원저 무증상뇌경색환자에서 Thymidylate Synthase Enhancer Region (TSER) 유전자의다형성 포천중문의과대학교신경과학교실, 임상의학연구소 a 김경근김현숙구영호김동건김원찬김옥준김남근 a Genetic Polymorphism of Thymidylate Synthase Enhancer Region (TSER) in Patients with Silent Brain Infarction Kyoung Keun Kim, M.D., Hyun Sook Kim, M.D., Young Ho Koo, M.D., Dong Gun Kim, M.D., Won Chan Kim, M.D., Ok Jun Kim, M.D., Ph.D., Nam Keun Kim, Ph.D. a Department of Neurology, Institute for Clinical Research a, Pochon CHA University College of Medicine, Seongnam, Korea Background: Hyperhomocysteinemia is an independent risk factor for silent brain infarction (SBI). The plasma homocysteine (phcy) level is influenced by the activities of enzymes such as 5,10-methylenetetrahydrofolate reductase (MTHFR). Thymidylate synthase (TS) also competes with MTHFR for their common cofactor, 5,10-methylenetetrahydrofolate (5,10-meTHF). The polymorphism of thymidylate synthase enhancer region (TSER) might affect homocystein metabolism by modulating the activity of TS, and may be a determinant of SBI by elevating phcy concentrations. Therefore, we studied the polymorphism of TSER in patients with SBI. Methods: 98 patients with SBI and 92 healthy controls were included in the study. The genotypes of TSER and MTHFR were identified with the PCR-RFLP methods. Results: The mean phcy level was significantly higher in SBI patients (13.5±8.5 μmol/l) than in controls (10.3± 4.1 μmol/l)(p<0.01). The frequencies of MTHFR C677T genotype and TSER 28 bp tandem repeat genotype were not different between the patients and the controls. The phcy concentrations were not considerably different between the 3R3R and 2R3R genotypes in the population as a whole (p=0.712), nor in subsets of patients with SBI (p=0.484). However, in cases with the TSER 3R3R genotype, the phcy level was significantly higher in patients (14.0±10.26 μmol/l) than in controls (9.9±3.1 μmol/l)(p=0.006). Folate and phcy was inversely correlated in the SBI patients with the TSER 3R3R genotype (r=-0.424, p=0.039). Conclusions: Our findings suggest that the TSER genotype is not a major determinant of phcy concentrations and is neither a risk factor for SBI in the Korean population. However, further study will be needed to confirm this findings. J Korean Neurol Assoc 25(3):338-343, 2007 Key Words: Thymidylate synthase, Silent brain infarction, Homocysteine, Thymidylate synthase enhancer region, Genetic polypmorphism 서론 Received January 23, 2007 Accepted April 16, 2007 *Hyun Sook Kim, M.D., Nam-Keun Kim, Ph.D. Department of Neurology, Institute for Clinical Research, Bundang CHA Hospital, 351 Yatap-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-712, Korea Tel: +82-31-780-5483 Fax: +82-31-780-6201 E-mail: hskim626@cha.ac.kr & nkkim@cha.ac.kr * 본연구는한국학술진흥재단과교육인적자원부학술연구비 (KRF- 2006-331-E00304) 에의해지원되었음. 