원 저 J Korean Neurol Assoc / Volume 22 / June, 2004 전조성편두통과호모시스테인및 MTHFR C677T 유전자다형성의상관관계 포천중문의과대학교신경과학교실, 이화여자대학교의과대학신경과학교실 * 서정호김현조이일형김옥준최병옥 * Association between Migraine with Aura and both Homocysteine and MTHFR C677T Polymorphism Jung-Ho Seo, M.D., Hyun-Jo Kim, M.D., Il-hyung Lee, M.D., Ok-Joon Kim, M.D., Byung-Ok Choi, M.D.* Department of Neurology, Pochon CHA University College of Medicine, Seongnam; Department of Neurology, Ewha Womans University College of Medicine*, Seoul, Korea Background: The pathophysiology of migraine is not yet fully understood. Homocysteine acts as an excitatory amino acid in vivo and may influence the threshold of migraine. The previous studies have revealed strong association between the homozygous C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene and hyperhomocysteinemia. In this study, we examined the association between both homocysteine and MTHFR C677T polymorphism and headache, such as migraine and tension headache. Methods: We enrolled 148 headache patients (75 tension headache, 34 migraine with aura [MWA], and 39 migraine without aura [MOA]) and 121 normal controls; checked their fasting homocysteine levels and analyzed for the MTHFR C677T polymorphism. For the multivariate analysis, we used logistic regression to adjust for age and sex. Results: Plasma homocysteine levels were significantly higher in patients with MWA than those in controls (adjusted odds ratio (AOR), 1.12; 95% confidence intervals (CI), 1.02~1.23). The AOR and 95% CI of the MTHFR 677TT genotype was significantly higher in patients with total migraine (AOR, 3.32; 95% CI, 1.30~9.55; p< 0.05) and with MWA (AOR, 4.70; 95% CI, 1.44~15.29; p<0.01) than the controls. However, the AOR was not significant in patients with tension headache (AOR, 0.93; 95% CI, 0.37~2.32) and with MOA (AOR, 2.77; 95% CI, 0.78~9.74). Conclusions: Our findings suggest that both elevated homocysteine level and the homozygous C677T mutation in the MTHFR gene are associated with migraine with aura. J Korean Neurol Assoc 22(3):200~205, 2004 Key Words: Migraine, MTHFR, Gene, Homocysteine, Risk factors 서론 편두통은재발성, 발작성두통으로박동성을특징으로 Received October 7, 2003 Accepted December 4, 2003 *Address for correspondence Byung-Ok Choi, M.D. Department of Neurology, Ewha Womans University College of Medicine, Dongdaemun Hospital 70, Jongno-6ga, Jongno-gu, Seoul, 110-783, Korea Tel:+82-2-760-5257 Fax:+82-2-760-5008 E-mail:bochoi@ewha.ac.kr * 본연구는한국과학재단목적기초연구 (R01-2003-000-10716-0) 지원으로수행되었음. 하고메스꺼움, 구토및광선공포증등의신경학적증상을동반한다. 1 편두통은가장활동적인연령인 20 대에서 50 대사이에호발하고, 두통이발작할때에는일상생활에지장을주는정도의심한통증을일으키므로삶의질에막대한지장을초래할수있다. 2,3 따라서편두통환자가정상적인일상생활기능을발휘할수있도록적절한예방과치료가이루어져야한다고생각한다. Kowa 등 4 은 MTHFR (methylenetetrahydrofolate reductase) C677T 동형접합성유전자변이가편두통, 특히전조성편두통 (MWA; migraine with aura) 에서유전적원인인자가됨을보고하였고, Evers 등 5 은전조성편두통이고호모시스테인혈증 (hyperhomocysteinemia) 과 200 대한신경과학회지 22 권 3 호
