Dementia and Neurocognitive Disorders 2013; 12: ORIGINAL ARTICLE 알츠하이머병뇌척수액생물표지자다기관연구를위한예비연구 박선아 * 김종헌

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1 Dementia and Neurocognitive Disorders 2013; 12: 1-8 ORIGINAL ARTICLE 알츠하이머병뇌척수액생물표지자다기관연구를위한예비연구 박선아 * 김종헌 김형준 * 김태은 * 김윤정 * 이동현 * 박정호 * 채원석 임수재 서상원 나덕렬 최성혜 순천향대학교부천병원신경과 *, 국민건강보험일산병원신경과, 순천향대학교부천병원마취과, 순천향대학교부천병원정형외과, 성균관대학교의과대학삼성서울병원신경과, 인하대학교의과대학신경과 Received: August 13, 2012 Revision received: February 12, 2013 Accepted: February 12, 2013 Address for correspondence Seong Hye Choi, M.D. Department of Neurology, Inha University School of Medicine, 366 Seohae-daero, Jung-gu, Incheon , Korea Tel: Fax: seonghye@inha.ac.kr * 본연구는보건복지부보건의료연구개발사업의지원에의하여이루어짐 (A 와 A102065). Preliminary Study for a Multicenter Study of Alzheimer s Disease Cerebrospinal Fluid Biomarkers Sun Ah Park, M.D.*, Jung Hun Kim, M.D., Hyeong Jun Kim, M.D.*, Tae Eun Kim, M.D.*, Yoon-Jeong Kim, M.S.*, Dong Hyun Lee, M.D.*, Jeong Ho Park, M.D.*, Won Seok Chae, M.D., Soo Jae Yim, M.D., Sang Won Seo, M.D., Duk L. Na, M.D., Seong Hye Choi, M.D. Departments of Neurology*, Anesthesiology, and Orthopedic Surgery, Soonchunhyang University Bucheon Hospital, Bucheon; Department of Neurology, Ilsan Hospital, National Health Insurance Corporation, Goyang; Department of Neurology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul; Department of Neurology, Inha University School of Medicine, Incheon, Korea Background: The usefulness of cerebrospinal fluid (CSF) concentrations of amyloid beta protein 1-42 (Aβ42), phosphorylated tau (ptau) and total tau (ttau) have been increasing in Alzheimer s disease (AD). However, the direct adoption of previously reported standard values is not appropriate due to interlaboratory variability. We started this study to set up an accessible system to measure CSF biomarkers in our country with high reproducibility and validity. Methods: Including CSFs from four different institutes the levels of Aβ42, ptau181 and ttau were measured in one lab. The intertest variability and difference in the levels of biomarkers depending on diseases were assessed. Through analysis of receiver operating characteristic cut points and binary logistic regression the cut-off values of Aβ42, ptau and ttau level were obtained, and their validity was evaluated. Results: The intertest