고호모시스테인혈증이죽상동맥경화증및혈전성질환의중요한인자로알려지면서뇌경색및무증상뇌경색의위험요소로고려되고있다. 1-5 특히혈중호모시스테인의농도가정상보다증가하면무증상뇌경색의발생빈도가 2.5 배에서 4.5 배까지증가된다는보고가있으며, 6,7 국내에서도고호모시스테인혈증을무증상뇌경색의독립적위험인자로보고하였다. 8 이로 338 대한신경과학회지제 25 권제 3 호, 2007
무증상뇌경색환자에서 Thymidylate Synthase Enhancer Region (TSER) 유전자의다형성 인해호모시스테인대사및이와관련된유전자연구가활발하게이루어지고있다. 9 호모시스테인은비필수아미노산의일종으로필수아미노산인메티오닌 (methionine) 의대사과정에서생성된다. 메티오닌이탈메틸화 (demethylation) 되어호모시스테인으로바뀐후에는 cystathionine β synthase 에의해황전환작용 (transsulfuration) 을거쳐시스테인 (cysteine) 으로대사되거나, 재메틸화 (remethylation) 의과정을거쳐다시메티오닌으로바뀌게된다. Methylenetetrahydrofolate reductase (MTHFR) 은 5, 10-methylenetetrahydrofolate (5,10- methf) 를 5-methyltetrahydrofolate (5-meTHF) 로환원시키며 5-meTHF 는호모시스테인을메티오닌으로재메틸화시키는탄소공여자로작용한다. 이러한대사과정중에효소나보조인자의결핍으로고호모시스테인증을일으킬수있으며관련유전자중 MTHFR 유전자가가장대표적이며 C677T 다형성에따라효소활성이변화되어고호모시스테인혈증이유발된다. 10,11 그러나 Thymidylate synthase (TS) 도 5,10-meTHF 에 MTHFR 과경쟁적으로작용하며호모시스테인대사에관계된다. 12 특히 TS 유전자의 enhancer 부위 (TSER, Thymidylate synthase enhancer region) 에 28 bp의염기가직렬반복되는유전자다형성이알려져있으며, 이중 3R3R 유전자형이고호모시스테인혈증및엽산농도감소와연관이있는것으로보고되었다. 13 이에저자들은대상군에서혈중호모시스테인및엽산의농도와 TSER, MTHFR C677T 의유전자다형성을분석하여, 유전자형에따른혈중호모시스테인및엽산농도의상관관계및 TSER 의유전자다형성이무증상뇌경색의독립적위험인자에해당하는지를알아보고자하였다. 2. 방법 1) Homocysteine 의생화학적분석혈액전처리는채혈전 8시간공복후아침식전에환자의상박에서정맥채혈을하였다. 전혈은 citrate salts 가포함된 vacutainer tube 에담아, 얼음으로냉각후즉시 2,000 rmp 으로 5분간원심분리하여혈장을 0~4 도로냉장보관하였다. 혈중호모시스테인의정량은혈장내호모시스테인의농도를 Ueland 등 14 이기술하고, Chadefauz 등 15 에의하여수정된효소방사선학적방법으로측정하였다. 2) MTHFR C677T 유전자및 TSER 유전자다형성분석 DNA 의분리는 DNA 추출 kit (extraction column, QIAmp blood kit, Qiagen) 에의해제조자의프로토콜에따라환자의정맥혈액에서추출한백혈구로부터분리하였다. MTHFR C677T 유전자다형성을분석하기위해서분리된 DNA 표본을이전에기술된바에따라 PCR-RFLP 을시행하고, 2.5% agarose-ethidium bromide gel 에전기영동하여다형성을확인하였다. 16 TSER 유전자다형성을분석하기위해 TS 유전자의 5 -untranslated region에서 28 bp tandem repeat를포함한부분을증폭할수있게제작된 primer (sense: 5 -GTGGCTC CTGCGTTTCCCCC-3 과 antisense:5 -GCTCCGAGCCGGCCA CAGGCATGGCGCGG-3 ) 를사용하여 GeneAmp PCR machine (Perkin Elmer 9600) 에서증폭시켰다. PCR 조건은 94도에서 40초, 62도에서 1분, 72도에서 40초간 30회반복후, 72도에서 5분간 elongation 시켰으며, 증폭된 DNA 단편은 3.5% agarose gel 에전기영동하여다형성을분석하였다. 대상과방법 1. 대상 본연구는 2003 년 8월부터 2004 년 7월까지한대학병원외래에방문한환자들중에, 과거력및신경학적검사상이상소견이없으나예방적인검사를원해뇌자기공명영상을시행하고, TSER 및 MTHFR 유전자다형성검사에동의한 198 명을대상으로하였다. 이중뇌자기공명영상중 T2 강조영상에서 3 mm 이상의크기로신호증가를보이고 T1 및 FLAIR 영상신호에이와부합되는병변을보이는 98명을무증상뇌경색의환자군에포함시켰다. Virchow-Robin space 나 leukoaraiosis 소견을보인 8명은제외하였다. 뇌자기공명영상에서이상이없는 92명을정상대조군에포함시켰다. 