術情報リポジトリ
Kyushu University Institutional Repository
クルー斑を有するクロイツフェルト・ヤコブ病患者 はゲルストマン・ストロイスラー症候群に特異的な 変異型プリオン蛋白を持つ
堂浦, 克美
九州大学医学系研究科病理系専攻
https://doi.org/10.11501/3054136
出版情報:Kyushu University, 1990, 医学博士, 課程博士 バージョン:
権利関係:
C r e u t z f e l d t ‑ J a k o b D i s e a s e P a t i e n t s w i t h C o n g o p h i l i c Kuru P l a q u e s Have
出 eM i s s e n s e V
紅i a n tP r i o n P r o t e i n C0 1 l 1 1 l 1 on t o G e r s t t n a n n ‑ S t r a u s s l e r S y n d r o l l l e
Ka紅ts山u1江m叩liDohι‑ur,a色M D
釦 dYoωs凶h立iI卯yukiSa辻ka辻ki,PhD↑
Congophilic kuru plaques, one of the pathological hallmarks in kuru and Gerstmann‑Straussler syndrome, are some‑ times present in patients with Creutzfeldt‑Jakob disease (CJD). The congophilic kuru plaques are composed part1y of a host‑encoded prion protein, and a missense variant prion protein with the codon 102 proline‑tcトleucinechange (Leu 102) is commonly present in patients with Gerstmann‑Straussler syndrome. To investigate the relationship between this syndrome and CJO with congophilic kuru plaques
,
we made a sequence analysis of the prion protein gene from patients with CJD, with or without congophilic kuru plaques. We found no alterations other than the Leul02 change, common to Gerstmann‑Straussler syndrome, in one of the prion protein alleles of the patient with congophilic kuru plaques. 1n the prion protein genotype analysis of other patients with CJO, the Leul02 allele was revealed to be carried heterozygously by 6 of 7 patients who had CJD with congophilic kuru plaques, yet no patient with CJO without congophilic kuru plaques had this allele. 1nterestingly,
the Leul02 allele was also carried by some unaffected relatives of 3 patients with CJO with congophilic kuru plaques but with no app紅 entfamilial occurrence of a similar neurological disorder. Our findings show that CJO with congophilic kuru plaques should be categorized as belonging to Gerst‑ mann‑Straussler syndrome, not CJO, and also suggest that the variant prion protein with Leu102 is c10sely related to the amyloidgenesis seen in subjects with congophilic kuru plaques.Doh‑ura K, Tateishi J, Kitamoto T, Sasaki H, Sakaki Y. Creut2feldt‑Jakob disease patients with congophilic kuru plaques have the missense variant prion protein common to Gerstmann‑Straussler syndrome. Ann Neurol 1990;27: 121‑126
Creutzfeldt‑]akob disease (C]D), Gerstmann‑Strauss‑
ler syndrome (GSS), and kuru in humans and scrapie in animals are progressive neurodegenerative disorders characterized by arnyloid deposits (kuru plaques) and spongiform changes in the brain and by transmissibility to experimental animals [1ーラ].The infectious agent has not been dearly identified, but an isoform of a protein called prion protein (PrP), a species‑specific host‑encoded protein [6‑10}, has been proposed to be a component of the agent, or the agent itself [11, 12]. PrP has been shown to a舘regateto form arnyloid ι brils [13]. Hsiao and associates [14} reported that a missense variant PrP with the codon 102 proline (Pro 102)̲to・leucine(Leu 102) change is tightly linked to ataxic GSS in two unrelated Caucasian families. They
From rhe ‑Departmenr of Neuroparhology, Neurological Insrirure, Faculry of Medicine, and rhe tResearch Laborarory for Genetic In‑ formarion, Kyushu Universiry, Fukuoka, Jap叩.
Received Aug 21, 1989, and in revised form OCt 4. Accepred for publicarion Ocr 12, 1989.