consistency was high in measuring CSF biomarkers. The value of Aβ42 was markedly decreased in AD (n = 17) and other dementia (n = 9) compared to normal control (n = 12). The levels of ptau181 and ttau were high in AD, but not in other dementia and normal control. The threshold values of Aβ42, ptau181 and ttau were pg/ml, 54.3 pg/ ml, and pg/ml in differentiating AD from normal control showing high sensitivity and specificity. Especially, the ratios of ptau181/aβ42 ( > 0.16) and ttau/aβ42 ( > 0.76) showed the prime validity. Conclusions: Our data of CSF Aβ42, ptau181, and ttau levels were highly reproducible. PTau181/Aβ42 and ttau/aβ42 ratios were the greatly helpful in differentiating AD from normal control. Key Words: Alzheimer s disease, Amyloid beta protein, Biomarker, Enzyme-linked immunosorbent assay, Cerebrospinal fluid, Tau 서론알츠하이머병의핵심병리인베타아밀로이드단백과과인산화 tau단백을표적으로하는신약들이예측과달리대규모임상연구에서연이어실패하면서, 이들약물의투약시점을알츠하이머병병리발생이후지만, 임상적으로는치매가발생하지않은 치매전단계 로앞당겨야한다는주장이높아지고있다 [1, 2]. 이를위해서는정확한알츠하이머병조기진단이필요하기에, 그어느때보다알츠하이머병병리진행을반영하는생물표지자 (biomarker) 의개발과 이용에대한관심이높다 [3]. 혈액, 뇌척수액, 뇌자기공명영상 (MRI), 그리고양전자방출단층촬영 (PET) 등을이용한알츠하이머병생물표지자국제공동연구를활발히진행하고있는 Alzheimer s Disease Neuroimaging Initiative (ADNI) 는이같은고양된국제적관심의대표적인예이다 [4]. 국내에서도국제적수준의뇌영상생물표지자연구가진행되고있고, 혈장생물표지자개발위한연구들도지속적으로보고되고있다 [5-8]. 그러나이에비해뇌에가까이있어뇌의대사물의변화를잘반영하는뇌척수액을이용한생물표지자연구는상대적으로적다 [9] Korean Dementia Association This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1

2 2 박선아 김종헌 김형준외 9 인 알츠아이머병과연관되어뇌척수액에서변화를보이는여러단백중, 베타아밀로이드 1-42 (Aβ42), 인산화tau단백 (ptau) 그리고총 tau단백 (ttau) 이가장유용한생물표지자로알려져있다 [10]. Aβ42는알츠하이머병뇌척수액에서질환초기부터약 50% 감소되고 [11], 반면 ttau 와이의인산화형태인 ptau 는약 2-3배증가된다 [12]. Aβ42의감소는알츠하이머병의초기진단에도움이되나, 다른퇴행성뇌질환에서도감소되는경우가있어, ptau 의증가의동반이다른퇴행성뇌질환으로부터알츠하이머병을감별하는데도움이된다 [13]. 따라서, Aβ42, ptau, ttau 각각보다, 이들의조합인 ptau/aβ42 혹은 ttau/a β42 비가알츠하이머병을진단하거나, 경도인지장애환자가알츠하이머병으로진행하는것을예측하는데유용하여, 민감도와특이도가 85-91% 에이른다 [14, 15]. 이처럼유용성이증명되어, 국제적으로널리이용되는뇌척수액 Aβ42, ptau, ttau 농도의실제이용이국내에서는매우드물게이루어져왔다 [16]. 그나마보고된연구에서도이용된측정방법이국제적으로널리사용하는방법이아니었고, 같은검체를이용한반복검사의일치도검증이없어, 향후국내연구데이터로지속적으로참조하는데한계가있다. 이에본연구자들은알츠하이머병진단을위한뇌척수액 Aβ42, ptau 및 ttau 의국내기준치마련및변동성이적고유용한측정값을제공하는시스템구축이향후이들생물표지자를이용한활발한국내연구를위해선행되어야한다고생각하여본연구를시작하였다. 알츠하이머병영상소견을추가로보이는경우로하여, 예비연구를위해엄격한알츠하이머병에의한치매진단기준을정하였다. 즉, MRI에서해마등내측두엽의위축이보이는경우또는 / 그리고, FDG-PET 이나 Amyloid PET에서양측측두-두정엽의포도당대사저하혹은 Aβ 리간드를이용한 PET에서알츠하이머병에합당한이상을보이는경우로하였다. 