3) 통계분석환자군과대조군의일반적인특징을분석하기위해서, 범주형자료 (categorical data; 성별, 고혈압, 당뇨 ) 분석에는 chisquare test 를사용하였고연속형자료 (continuous data; 연령, 공복혈중총콜레스테롤농도, 호모시스테인농도, 엽산농도및비타민 B12 농도 ) 에는 two-sample t-test 를사용하였다. 환자군과대조군사이에서무증상뇌경색의비교위험도를측정하기위해서 crude odds ratio (COR) 를구하였다. 환자군과대조군에서유전자형의차이및각 TSER 유전자다형성에따른환자군과대조군간의혈중호모시스테인, 엽산, 비타민 B12 농도의차이를비교하기위해나이, 성별, 고혈압, 당뇨, 총콜레스테롤농도를보정한 multiple logistic regression test 를시행하였다. 연구대상전체, 환자군및대조군에서 TSER 유전자형에따른호모시스테인, 엽산, 비타민 B12 농도차이를분석하기 J Korean Neurol Assoc Volume 25 No. 3, 2007 339
김경근김현숙구영호김동건김원찬김옥준김남근 위해 independent t-test 를사용하였다. 각유전자군에서호모시스테인과엽산의농도의상관관계를분석하기위해 correlation analysis 를시행하였다. 모든통계적분석은 SPSS for windows, version 11.0 (SPSS Inc., Chicago, Illinois, USA) 을이용하였다. 결과 1. 환자군과대조군의임상적특징 본연구에포함된무증상뇌경색환자군은 98명 ( 남자 46명, 여자 52명 ) 이며평균연령은 64.4±10.5 세였으며, 대조군은 92 명 ( 남자 45명, 여자 47명 ) 이며평균연령은 56.4±12.0 세로성별의차이는없었으나, 환자군이대조군에비해나이가많았다 (p<0.001). 고혈압, 당뇨, 혈중총콜레스테롤농도가환자군과대조군에서통계적으로유의하게차이를보였다 (Table 1). 혈중호모시스테인치는환자군 (13.5±8.5 μmol/l) 에서대조군 (10.3 ±4.1 μmol/l) 보다높았고 (COR, 1.12; 95% CI, 1.03 to 1.22, p=0.001), Vit. B12는환자군 (550.2±264.4 pmol/l) 에서대조군 (643.1±270.6 pmol/l) 에비해유의하게낮았으나 (COR, 0.99; 95% CI, 0.99 to 1.00, p=0.021), 엽산의농도는차이가 없었다. 2. 환자군과대조군에서의 MTHFR C677T 과 TSER 유전자다형성분포 MTHFR C677T 유전자형은환자군에서정상 (C/C) 은 24명 (24.5%), 이형접합성변이 (C/T) 는 56명 (57.1%), 동형접합성변이 (T/T) 는 18명 (18.4%) 을보였고, 대조군에서는정상 (C/C) 은 25명 (27.2%), 이형접합성변이 (C/T) 는 54명 (58.7%), 동형접합성변이 (T/T) 는 13명 (14.1%) 으로두군간에통계적으로유의한차이는없었다 (AOR, 0.633; 95% CI, 0.205 to 1.957, p=0.427). TSER 의 28 bp의반복횟수는 2R2R, 2R3R, 2R6R, 3R3R, 3R5R 로관찰되어, 이를호모시스테인대사능력에따라 2R3R군 (2R2R, 2R3R,2R6R) 과 3R3R군 (3R3R, 3R5R) 으로분류하여분석하였다. TSER 유전자다형성분포는환자군에서 2R3R 이 36 명 (36.7%), 3R3R 이 62 명 (63.3%) 이었고, 대조군에서 2R3R 이 40명 (44.4%), 3R3R 이 52명 (55.6%) 으로, 환자군에서정상대조군에비해 3R3R 유전자형이많은경향이었으나통계적인유의성은없었다 (COR, 0.773; 95% CI, 0.312 to 1.913, p=0.577)(table 2). Table 1. Characteristics in patients with silent brain infarction and controls Controls (n=92) Patients (n=98) COR 95% CI p* Age (yrs) 56.4±12.0 64.4±10.5 1.06 1.04-1.09 <0.001 Sex (M/F) 45/47 46/52 1.10 0.63-1.95 0.773 Hypertension 33 74 5.42 2.89-10.16 <0.001 Diabetes mellitus 8 19 2.50 1.03-6.03 0.040 Total cholesterol 194.4 215.9 1.01 1.06-1.25 0.001 Homocysteine 10.3±4.1 13.5±8.5 1.12 1.03-1.22 0.001 Vit. B12 643.1±270.6 550.2±264.4 0.99 0.99-1.00 0.021 Folate 10.2±6.8 9.1±4.6 0.97 0.92-1.02 0.193 COR; crude odds ratio, 95% CI; 95% confidence intervals, *Chi-square test for the categorical data and two-sample t-test for the continuous data. Table 2. Frequencies of TSER and MTHFR genetic polymorphisms between patients with silent brain infarction and controls Controls (n=92) Patients (n=98) COR 95% CI p* TSER (%) 2R3R 40 (44.4) 36 (36.7) 3R3R 52 (55.6) 62 (63.3) 2R3R vs 3R3R 0.773 0.312-1.913 0.577 MTHFR (%) CC 25 (27.2) 24 (24.5) CT 54 (58.7) 56 (57.1) TT 13 (14.1) 18 (18.4) CC/CT vs TT 0.633 0.205-1.957 0.427 COR; crude odds ratio, 95% CI; 95% confidence intervals, *Chi-square test. 340 대한신경과학회지제 25 권제 3 호, 2007