전조성편두통과호모시스테인및 MTHFR C677T 유전자다형성의상관관계 연관된다고주장하였다. 뇌혈관질환, 관상동맥질환및말초동맥질환등에서고호모시스테인혈증은독립적인위험인자로알려져있으며, MTHFR 677TT 유전자형은고호모시스테인혈증을일으키는중요한원인으로보고되어있다. 6-9 또한고호모시스테인혈증은엽산등의섭취를통해서혈중농도를감소시킬수있는것으로알려져있다. 10,11 따라서혈중호모시스테인농도및 MTHFR 의유전자형과이들두통과의관계에대해서알아보는것은편두통및긴장형두통 (tension headache; TH) 의병인을찾아내는것뿐만아니라예방에도도움이될수있을것으로생각된다. 그런데아직국내에서는편두통및긴장형두통과 MTHFR 유전자형및고호모시스테인혈증에대한보고는없었다. 이에저자들은한국인편두통및긴장형두통환자군에서혈중호모시스테인농도및 MTHFR C677T 유전자다형성이편두통및긴장성두통의위험인자에해당하는지를알아보고자하였다. 대상과방법 1. 대상 2003 년 1 월부터 2003 년 9 월까지이화여자대학교의과대학동대문병원에두통을주소로내원한환자들을본연구의대상으로하여 hospital based case-control study 를하였다. 두통의진단은국제두통학회 (International Headache Society) 에서 1988 년에제안한진단기준에따라분류하였다. 1 148 명의두통환자군중긴장형두통환자 (TH) 는 75 명이었고전체편두통환자 (TM; total migraine) 는 73 명이었다. 전체편두통환자들은전조성 (aura) 의유무에따라전조성편두통 (MWA) 과무전조성편두통 (migraine without aura; MOA) 으로나누었는데, 각각 34 명과 39 명이었다. 연구대상환자들중에서뇌혈관성및심장혈관성, 말초혈관성질환을가진환자들은제외하였다. 정상대조군은같은기간에같은병원에건강검진을위해내원한사람들로서, 뇌혈관성, 심장혈관성및말초혈관성질환을가지지않았을뿐만아니라, 과거력상편두통의병력및가족력이없는건강한사람들을대상으로하였다. 그외의기준은환자군과동일하게적용하였다. 연구대상군에서고혈압, 당뇨및고지혈증등의진단은검사할당시의상태를기준으로하였다. 2. 생화학적분석 채혈전 12 시간이상공복후아침에환자의상박에서정맥채혈을하였다. 전혈은 citrate salts 가포함된 vacutainer tube 에담아, 얼음으로냉각후즉시 2,000 rpm 으로 5 분동안원심분리하여혈장을 0~4 로냉장 보관하였다. 혈중호모시스테인치의정량은혈장내호모시스테인치를 IMx kit (Abbott Laboratories) 를사용하여형광편광면역검사법 (fluorescence polarization immunoassay; FPIA) 으로측정하였다. 엽산 (folate) 과비타민 B12 는같은혈액에서채취하여자동화학발광분석기인 ACS: 180 (Bayer) 을이용하여경쟁적면역검사 (competitive immunoassay) 의원리로측정하였다. 3. 유전자분석 MTHFR 의유전자변이는다음과같은방법으로검사하였다. 먼저 DNA 분리는 DNA 추출 kit (extraction column, QIAmp blood kit, Qiagen) 에의해제조자의프로토콜에따라환자의혈액에서추출한백혈구로부터분리하였다. 분리한 DNA 표본은시발체세트 (primer set) 인 sense primer (5'-TGAAGGAGAAGGTGTCTG CGGGA-3') 와 antisense primer (5'-AGGACGGTGCG GTGAGAGTC-3') 를사용하여 GeneAMp PCR machine (Perkin Elmer 9600) 으로증폭시켰다. 198 bp 생성물을증폭시키기위해 95 에서 60 초동안변성시킨후 62 에서 90 초동안시발체를아닐링 (annealing) 한다음 72 에서 60 초동안시발체연장반응을시행하는과정을 35 회반복하였다. 증폭된단편들에서 677C T 변이를인식할수있는제한효소 HinfI (10 unit/reaction mixture) 으로 37 에서 3~4 시간동안분해하였다. A (Ala) 대립유전자 (allele) 에서얻어진 198 bp 단편은 HinfI 로분해되지않은반면에 V (val) 대립유전자에서얻어진같은길이의단편들은 175 bp 와 23 bp 단편으로분해되었다. 이후 HinfI 으로처리한단편들은 2.5% agarose gel 로전기영동한후 ethidium bromide 로염색하여유전자형을관찰하였다. 4. 통계분석 통계패키지로는 SPSS for windows, version 11.0 통계프로그램을이용하여자료를분석하였으며환자군과대조군의일반적인특성을알아보기위하여기술통계량을구하였다. 대조군과각질환군사이에유전자형이나대립유전자의빈도차이는 chi-square 검정을하여 p 값이 0.05 미만인경우를통계학적으로유의한것으로판정하였다. 연속형자료 ( 혈중호모시스테인, 엽산, vitamin B12, total cholesterol, triglyceride 농도 ) 에는 twosample t-test 을사용하였다. MTHFR 유전자다형성에따른각질환군과대조군에서호모시스테인과의관련성을알아보기위하여상관분석을하였으며, 비교위험도를측정하기위한다변량분석을위해서로지스틱회귀분석을하였고가능한연령과성별에대해서는조정을하여 odds ratio 와 95% 신뢰구간을구하였다. 모든자료는평균 ± 표준편차로표시하였다. 22 권 3 호대한신경과학회지 201