정상인은치매임상증상이없고, 인지기능검사에서정상 (MMSE인경우는 27점이상 ), 그리고, 뇌 MRI 혹은컴퓨터단층촬영 (CT) 에서뇌위축을포함한기타특기할이상이없는경우로하였다. 기타질환의경우는임상적으로인지기능저하를보였으나, 진단기준, 뇌영상, 그리고추적임상경과를통해알츠하이머병이아닌타질환으로진단된경우로하였다. 파킨슨병치매는 Emre 등 [19] 의진단기준에따라, 레비소체치매는 McKeith 등 [20] 의진단기준에따라, 전두측두엽치매는 1998년합의된진단기준에따라 [21], 진행핵상마비는 National Institute of Neurological disorders and Stroke (NINDS)-Society for Progressive Supranuclear Palsy (SPSP) 진단기준에따라 [22], 혈관성치매는 NINDS-Association Internationale pour la Recherche en l Enseignement en Neurosciences (AI- REN) 진단기준 [23] 에따라정하였다. 그외정신과적질환은진료초반에치매가의심되었지만, 여러진단기준에맞지않고, 정신과의사에의한약물치료로증상이호전된경우로정하였다. 뇌척수액채취및분석 대상과방법대상노인성치매임상연구센터의다기관시료연구를위해해당기관의임상시험심사 (Institutional Review Board, IRB) 의승인을얻은국내 4기관에서이루어졌다. 여러임상목적으로시행된뇌척수액채취가본연구에적합하게이루어지고, 뇌척수액공여자가 Aβ42, ptau181, ttau 측정에동의한경우연구에포함시켰다. 추가로뇌척수액의채취와인지장애유무를판단하기위한간이정신상태검사 (Korean version of the minimental state examination, K-MMSE) 및임상치매평가척도 (Clinical dementia rating, CDR) 검사사이의간격이 2개월이내이고, 병력채취, 신경학적진찰, 그리고뇌영상이있고, 뇌척수액채취후임상적추적이 6개월이상이루어진경우로대상을제한하였다. 알츠하이머병에의한치매진단은 National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer s Disease and Related Disorders Association criteria for AD (NINCDS- ADRDA) 의진단기준에근거하여 probable/definite 알츠하이머병 [17] 이면서, revised academic criteria [18] 에따라 MRI 혹은 PET에서 뇌척수액채취는 8시간이상금식후오전 8시를가장권장하였으나, 오전 10시까지포함하였다. 23G needle로시행하였고, 첫 2 ml 는임상적필요에의한검사 ( 화학성상및세포수분석등 ) 를시행하는데사용하였고, 그후배출되는뇌척수액 5 ml를 polypropylene 재질의튜브에받아 2시간이내에 4 C, 4,000 g에서 10분간원심분리하였다. 그후 polypropylene 재질의 1.5 ml frozen tube 혹은 eppendorf tube 에 70% 차도록나누어담은뒤밀봉후 -70 ~ -80 C 초저온냉동고에검체분석하기전까지보관하였다. 분석기관에서가까운외부기관시료의경우채취당일날실온에서 2시간이내에분석기관으로운반후원심분리부터같은과정으로검체를분하여보관하였다. 거리가있어 2시간이내운반이어려운외부기관에서는같은프로토콜에따라검체분리후초저온냉동보관후드라이아이스가채워진보온상자를이용하여분석기관으로운반되었다. 이같은검체체취, 분리및보관기준은 Dominantly Inherited Alzheimer Network (DIAN) ( 와 ADNI [14] 의뇌척수액수집프로토콜을따른것이다. 초저온냉동고에저장된분주된뇌척수액은분석당일 4 C에서해동후항원의서로다른부위를인식하는두개의항체를이용한 INNOTEST ELISA kit (Innogenetics, Ghent, Belgium) 에명시된대로

3 알츠하이머병뇌척수액바이오마커 3 진행하였다. 표준농도샘플및검체샘플모두한판내에이중 (duplicate) 으로측정하였고, 두값의차이는 cost variance percent (CV%, = 표준편차 / 평균 %) 로계산하였다. 또한, Aβ42는 1개월의간격으로 35개샘플에대해, ptau181 과 ttau 는 7개샘플에대해 9개월간격으로두차례 ELISA 측정하여, 같은샘플의 ELISA 검사에따른측정값차이를분석하였다. 모든 ELISA 검사는동일한연구자에의해동일한장소에서시행되었다. 통계통계분석을위해서는 SPSS 13.0 윈도우버전을이용하였다. 세군사이비교를위해 ANOVA test와 Chi-square 를시행하였다. 사후검증으로 Tukey test를이용하였다. 