suggested that this missense variant does not merely represent a genetic marker
,
but rather might provoke development of the il1ness. This has been supported by our findings that the Leu 102 change is the most cornmon mutation found in patients with GSS, irre‑ spective of their ethnic backgrounds,
but it is not the sole mutation related to GSS [1う].In arnyloid deposits or accumulations immunohistochemically evidenced to be partly composed of PrP, there are deposits detected by congo‑red stain and some that are unstainable [16]. The former, congophilic amyloid deposits or kuru plaques (KPs), which are frequently seen in brains affected by kuru or GSS and are regarded as being pathological hallmarks of these diseases, are present in some brains affected by C]D. To clarify the relation‑Address correspondence ro Dr Doh‑ura, Departmenr of Neuro‑ parhology, Neurological Insrirute, Faculry of Medicine, Kyushu Universiry, 3‑1‑1 Maidashi, Higashi‑ku, F叫woka812, Japan
Reprin,ed from Annals of Neurolc事Y.Vol 27, No 2, Febcua巧1990.Published by ,j{de, Brown and Company. Copyrighl ¥C 1990 by thc Ameri.can Ncurolog日IAssocia,ion. AII r唱h目 同 日 刊ed.No part of this reprin, may Ix陀produ日din any form or by any electronic or mechanical means, including info口nationsrorage and re,rieval sySlems, wi,hoUl the publisher's wntten permlSSlon
/ 1¥¥¥
Fig 2. Sequence anafysis %ne
0 /
the prion protein alfefes /rom a patient with Creut功ldt‑jakobdisease with congoph;lic kllT'llpl軍qll釘 (Case2). The second Letter
0 /
codon 102打notC but T, res ufting in a changeJ
トompro仰eto !eucine.O
〈
22
官 官
EH OZ
υ
ト
出e chain reacticin (PCR) using a pair of primers:う
GATGCTGGTTCTCTTTGTGG‑3' is complementary to 出eantisense strand around the initiation codon andう
TCCCACTATCAGGAAGATGA‑3' is complementary to the sense strand at the termInus of the coding region. PCR was performed as described by Saiki釦dassociates [18], with slight modmcation. One microgram of high‑molecular‑mass DNA was mixed withう
o
pmol of each primer and 200μM of each deoxyribonucleoside 5' ‑triphosphate in 100 μ1 of reaction buffer containing 10 m M Tris hydrochloride (pH 8.3),うo
m M potassium chloride, 1.う m Mmagnesium chloride, and 0.01% gelatin. The mixture was heated to 940C for 7 minures for strand separation before annealing for 2 minutes at room temperature. Five units of Taq poly‑ merase (Perkin‑Elmer Cetus, Norwalk, CT) was then added. The cycle of denaturation (940仁,1 minure), annealing (550C,1 minure), and extension (730C, 1 minute) was carried out on a DNA thermal cycler (Perkin‑Elmer Cetus) for 30 cycles Transmission
to Rodentsb
Cloning and Sequencing
0 /
Amplified DNAPCR‑amplified products of Case 2 (C]D‑KP) and Case 9 (C]D) (see Table 1) were doubly digested with Bal 1 and Sau3AI and then cloned intd pUC18. Nucleotide sequence of the cloned fragments was determined by the dideoxy chain termination method [19]
+ +
+
almmunostaining of amyloid deposirs was performed wirh anrj‑Gersrmann‑Straussler syndrome amyloid plaque core antiserum, as described (16]
bThe transmission experiment was performed as previously described {3. 5}
CRerrospecrive examinarion revealed congophilic kuru plaques
S‑C spinocerebeUar signs; NE nor examined; ? quesrionable dara; PSDs periodic synchronous discharges; C]D‑KP = Creurzfeldt Jakob disease with congophilic kuru plaques; C]D = Creutzfeldt‑Jakob disease wirhour congophilic kuru plaques
+ Ongoing Ongoing Ongoing NE Ongoing
+
Ongoing
+
Ongoing Ongoing Ongoing Ongoing
+ Ongoing Immuno‑
stained Depositsa
+ + + + +
+
++
Congo‑
philic Plaques
+ +
+C + + + + PSDs+ ﹀
・2・
+
+ + + MyocJonus+
++ +
+
+ +
+
Onset with S‑C
+
+ + + +
++ Diagnosis
+ +
+
+ +
++
++
+ + + C]D‑KPC)D‑KP C]D‑KP CJD‑KP CJD‑KP C)D‑KP CJD‑KP C]D C]D C]D C]D C]D 仁JD CJD C]D Clinical
Duration (Mo)
守LQud
U1 nu パU
/O只u
﹃ ノ ハ
U勺L
司 令J
'I
勺Ld叫A
nu
‑‑d
﹃ ペ ノ
/O n y n y n U T i
‑
‑ 1 i
守4
吋ノ
﹄ 守
L弓J
. ︐ .