서정호김현조이일형김옥준최병옥 결과 두통환자군은모두 148 명 ( 남자 56 명, 여자 92 명 ) 이었고이중편두통은 73 명 ( 남자 25 명, 여자 48 명 ), 긴장형두통은 75 명 ( 남자 31 명, 여자 44 명 ) 이었으며, 정상대조군은 121 명 ( 남자 52 명, 여자 69 명 ) 이었다. 이들의일반적인특성은 Table 1 에나타나있는데, 혈액학적검사상편두통및긴장형두통환자군과정상대조군에서엽산, 비타민 B12, total cholesterol 과 triglyceride 의농도사이에는유의한차이를보이지않았다. MTHFR C677T 유전자다형성을분석하였을때, 동형접합성변이유전자형 (677TT) 은편두통환자군에서 30.1%, 긴장형두통 13.3%, 그리고대조군에서는 12.4% 의빈도를보여, 편두통환자군에서대조군에비해유의하게높은빈도를보였고긴장형두통환자군에서는유의한차이를보이지않았다 (p<0.05). 혈중호모시스테인의농도는두통환자군 ( 긴장형두통및전체편두통, 전조성편두통, 무전조성편두통 ) 을정상대조군과비교하였을때전조성편두통환자군 (AOR, 1.12; 95% CI, 1.02~1.23) 에서만유의한차이를보였다 (Table 2). 저자들은긴장형두통환자군을대조군으로하여편두통환자군과비교하는시도를해보았다. 이때전체편두통환자군 (AOR, 1.13; 95% CI, 1.01~ 1.26) 과전조성편두통환자군 (AOR, 1.39; 95% CI, 1.15~ 1.69) 에서긴장형두통환자군에비해유의한차이를보인다는사실을발견하였다. 편두통및긴장형두통환자군과정상대조군에서 MTHFR 유전자다형성을비교하였다 (Table 3). MTHFR 이형접합성변이유전자형 (677CT) 과정상유전자형 (677CC) 을비교해보았을때는유의한차이를발견할수없었다. 그러나동형접합성변이유전자형을정상유전자형과비교하였을때, 전체편두통환자군 (AOR, Table 1. Baseline characteristics in patients with migraine and tension headache, and genotype distributions and allele frequencies of the MTHFR gene Controls TH Migraine TM MWA MOA Age (years) 49.3±8.1 48.8±14.3 40.7±9.9 41.7±11.5 40.7±10.0 No. of patients 121 75 73 34 39 Female (ratio) 0.57 0.59 0.66 0.65 0.67 Cholesterol (mg/dl) 197±44 197±41 204±38 217±33 198±35 Triglyceride (mg/dl) 138±83 148±86 145±93 167±113 134±77 Vit.B12 (pg/dl) 683±591 678±410 601±193 622±231 589±145 Folate (ng/dl) 10.6±8.2 11.2±6.5 9.4±4.3 10.5±5.6 7.8±3.3 MTHFR genotype (%) 677 CC 677 CT 677 TT 46 (38.0) 60 (49.6) 15 (12.4) 31 (25.6) 34 (45.3) 10 (13.3) 27 (37.0) 24 (32.9) 22 (30.1) 10 (29.4) 11 (32.4) 13 (38.2) 17 (43.6) 13 (33.3) 9 (23.1) t allele ratio 0.37 0.36 0.47 0.54 0.40 Values are mean±sd, TH; tension headache, TM; total migraine, MW; migraine with aura, MOA; migraine without aura Table 2. Homocysteine levels in controls and patients include TH, TM, MWA, and MOA Controls (n=121) TH (n=75) TM (n=73) MWA (n=34) MOA (n=39) Homocysteine (µmol//l) OR (95% CI)* OR (95% CI) 9.15±4.46 9.02±2.29 10.24±5.69 10.98±4.05 9.59±6.78 1.00 0.99 (0.91~1.07) 1.07 (0.99~1.15) 1.12 (1.02~1.23) 1.01 (0.94~1.09) 1.00 1.13 (1.01~1.26) 1.39 (1.15~1.69) 1.04 (0.94~1.15) Values are mean±sd, Two-sample t-test for the continuous data *OR (95% CI); odds ratio and 95% confidence intervals, adjusted for age and sex, comparisons of homocysteine levels between controls and patients (TH, TM, MWA, MOA), OR (95% CI); odds ratio and 95% confidence intervals, adjusted for age and sex, comparisons of homocysteine levels between tension headache and migraine patients (TM, MWA, MOA), p<0.01 202 대한신경과학회지 22 권 3 호