기간을두고같은샘플을이용하여시행한 ELISA 측정치사이의차이를 CV% 로분석하였고, Pearson s correlation test 이용한상관도분석도하였다. Aβ42, ptau181, ttau, ptau/aβ42, 그리고 ttau/aβ42 값의알츠하이머병진단효용에대해서는정상군을대조군으로하여이항로지스틱회귀분석 (binary logistic regression) 을통해검증하였고, receiver operating characteristic (ROC) curve 분석을통해 cut-off value, 민감도및특이도를산출하였다. 결과대상군분석연구참여에동의하면서연구기준에합당하게뇌척수액채취및보관을하였고, 임상정보를얻을수있었던대상군은알츠하이머병에의한치매 17명, 정상 12명, 기타치매 9명이었다 (Table 1). 알츠하이머병에의한치매군이정상및기타치매군에비해의미있게연령이적었고 (p < 0.05), MMSE와 CDR 점수는정상군은정상범위였으나, 알츠하이머병에의한치매와기타치매군에서는저하되어있었다 (p < 0.05). 교육연령은알츠하이머병에의한치매환자가나머지두군에비해높았다 (p < 0.05). 기타치매군은파킨슨병치매 2 명, 레비소체치매 1 명, 전두측두엽치매 1 명, 진행핵상마비 1 명, 혈관 성치매 2 명, 그리고정신질환 2 명이었다. 뇌척수액반복측정사이의일치도 한 ELISA 판내에서이중으로측정된동일샘플의측정값차이 (CV%) 는 Aβ42 의경우 5.7± 4.4%, ptau181 은 2.3± 2.8%, 그리고 ttau 는 5.8 ± 5.2% 였고, 모두 INNOTEST ELISA kit 에서허용하는 CV% 한 도 20% 이내였다 (Table 2). 1 개월의간격으로두차례시행한 Aβ42 ELISA 검사에서중복측 정된 35 개샘플의측정값차이는 48.3 ± 44.1 pg/ml 로, 14.5 ± 14.6% 의 CV% 를보이면서, 높은수준의일치도를나타냈다 (r 2 = 0.834, p = 0.000). 9 개월간격으로두차례시행한 ptau181 과 ttau ELISA 에서중 복측정된 7 개샘플의측정값도각각 6.1± 5.0 pg/ml 과 36.2 ± 35.8 pg/ ml 의차이를보이면서, CV% 가 5.9 ± 4.2% 와 7.0 ± 7.1% 정도로좋았고, 높은수준의일치도를나타냈다 (r 2 = 0.977, p= for ptau181, r 2 = 0.974, p = for ttau). 질환군별뇌척수액 Aβ42, ptau181, ttau 첫 ELISA 검사에는본연구에해당되지않는환자의샘플이같이 있었기에, 질환군별뇌척수액생물표지자분석은두번째시행한 ELISA 에서얻은수치로하였다 (Table 3). 알츠하이머병에의한치매 군이나기타치매군의 Aβ42 농도는정상군에비해의미있게낮았 Table 1. Demographic characteristics of the subjects (mean± SD) Characteristics AD NC Other D Number Gender F:6, M:11 F:8, M:4 F:3, M:6 Age, year-old 59 ± 8* 63 ± 9 70 ± 9 MMSE 15 ± 7 28 ± 1* 18 ± 8 CDR (CDR SOB) 1.3±1.2 (7±7) 0±0 (0±0)* 1.3±1.6 (7±9) Education, year 12 ± 4* 6 ± 4 8 ± 5 *p< AD, Alzheimer s disease; CDR, clinical dementia rating; D, dementia; F, female; M, male; MMSE, mini-mental state examination; NC, normal control; SD, standard deviation; SOB, sum of box. Table 2. The coefficient of variance percent (CV%) within a test and intertest correlations Aβ42 ptau181 ttau Intratest CV% (range) 5.7±4.4 ( ) 2.3±2.8 (0-17) 5.8±5.2 ( ) Intertest variance (pg/ml) 48.3±44.1 ( ) 6.1±5.0 ( ) 36.2±35.8 ( ) Intertest CV% (range) 14.5±14.6 ( ) 5.9±4.2 ( ) 7.0±7.1 ( ) Intertest correlations r 2 = 0.834, p= r 2 = 0.977, p= r 2 = 0.974, p= Mean±standard deviation. CV%, coefficient of variance percent.