Age at Onset (yr) T able 1. Patient Data
Q U Q U 2
︐4 Z JRノ7
1 7
ハUQU
︑ コ
ny
句 コ
' 3
RJR
ノ〆Or
4 4 J /O Z J /0 7 '7F/
O/OfO/O︽J
Sex M M M F M F F M M F F F M F F Case No.
1 2 3 4 3 6 7 8 9 0 1 2 3 4
う
‑ EA
唱Ea‑‑ι2・a
'E
a'
EA
respectively). The coding region of PrP gene was ampli五edby P仁R,cloned into plasmid pUC18, and then sequenced. As a result, several clones from the pacient with C]D‑KP were found to contain a C to T transition at the second letter of codon 102, resulting in a proline‑to‑leucine change (Leu 102) (Fig 2). This substirurion is rhe same change rhar has been com‑
monly found in parienrs with GSS regardless of ethnic origins [1う].1n contrast, 20 of rhe clones from the patient with C]D showed no base changes other th釦
arcifacts occurring during PCR or cloning experiments, as confirmed by resrriction fragmenr length polymor‑
phism with appropriate resrriction enzymes or by dot differential hybridizarion wirh appropriare pairs of allele‑specific oligonucleotide probes.
PrP Polymorphism Genotype Analysis
W e determined genotypes of 7 patients with C]D‑KP and 8 patienrs wirh C]D for the polymorphism in co‑ don 102. As shown in Figure 3 and summarized in Table 2, 6 of 7 parients wirh仁JD・KPcarried a Leu102 allele, heterozygously. In contrast, none of the 8 pa‑ tients without congophilic KPs carried this allele. The only patient (Case 7) with C]D‑KP who was negative for the Leti 102 allele w出 a9・ye紅 survivor,presencing with an acutely progressive gair disturbance, visual im‑ pairment, disorientacion, and dementia preceding a 7・
year apallic stare and pathologically showing severe cerebral atrophy and a few congophilic KPs. Since clinical signs and symptoms progressed acutely at rhe Dot Hybridization Analysis
Dot blot differential hybridization of PCR‑ampli五edDNA was performed with a pair of allele‑specmc oligonucleo‑ tide probes:,う‑ACAAGCCGAGTAAGC仁A‑3'for ProlO2 (normal);デーTGGCTIACTC
d .
GCTIGT‑3'for LeulO2 (vari‑ ant), which were designed not to form stable G: T mis‑ matches in hybrids [20]. Ten microliters of PCR‑amplified DNA was alkaline‑denatured with 0.2 N sodium hydroxide at room temperature for 10 minutes and then neutralized to 1 M ammonium acetate before being dot‑blotted onto nylon membranes. The membrane was prehybridized for 30 min‑ utes at 420C in 10 x SSC (1.うMsodium chloride, 1うOmM sodium citrate; pH 7.0), 20 x Denhardt's solution (0.4% bovine serum albumin, 0.4σ% polyvinylpyrolidone, 0.4% Ficoll), 1σ% sodium dodecyl sulfate, and 2 mglml herring sperm D NA. Each allele‑specific oligonucleotide probe, the う,‑end of which was labeled with [γ̲32p]ATP, was then added to出esame bu仔erand hybridization was performed at 420C for 1 hour. The membrane was rinsed twice with 6.67 x SSC (1.0 M sodium chJoride, 100 m M sodium citrate; pH 7.0) and 0.1 % sodium dodecyl sulfate and washed once wi出
the same solution atう
rc
for the normal probe and 500C for the variant, which is about 20C below the melting tempera‑ ture of each of the probes. The membrane was then exposed for several hours to x‑ray五1mat ‑700C with intensifying screens.female male deceased
102 affected, with Leu
102 not affected, with Leu not affected, without LeJ02
not examined 111
O
口
五ざ,回
・ ,
目‑
e
目 0, 口
36
11
35
Fぽ1.Pedigrees 0/3 /amilies affected by Creut
ψ
ldt‑jakob dis‑ ease with congophilic kuru plaque s.Numbers indicate age (years)0 /
the /iving individuals. 1, 11, 111 indicate Pedigrees0 /
/amilies0 /
Cases 1, 2, and 5 with Creutz/eLdt‑jakob disease with con‑ gophilic kuru plaques, respectively.Res叫ts
PrP Gene Analysis
To investigate the riature of PrP 丘ompatients who had C]D wirh or withour congophilic KPs, we analyzed the PrP genes of a pacient with C]D‑KP and of a pacient with C]D who was negacive for congophilic KPs and immunostained deposits (Cases 2 and 9 in Table 1, The subjects analyzed in the family study of CJD‑KP are
listed in Fi伊re1.