무증상뇌경색환자에서 Thymidylate Synthase Enhancer Region (TSER) 유전자의다형성 (A) r=-0.015, p=0.913 (B) r=-0.424, p=0.039 Figure 1. Correlation between folate and phcy level in controls (A) and patients with TSER 3R3R genotype (B). r ; correlation coefficient with using the correlation analysis. 3. 환자군 -대조군및 TSER 유전자형에따른호모시스테인, 비타민 B12 와엽산의농도비교전체연구대상및환자군, 대조군에서 TSER 유전자다형성에따라혈중호모시스테인, 비타민 B12, 엽산의농도는차이가 없었다 (Table 3). TSER 2R3R 유전자군에서환자군과대조군을비교한결과혈중호모시스테인, 비타민 B12, 엽산의농도차이가없었다. 그러나 TSER 3R3R 유전자군에서는혈중호모시스테인농도가대조군 (9.9±3.1 μmol/l) 에비해환자군 (14.0 ±10.3 μmol/l) 에서유의하게높았고 (p=0.006), 혈중비타민 Table 3. Homocysteine, Vit. B12 and folate levels according to TSER polymorphism Population All (n=190) Patients (n=92) Controls (n=98) Analyte TSER genotype 2R3R 3R3R OR 95% CI p* Homocysteine 11.7±4.7 12.1±8.1 1.008 0.965-1.054 0.712 Vit. B12 587.6±254.9 599.9±282.4 1.000 0.999-1.001 0.761 Folate 9.0±5.5 10.0±5.9 1.032 0.978-1.090 0.246 Homocysteine 12.7±4.1 14.0±10.3 1.021 0.963-1.084 0.484 Vit. B12 543.3±255.3 555.3±273.4 1.000 0.999-1.002 0.829 Folate 8.5±4.1 9.5±4.9 1.051 0.955-1.156 0.307 Homocysteine 10.8±5.1 9.9±3.1 0.942 0.896-1.049 0.278 Vit. B12 629.5±250.7 653.1±286.3 1.000 0.999-1.002 0.682 Folate 9.5±6.6 10.7±7.0 1.027 0.962-1.095 0.427 Values are mean±s.d. Units are μmol/l for homocysteine, pmol/l for Vit. B12 and nmol/l for folate. *Independent t-test for continuous variables. Table 4. Plasma homocysteine, Vit. B12 and folate levels between patients and controls according to TSER genotype TSER genotype Analyte Controls (n=92) Patients (n=98) AOR 95% CI p* 2R3R Homocysteine 10.8±5.1 12.7±4.1 0.893 0.699-1.142 0.368 Vit. B12 629.5±250.7 543.3±255.3 1.000 0.995-1.005 0.973 Folate 9.5±6.6 8.5±4.1 0.824 0.646-1.050 0.117 Homocysteine 9.9±3.1 14.0±10.3 0.621 0.444-0.870 0.006 3R3R Vit. B12 653.1±286.3 555.3±273.4 1.006 1.001-1.010 0.010 Folate 10.7±7.0 9.5±4.9 0.184 0.958-1.250 0.184 Values are mean±s.d. Units are μmol/l for homocysteine, pmol/l for Vit. B12 and nmol/l for folate. AOR; adjusted odds ratio, adjusted for age, sex, hypertension, diabetes mellitus and hypercholesterolemia, *multiple logistic regression test. J Korean Neurol Assoc Volume 25 No. 3, 2007 341