4 4 박선아 김종헌 김형준외 9 인 Table 3. The concentrations of Aβ42, ptau181, ttau in cerebrospinal fluid Aβ42 (pg/ml) ptau181 (pg/ml) ttau (pg/ml) ptau181/aβ42 ttau/aβ42 NC M ± SD ± 101.8* 46.5 ± ± ± ± 0.13 [95% CI] [ ] [ ] [ ] [ ] [ ] AD M ± SD ± ± 31.0* ± 254.1* 0.47 ± 0.23* 3.52 ± 2.44* [95% CI] [ ] [ ] [ ] [ ] [ ] Other D M±SD 184.5± ± ± ± ±3.18 [95% CI] [ ] [ ] [ ] [ ] [ ] *p< AD, Alzheimer s disease; CI, confidence interval; D, dementia; M, mean; NC, normal control; SD, standard deviation. Table 4. Receiver operating characteristic (ROC) curve parameters Aβ42 (pg/ml) ptau181 (pg/ml) ttau (pg/ml) ptau181/aβ42 ttau/aβ42 Test accuracy 84.00% 86.20% 86.20% 96.60% 96.60% Positive predictive value 88.20% 93.30% 93.30% % % Negative predictive value 83.30% 78.60% 78.60% 92.30% 92.30% ROC AUC Cut-off value (pg/ml or ratio) > > >0.163 >0.758 Sensitivity 88.20% 82.40% 82.40% 94.10% 94.10% Specificity 83.30% 91.70% 100% 100% 100% ROC, receiver operating characteristic; AUC, area under the curve. 다 (p < 0.05). 알츠하이머병에의한치매군이나, 치매증상을보인기타치매군사이에의미있는 Aβ42 농도차이는없었다. 반면, ptau181 은알츠하이머병에의한치매군에서정상군이나기타치매군에비해의미있게증가되어있었다 (p < 0.05). 기타치매군에서는 ptau181 농도가정상군과비슷한수준으로낮았다. ttau 농도도 ptau181 과같은경향을보여알츠하이머병에의한치매군에서의미있게증가하였고 (p < 0.05), 정상군과기타치매군에서는비슷한수준으로낮았다. 추가로 ptau181/aβ42 및 ttau/aβ42 의질환군별차이를분석하였다. 모두알츠하이머병에의한치매군이정상군에비해의미있게증가되었다 (p < 0.05). 그러나, 기타치매군은알츠하이머병에의한치매군과정상군사이의농도를보였는데, 사후검증에서정상군이나알츠하이머병에의한치매군에비해의미있는차이가없었다. 병에의한치매로진단하였고, 정상군의 78.6% 를정상이라고진단하여모두 86.2% 정도의검사정확도를보였다. 추가로분석한 ptau181/ Aβ42 비및 ttau/aβ42 비는 96.6% 높은수준의검사정확도를보였는데, 알츠하이머병에의한치매군의 100% 를알츠하이머병에의한치매로진단하였고, 정상군의 92.3% 를정상으로진단하였다. ROC curve 분석으로구한알츠하이머병에의한치매진단을위한생물표지자의 cut-off 농도는 Aβ pg/ml, ptau pg/ml, ttau pg/ml이었고, ptau181/aβ42 및 ttau/aβ42 는각각 과 이었다. 이들 cut-off 농도의민감도와특이도는 Aβ 42 경우, 88.2% 와 83.3%, ptau181 은 82.4% 와 91.7%, ttau는 82.4% 와 100% 였다. ptau181/aβ42 와 ttau/aβ42 의 cut-off 값은동일하게 94.1% 의민감도와 100% 의특이도를나타냈다. 뇌척수액생물표지자의알츠하이머병진단유용성분석기타치매군에는치매증상을보이는다양한뇌질환이포함되었고, 대상환자수가충분치않아뇌척수액생물표지자의알츠하이머병에의한치매진단유용성을위한분석에서제외하였다. 정상군과알츠하이머병에의한치매군사이에이항로지스틱회귀분석을시행하였고, 더불어 ROC curve 분석을하여 cut-off value를구하고, 이의유용성을확인하였다 (Table 4). Aβ42 농도로알츠하이머병에의한치매군의 88.2% 를알츠하이머병에의한치매로진단하였고, 정상군의 83.3% 를정상으로진단하여, 검사정확도는 84% 였다. ptau181 와 ttau 농도로알츠하이머병에의한치매군의 93.3% 를알츠하이머 고찰본연구에서는 17명의알츠하이머병에의한치매, 12명의정상, 그리고 9명의치매증상을보인기타치매를대상으로뇌척수액의 Aβ 42, ptau, ttau 농도를측정하여, ELISA 검사간측정값의일치도, 질환군별차이그리고, 정상-알츠하이머병에의한치매사이의감별진단을위한각생물표지자농도의 cut-off 값및이들의진단유용성에대한결과를얻었다. 뇌척수액 Aβ42, ptau181, ttau 농도의알츠하이머병진단효용성및진단기준치가보고한센터마다차이가크다 (Table 5). 이의원인