Polymerase Chain Reaction
High‑molecular‑mass DNA was prepared from frozen brain tips (from all CJD and C]D‑KP subjects except for Case 5), formalin‑fixed brain tissues (Caseう), or pe‑ ripheral blood cells (all subjects tested in the family study). The PrP coding region was amplified by polymer‑
39
ship between GSS and C]D with congophilic KPs
,
we iiwestigated the PrP gene from ]apanese parients who had C]D with or wIthout congophilic KPs.Materials and Methods Subjects
Specimens from 7 patients with sporadic CJD with con‑ gophilic KPs (CJD‑KP) and 8 patients with sporadic CJD without congophilic KPs were analyzed (Table l). The pa‑ tients with CJD‑KP had no known affected family members, but the clinicopathological features resembled those of GSS rather than C)D. Generally, the patients with C)D‑KP pre‑ semed with spinocerebellar signs and had an insidious clinical course with amnesia and disorientation for over 1 year be‑ fore lapsing into a bedridden state. Pathologically, they showed numerous congophilic KPs. On the other hantl, pa‑ tients with C]D without congophilic KPs started with acute or subacute psychiatric signs, behavior abnormality, character change, visual impairment, memo円 and仁alculationdisrur‑ bance, and gait disturbance, and had an acute progression for several months, proceeding to an apallic state with remark‑ able myoclonus and periodic synchronous discharges on electroencephalogram. There were remarkable brain atrophy and spongiform changes but no congophilic KPs. The diag‑ noses of C]D and C)D‑KP were con五rmedhistOpathologi‑ cally, immunohistochemically, and by kuru plaque core purmcation and PrP detection using Western blots and, in some cases, by experimemal transmission to small rodems [3,う,16, 17]. All of these patients were nonconsanguineous.
123 Doh‑ura et al: Creutzfeldt‑)akob Disease with Congophilic Kuru Plaques February 1990
No 2 Vol27 Annals of Neurology
122
102 102
Pro Leu
2 3 4 5 6 2 3 4 5 6 CJD‑KP
and control
7 P L 7 P L
8 9 10 II 12 13 8 9 10 II 12 13 CJD
and control
14 15 P L 14 15 P L Fig 3. Examples /or dot blot analysis /or the prion protein poly‑ moゅhismat codon 102 with a pair
0 /
al1ele‑stecificρrobes. Data on all patients with Creutz/eldt‑]akob disease (C}D) or。
Dwith congophilic kuru plaq間 (C}D‑KP)who were testedare shown. Numbeηindicate the case numbers
0 /
su印刷m Table 1. Case 5 was derived jトoma formalin‑exposed DNA sample. P = c/oned prion protein gene with Pro102, L = c/oned prion protein gene with LeuJ02.beginning of the clinical course and the congophilic KPs were fewer than in other patients with C]D‑KP, this case seems to differ from the other cases of C]D‑
KP.
Family Anaか'5t5
Eight individuals, with no signs or symptoms of GSS or C]D, from 3 families of patients with C]D‑KP who had a Leu 102 allele were examined concerning substiru‑ tion in codon 102, and the results are shown in Figure l. The Leu 102 allele was carried by a child (pedigree 1), a brother (pedigree II), and three children (pedigree III) of patients with C]D‑KP.
Fig 4. The predicted secondaワstructure
0 /
prion protein with pro102 (1) and prion protein with LeuI02ロ1).102 Pro
1.
11.