김경근김현숙구영호김동건김원찬김옥준김남근 B12 농도는정상대조군 (653.1±286.3 pmol/l) 에비해환자군 (555.3±273.4 pmol/l) 에서유의하게낮았다 (p=0.01)(table 4). 호모시스테인과비타민 B12, 엽산의상관관계를각군에서분석한결과, TSER 3R3R 유전자형을가진환자군에서대조군과는달리호모시스테인과엽산사이에통계적으로유의한역함수관계가있었다 (r=-0.424, p=0.039)(fig. 1). 고찰 무증상뇌경색이혈중호모시스테인농도와관련이있다고제시되면서, 고호모시스테인혈증을일으킬수있는유전적요인으로 MTHFR C677T 유전자다형성이연구되었으나, 현재까지환자군과대조군간에통계적으로유의한유전자다형성의분포차이를보이지는않았다. 6-8 TS는 MTHFR 과같이호모시스테인대사에관여하며, MTHFR 과유사하게유전자다형성에따라효소활성도가달라진다. 17 TS의효소활성도를결정하는유전자다형성은 TSER 에존재하며 28 bp의 tandem repeat number 에따라 2R, 3R, 4R 등으로구분된다. 생체내에서 TSER 의 tandem repeat number 가증가할수록 TS의활성도가더높아져 cofactor 인 5,10-meTHF 의농도가감소된다. 18,19 이에따라 TSER 3R3R 형은 2R2R 또는 2R3R 유전자형에비해 5,10-meTHF 를 cofactor 로사용하는 MTHFR 의활성도를떨어뜨려최종적으로고호모시스테인혈증을일으킬것으로생각되어최근연구되고있다. 현재까지의연구에서 TSER 3R 유전자다형성의빈도는동양인 ( 중국, 일본, 한국 ) 이백인보다약 2배높고, 20,21 이는지역적인식이의다양성에의해유발되었을가능성이있다. 20,22 이러한이유로 TSER 유전자다형성이동양인에서혈중호모시스테인및엽산대사에더중요한요소로고려될수있을것이다. 실제로중년의동양인들에서 TSER 3R3R 유전자형을가진군에서다른유전자형을가진군에비해혈중엽산농도가통계적으로유의하게낮았고, 엽산의섭취가평균이하일경우에 3R3R 유전자형을가진군에서유의하게혈중호모시스테인농도가높은것으로나타났다. 11 반면에백인성인에서는 TSER 유전자다형성에따른혈중호모시스테인과엽산의농도의차이는없는것으로보고되어인종적차이를강하게시사하였다. 23 본연구에서는무증상뇌경색환자군에서혈중호모시스테인의농도가대조군에비해서통계적으로유의하게높아기존의연구결과들과일치하였다. 5,6 하지만 TSER 유전자다형성의분포는무증상뇌경색을가진환자군과정상대조군간에통계적으로유의한차이가없었고, 연구대상전체, 환자군및대조군에서 TSER 유전자형에따른혈중호모시스테인농도, 엽산 농도, 비타민 B12 의농도또한통계적으로차이를보이지않았다. 이는 TSER 의유전자다형성이혈중호모시스테인의농도결정에주요한역할을하지않아, 무증상뇌경색의주요한위험인자로서기능하지않을것을시사한다. 이러한결과는본연구대상자의표본수가제한적이고, 연구대상자들의엽산평균섭취량연구가이루어지지않았기때문에생겼을가능성을완전히배제할수는없다. 특히 TSER 3R3R 형을가진환자군에서는대조군에비해혈중호모시스테인의농도가통계적으로유의하게높고비타민 B12 의농도가낮은결과를보였고, 이군에서호모시스테인과엽산의농도사이에유의한역함수관계가있는것이관찰되었다. 이는기존의 MTHFR 677TT 유전자형을가진환자군에서보였던호모시스테인과엽산농도사이의역함수관계와유사한결과로, TSER 3R3R 유전자형이무증상뇌경색발생의주요한위험예측인자는아니지만, 무증상뇌경색환자에서대조군에비해고호모시스테인혈증을일으키는부차적요소일수있을것으로사료된다. 하지만 TSER 3R3R 유전자형을가진연구대상의표본수가적어, 가양성의결과의가능성이있어보다많은수의환자를대상으로한연구가필요하리라생각된다. 결론적으로본연구에서는한국인에서호모시스테인대사에불리한 TSER 3R3R 유전자형이많고, TSER 유전자형의차이가무증상뇌경색의독립적위험인자는아닌것으로확인되었다. 하지만 TSER 3R3R 유전자형을가지는무증상뇌경색환자에서정상대조군에비해통계적으로유의하게높은혈중호모시스테인농도를보이고, 엽산농도와역함수관계를보여, 무증상뇌경색을가진환자중 TSER 3R3R 유전자형을가지는경우추가적인엽산섭취가도움이될것으로추정된다. 이를확인하기위해서는향후더많은수의표본을대상으로엽산의섭취량을고려한환자-대조군연구와 TSER 유전자다형성에따른엽산등의비타민의보충이무증상뇌경색의발생에미치는영향에대한후향적연구가더필요하리라생각된다. REFERENCES 1. Selhub J, Jacques PF, Wilson PW, Rush D, Rosenberg IH. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 1993;270:2693-2698. 2. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, et al. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991;324:1149-1155. 3. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitativeassessment of plasma homocysteine as a risk factor for vascular disease: probable benefits of increasing folic acid intakes. JAMA 1995; 274:1049-1057. 342 대한신경과학회지제 25 권제 3 호, 2007
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