5 알츠하이머병뇌척수액바이오마커 5 Table 5. Brief summary of the previous literatures Study group Subjects Methods Results (pg/ml*) Galasko [42] AD, 36; CON, 14; INNOTEST kit ttau (mean); AD, 509; CON, 177 Hulstaert [43] AD, 150; MCI, 4; CON, 100; Other D, 79 INNOTEST kit Aβ42 (mean); AD, 487; MCI, 494; CON, 849; Non-AD D, 603; ttau (mean); AD, 425; MCI, 710; CON, 195; Non-AD D, 220; Aβ42 (cut-off); AD vs. CON, 643 (S 78%, Sp 81%); AD vs. Other D, 551 (S 71%, Sp 63%); ttau (cut-off); AD vs. CON, 252 (S 79%, Sp 70%); AD v s. Other D, 293 (S 71%, Sp 89%) Andreasen [44] Probable AD, 274; Depression, INNOTEST kit ttau (mean); Probable AD, 690; Depression, 231; CON, 227; ttau (cut-off); 302 (S 93% Sp 86%) 28; CON, 65 Riemenschneider AD, 74; FTD, 34; CON, 40 INNOTEST kit Aβ42 (mean); AD, 394; FTD, 835; CON, 1076; ttau (mean); AD, 540; FTD, 282; CON, 152 [45] Lee EH [16] AD: 16, CON, 14 Biosource ELISA kit (USA) Aβ42 (mean); AD, 391; CON, 827; ptau199 (mean); AD, 283; CON, 277; ptau/aβ42 (mean); AD, 1.01; CON, 0.4 Shaw et al. [14] Schoonenboom et al. [27] AD, 100; MCI, 196; CON, 114 AD: 512, FTD: 144, DLB: 52, VD: 34, CBD: 16, PSP: 20, CJD: 6, PSY: 135, SMI: 275 Multiplex xmap Luminex platform (Luminex Corp, Austin, TX) with Innogenetics kit reagents (INNO-BIA AlzBio3) INNOTEST kit Aβ42 (mean); AD, 138; MCI, 146; CON, 217; ptau181 (mean); AD, 36; MCI, 32; CON, 20; ttau (mean); AD, 110; MCI, 86; CON, 61; ptau181/aβ42 (mean); AD, 0.29; MCI, 0.23; CON, 0.10; ttau/ Aβ42 (mean); AD, 0.86; MCI, 0.62; CON, 0.31; Aβ42 (cut-off); AD vs. CON, 192 (S 96%, Sp 77%); ptau181 (cut-off); AD vs. CON, 23 (S 68%, Sp 73%); ttau (cut-off); AD vs. CON, 93 (S 70%, Sp 92%); ptau181/aβ42 (cut-off); AD vs. CON, 0.1 (S 91%, Sp 71%); ttau/aβ42 (cut-off); AD vs. CON, 0.39 (S 86%, Sp 85%) Aβ42 (mean); AD, 447; FTD, 741; DLB, 638; VD, 627; CBD, 681; PSP, 767; CJD, 755; PSY, 906; SMI, 863; ttau (mean); AD: 604, FTD, 350; DLB, 305; VD, 238; CBD, 262; PSP, 203; CJD, 2060; PSY, 213; SMI, 245; ptau (mean); AD, 83; FTLD, 47; DLB, 52; VD, 35; CBD, 50; PSP, 36; CJD, 54; PSY, 41; SMC, 45 *Unit of results. However in case of ratio, there is no unit. AD, Alzheimer s disease; CBD, corticobasal ganglionic degeneration; CI, cognitive impairment; CON, control; CJD, Creutzfeldt Jacob disease; D. dementia; DLB, diffuse Lewy body dementia; EOAD, early-onset AD; FTD, frontotemporal dementia; LOAD, late-onset AD; MCI, mild cognitive impairment; PSP, progressive supranuclear palsy; SMI, subjective memory impairment; SVD, subcortical vascular dementia; PD, Parkinson s disease; PSY, psychiatric disorder. 