sequence
ヤ
│N .102 Leu
124 Annals of Neurology Vol 27 No 2 February 1990
Table 2. Resu/ts
0 /
Prion P仰teinPolymorphおmGenotype AnaかsisSummarized in Relation to Diagnosis Pro̲Leu102
Genotype No Pro/Pro ProlLeu LeulLeu C]D‑KP 7 l 6 O
CJD 8 8 O O
CJD‑KP=CrELrIetzuftezlfdetlJdtaJkaokbo diSEasE with congophilic Kum plaques; C]D = Creurzfeldr‑)akob disease wirhour congophilic kuru plaques
Discussion
One of the main findings in this work is that the GSS‑
common mutatIon of PrP, the codon 102 proline‑to‑ leucine change, also exists in apparently sporadic cases of C]D‑KP. It is di伍cultto reconcile our findings with those of Hsiao and colleagues [14}, who provided strong evidence that the linkage of the codon 102 leucine substirution to GSS indicates that GSS is an inherited illness. However, we found that 3 patients with C]D‑KP had one or more unaffとcted family members with the PrP mutation, despite lack of similar neurological disease in the farnilies. The lack of other affected family members can be variously explained. In pedigree II, one of the parents of the patient (Case 2) must have had a Leu 102 allele, since both the patient and his brother had this allele. Hence, clinical evalua‑ tion of the disease may not have been thorough in the mother, who died at the age ofう4ye紅sof undeter‑ mined cause, and in the father, who died of gastric cancer at age 8う.They may have died before develop‑ ment of the illness, or they were not affected because of absence of other factors necessary for development of the illness. These explanations for pedigree II can also be applied to Case 1 (pedigree 1) and Caseう
̲ alpha‑helix
̲ beta‑sheet
‑COOH
‑ ・ ・ ・ ・ L
-..'l.~J・11 ・・・ -COOH(pedigree III). lf their p訂entsand siblings are free of the Leu102 allele, these patients might represent indi‑ viduals with de novo germline mutations. If this is the case, one would not expect to discover a history of neurodegenerative disease in the previous generation. However, one might well expect that the 4 offspring of C出es1釦 dう, aged 36, 46, 41, and 39 ye訂 丸 紅estill young enough to be at risk for the disease, and that all who possess the codon 102 leucine substitution will eventually develop neurological disorders. Older indi‑ viduals, such as Case 2's unaffected brother, who also h出 theleucine variam, are at risk to develop the ill‑ ness. Thus, these Leu 102 ‑positive patients with仁JD‑
KP, who showed clinicopathological features sugges‑ tive of GSS, may well be regarded as cases of GSS with no other app紅entfamilial occurrences.
W e do not know the exact role(s) of the variant PrP in the disease process of GSS or C]D‑KP, but it might (1) be a constituent of a causative agent or the agent itself, (2) increase susceptibility of the host to the agent, or (3) change the nature of PrP to aggregate to form amyloid. The first and the second possibilities can be clarified by follow‑up of Leu 102̲positive unaffected individuals. The third is now supported by the品ndings that the leucine variant was found in all congophilic KP‑positive patients with GSS [1う}or C]D‑KP except for one, and is a major component of congophilic KPs (data not shown). Interestingly, the secondary struc‑ ture of PrP with Leu 102 predicted by the method of Chou and Fasman [21} signi五cantlydiffers from that of PrP with the codon 102 proline (Pro102) (Fig 4). The change of proline to leucine in codon 102 predicts abolition of a beta‑turn structure at that position. This change might facilitate a.銘regationof the variant PrP to form amyloid, similarly to variant transthyretins in familial amyloidotic polyneuropathy [22, 23}. How‑
ever, there is a fundamental distinction between far叫 ‑ ial amyloidotic polyneuropathy and prion diseases in that the former has systemic deposition of amyloid fibrils and transmission is not horizontal. As for amy‑
loid deposits, immunostaining has facilitated detection of smaller cerebral amyloid accumulations that cannot be detected by congo‑red stain [16}. We出ked whether the amyloidgenesis seen in congophilic KPs is the same出 thatin the immunostained deposits unde‑
tectable by congo‑red stain.事rewish t
sumably an inherited disorder. It is also necessary to follow up Leu 102‑positive, unaffected family members of patients with C]D‑KP to elucidate and resolve the disease process, in relation ro the variant PrP. The variant PrP is involved in the amyloidgenesis seen in congophilic KPs.
Since Hsiao and associates [14} poimed out that出e use of D N A extracted from forma1in‑fixed tissues can alter base composition, the data on Case,うas derived from forma1in‑exposed DNA samples, are not abso‑ lute. Nevertheless, data on the children (see Fig 1, pedigree III) are interesting釦 dimPOrt釦 tand suPPOrt our proposal.
This work was supporred by a gram from rhe Science and Technol‑ ogy Agency, Japan.
We rhank Drs H. Hashiguchi, S. Kuz山ara,Y. Kurod ,aA. Mori‑ sada, S. Moromura, T. Muro, H. Nishirani, Y. Saro, Y. Shii, M Suersugu, H. Umezaki, S̲ Yagishira, N. Yamamur ,aT. Yosrumura,
and T. Waranabe for providing blood and rissue samples; Drs T. Ya‑ mada叩 dH. Furuya for rheir helpfuJ suggesrions and discussions; Ms H. Ohgusu for preparing oligonucleorides; and M. Ohara for commems
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