으로사용한 kit 마다의차이, 뇌척수액보관방법의차이, 측정전까지해동 / 동결횟수, 측정시조건의차이등이있지만 [24], 최근 Bateman 등 [25] 이제기한것처럼 Aβ42의생성과배출이하루중시간이나식사등활동에따라차이가있어, 채취시간및금식여부등이연구들마다서로일치하지않았던것도원인으로생각된다. 본연구에서는대상환자의채취시간을일정하게하였고, 공복상태에서채취하여, 이같은원인으로인한오류를최소화하였다. 한환자에서채취한동일한뇌척수액을동일한조건에서보관후, 이를나누어서로다른센터에서측정하면, 측정센터에따라차이가크다 [26]. Aβ42가가장높아 31-37% 이고, INNOTEST kit 사용으로 ELISA 측정방법을동일하게하더라도센터간의차이가 22% 정도이다. ptau181 와 ttau 경우는 Aβ42보다적어, 각각 13-15% 와 16-21% 이다. 이같이동일샘플을이용하더라도측정센터간차이가상당하여, ADNI 등국제공동연구에서는, 뇌척수액채취- 보관을공통프로토콜에따라일정하게한뒤, 뇌척수액분석은동결상태로드라이아이스상자에넣어생물표지자 core 로지정된센터로운반후이곳에서분석하게하여 ( biomarker/) 센터간측정값차이에따른오류를피하고있다. 따라서, 본연구자들도검체수집후분석은한센터를정하여그곳에서시행하였다. 동일센터내에서같은뇌척수액을반복해서측정할때생기는차이 (intertest variance, CV%) 는해외여러센터가공동으로한연구에서는 Aβ42, ptau181, ttau 가각각 21%, 9%, 15%, 혹은 25%, 7%, 18% 였다 [26]. 그러나, 최근대규모코호트대상연구에서보고된것처럼센터를지정하여그곳에서만지속적으로시행하게한경우에는 A β42는 11.3%, ptau181 은 9.4%, ttau 는 9.3% 정도로감소한다 [27]. 본연구의 intertest variance 는 Aβ %, ptau %, 그리고 ttau 7% 로보고된국제적센터의수준에근접한반복측정일치도를보였다. 본연구에서알츠하이머병에의한치매환자가이의비교군인정상군에비해학력이의미있게높았다 (12 ± 4년 vs. 6 ± 4년 ). 학력이높은경우뇌병리가있더라도치매증상발현까지버틸수있는인지기능저장력 (cognitive reserve) 이있어, 같은수준의치매단계저학력환자에비해뇌병리진행이심하다 [28]. 따라서, 본연구대상알츠하이머병에의한치매인경우동일치매단계중에서도비교적뇌병리가심한군이포함되었을가능성이높다. 반면정상군은학력이낮았기에, 알츠하이머병에의한치매의병리를가졌으나, 인지기능저장력으로버티고있던알츠하이머병에의한치매전단계 ( 혹은초기 ) 환자들이포함되었을가능성이고학력정상인을포함한연구에비해적다. 그리고, 정상군이오히려나이가많아, 연령이젊어초기알츠하이머병이더라도인지기능장애를보이지않은환자들이포함될가능성도줄었다. Aβ42는노년판 (senile plaque) 의주요성분으로알츠하이머병발생에핵심역할을하며, 뇌의실질로유리되어있다가이중일부가뇌척수액으로나온다. 이중가장응집력이높고, 세포독성이높은 42 개의펩타이드로구성된 Aβ42의농도가알츠하이머병뇌척수액에서질환초기부터감소하는데, 정상인에비해약 50% 정도감소한다

6 6 박선아 김종헌 김형준외 9 인 [29, 30]. Pittsburgh compound B (PIB) 를이용한 PET와뇌척수액 Aβ 42 농도를비교하면뇌척수액농도변화가 PIB-PET 보다이른시기에오고 [31, 32], 이둘사이에는높은수준의음의상관도가있다 [33]. 이는뇌척수액의 Aβ42 농도감소가뇌침착증가로인해뇌척수액으로의 Aβ42 배출이감소하기때문이라는가설을뒷받침한다 [10]. 그러나, 알츠하이머병뇌척수액의 Aβ42 농도감소가뇌척수액농도측정에이용하는대부분의 ELISA kit 가 monomer 형태의 Aβ42를측정하는것이라, 알츠하이머병에서 Aβ42가 monomer 상태로있기보다서로응집하여쉽게 oligomer 형태이기에측정되는 Aβ42의양이감소되게나타나는것이지, oligomer 형태의 Aβ42 농도를측정해보면오히려알츠하이머병에서증가된다는주장도있다 [34]. 미세관 (microtubule) 을안정시키는정상단백인 tau는과인산화되면서미세관으로부터유리되어망가진신경돌기 (neurite) 들과결합하여세포내신경원섬유매듭 (neurofibrillary tangle) 을형성한다. 이로인해신경세포기능이상이신경세포사로이어져세포가파괴되고, 그안에있던 tau 단백이세포밖으로유출된후다시뇌척수액으로나오기에알츠하이머병환자뇌척수액에서 2-3배농도증가가관찰된다. 알츠하이머병에서인산화가증가되는 tau 단백의위치에따라 ptau181, ptau , 그리고 ptau 의농도를구분하여측정한연구들이있는데 [13, 35, 36], 뇌척수액내의 ptau 의증가가알츠하이머병과다른퇴행성뇌질환사이에감별진단에도움이된다고알려져있다. Aβ42, ptau181 그리고 ttau 의이상은알츠하이머병환자에서단편적비교를해볼때, 인지기능장애정도등치매정도와상관성을보이지않으나, 경도인지장애환자가알츠하이머병에의한치매로진행되는것을예측하는데도움이된다 [37,38]. 즉, 이들단백의변화는알츠하이머병초기에변화한후에는그이상정도가일정한범위내에서유지되는것으로생각된다. 사후부검뇌와생전뇌척수액의변화를비교하여보고한기존보고들을종합해볼때 Aβ42, ptau181, ttau 각각보다, 이들의조합인 ptau181/aβ42 혹은 ttau/aβ42 가알츠하이머병을진단하거나, 경도인지장애환자가알츠하이머병으로진행하는것을예측하는데유용하여, 민감도와특이도가 85-91% 에이른다 [14, 15]. 본연구에서는대상환자수는적었지만, 알츠하이머병에의한치매군과정상군을기존연구들에비해엄격한기준으로선별하였고, 알츠하이머병에의한치매군은정상군에비해학력이높고, 나이는많아, 확실한알츠하이머병리를가지는군과정상병리를가지는군을비교하고자노력하였다. 이를통해얻은 Aβ42, ptau181 및 ttau 측정값의양성 ( 음성 ) 예측도는각각 88.2% (88.3%), 93.3% (78.6%), 93.3% (78.6%) 였고, 검사정확도는각각 84%, 86.2%, 86.2% 였다. 이전보고들처럼추가분석한 ptau181/aβ42 및 ttau/aβ42 비의양성 ( 음성 ) 예측도가 100% (92.3%) 였고, 검사정확도가높아 96.6% 였다. 이는 ADNI에서사후부검으로알츠하이머병을확진한환자의생전뇌척수액과인지기능정상인의뇌척수액을 이용해서진행한연구에서보고한 Aβ42, ptau181, ttau의양성 ( 음성 ) 예측도, 81.8% (95.2%), 73.1% (67.9%), 90.7% (73.8%) 및검사정확도 87%, 70.4%, 80.6% 와비슷한수준이다 [14]. Aβ42, ptau181 그리고 ttau 의알츠하이머병에의한치매군과정상군에서의평균농도및 ROC curve 분석으로얻은 cut-off 값은이전결과들과비슷하거나차이가있는데 (Table 5), 워낙분석센터마다차이가있을수있는수치이기에본연구에서분석이시행된센터의측정치로향후이용하면될것이다. 얻은 cut-off 농도로분석한알츠하이머병에의한치매진단의민감도와특이도는 Aβ42 경우, 88.2% 와 83.3%, ptau181 은 82.4% 와 91.7%, ttau 는 82.4% 와 100%, ptau181/aβ42 비와 ttau/aβ42 비는동일하게 94.1% 와 100% 로이전보고들중높은수준의민감도와특이도를보고한그룹들과비슷한수준이었다 [14, 15]. 본연구에서알츠하이머병에의한치매군과기타치매군사이의감별에서뇌척수액생물표지자들의유용성에대한분석은앞서언급한이유들로인해시행하지않았다. ptau181 과 ttau 농도는알츠하이머병에의한치매군에비해감소되어정상군과비슷한수준이었으나, Aβ42 농도는알츠하이머병에의한치매군과비슷한수준으로감소되어있었다. 포함된기타치매환자들은임상적치매증상을보인파킨슨병치매, 레비소체치매, 전두측두엽치매, 진행핵상마비, 혈관성치매, 그리고정신질환이었다. 파킨슨병이나레비소체치매에서 Aβ침착이흔히관찰되는데, 치매로진행된경우더욱그렇고 [39, 40], 혈관성치매에서도드물지않게관찰된다 [41]. 이같은이유가기타치매군에서 Aβ42 농도감소의원인이었을가능성이크다. 그러나, 6개월이상임상적추적을하였더라도, 기타치매의경우임상진단기준에의지하여서질환분류를하였기에, 알츠하이머병에의한치매환자가일부포함되었을가능성도배제할수는없다. 결론적으로, 본연구자들은알츠하이머병진단에유용한 Aβ42, ptau181 및 ttau 의뇌척수액농도의기준치마련을위한예비연구를통해정상인과알츠하이머병에의한치매를감별하는데도움이될 Aβ42, ptau181, ttau, ptau181/aβ42 및 ttau/aβ42 에대한수치를제시하였다. 반복측정사이의일치도및 cut-off 농도의민감도와특이도가높아뇌척수액생물표지자를이용한다기관국내연구에이용할만큼신뢰도있는뇌척수액 Aβ42, ptau181, ttau 농도측정위한국내네트워크를만들었다는데본연구의의의가있다. 향후치매를보이는여러기타치매와알츠하이머병에의한치매의감별유용성및더많은환자수를대상으로한검증등이필요할것으로생각된다. 참고문헌 1. Asien PS, Andrieu S, Sampaio C, Carrillo M, Dhachaturian ZS, Dubois B, et al. Report of the task force on designing clinical trials in early (pre-

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