전조성편두통과호모시스테인및 MTHFR C677T 유전자다형성의상관관계 Table 3. Risk assessment of the MTHFR C677T polymorphism compared with between patients, such as TH, TM, MWA, MOA and controls Controls TH Migraine TM MWA MOA 677 CC vs CT OR (95% CI) 1.00 0.70 (0.38~1.30) 0.70 (0.34~1.45) 0.77 (0.29~2.07) 0.64 (0.26~1.57) 677 CC vs TT OR (95% CI) 1.00 0.93 (0.37~2.32) 3.52 (1.30~9.55)* 4.70 (1.44~15.29) 2.77 (0.78~9.74) OR (95% CI); adjusted odds ratio and 95% confidence intervals, adjusted for age and sex. *p<0.05, p<0.01. Table 4. Risk assessment of the MTHFR C677T polymorphism compared with between migraine and tension headache patients TH Migraine TM MWA MOA 677 CC vs CT OR (95% CI) 1.00 1.04 (0.48~2.26) 1.19 (0.43~3.28) 0.91 (0.36~2.30) 677 CC vs TT OR (95% CI) 1.00 2.77 (1.04~7.37)* 4.01 (1.29~12.44)* 1.72 (0.52~5.69) OR (95% CI); adjusted odds ratio and 95% confidence intervals, adjusted for age and sex. *p<0.05. 3.52; 95% CI, 1.30~9.55; p<0.05), 특히전조성편두통환자군 (AOR, 4.70; 95% CI, 1.44~15.29; p<0.01) 에서는뚜렷하게유의한차이를보였으나긴장형두통및무전조성편두통환자군에서는유의한차이가없었다. 저자들은정상대조군대신긴장형두통환자군을대조군으로하여편두통환자군에서 MTHFR 유전자다형성을비교해보았다 (Table 4). 동형접합성변이유전자형을정상유전자형과비교하였을때무전조성편두통환자군에서는유의한차이가없었으나전조성편두통환자군 (AOR, 4.01; 95% CI, 1.29~12.44) 과전체편두통환자군 (AOR, 2.77; 95% CI, 1.04~7.37) 에서유의한차이가관찰되었다. 이형접합성변이유전자형과정상유전자형을비교하였을때에는정상대조군을대상으로한경우와같이유의한차이를보이지않았다. 또한전조성편두통환자군과정상대조군의비교에서성별과연령을조정하지않은것과각각을조정한경우를 Table 5 에나타내었다. 동형접합성변이유전자형과정상유전자형과비교에서연령과성별을조정하지않은것, 연령만조정한것, 성별만조정한것, 그리고성별과연령을조정한모두에서유사한결과를보여주고있다 (p<0.01). 그리고동형접합성변이유전자형과동형접합성변이가아닌모든경우, 즉정상유전자형과이형접합성변이형을합한경우의비교에서도모두유의한차이를보였으며, 이결과는동형접합성변이유전자형과정상유전자형을비교한결과와같았다. 고찰 편두통환자군과정상대조군에서 MTHFR C677T 유전자다형성및혈중호모시스테인농도의상관관계를알아보기위해서편두통환자군을전조성편두통과무전조성편두통으로분류 비교해보았을때, 전조성편두통환자군에서는동형접합성변이유전자형및혈중호모시스테인농도가정상대조군에비해서유의하게높다는사실을알수있었다. 그런데무전조성편두통환자군에서는정상대조군에비해 MTHFR 유전자다형성및혈중호모시스테인농도에서유의한차이를보이지않았다. Hering-Hanit 등 12 은전체편두통환자군과정상대조군을비교하였을때혈중호모시스테인의농도에차이가없다고하였으며, Evers 등 5 은무전조성편두통과긴장형두통에서호모시스테인이증가되지않은데반하여전조성편두통환자에서증가된소견을발견하고전조성편두통과고호모시스테인혈증과연관될것으로보고하였다. 또한 Kowa 등 4 은 MTHFR 동형접합성유전자변이가편두통, 특히전조성편두통에서하나의유전적요인임을주장하였으며, Russel 과 Olesen 13 은가족연구를통해서전조성편두통과무전조성편두통이서로다른원인에의해발생될수있음을시사하였는데이들은모두본연구의결과와일치하고있다. 이와같은사실들은전조성편두통과무전조성편두통이서로다른유전적및생화학적병인을가지는질환일가능성을시사하고있다. 22 권 3 호대한신경과학회지 203
서정호김현조이일형김옥준최병옥 Table 5. Association in each MTHFR polymorphism between migraine with aura and controls MTHFR genotype 677 CC vs CT 677 CC vs TT 677 CC+CT vs TT Crude OR Model 1 Model 2 Model 3 0.84 (0.33~2.16) 0.78 (0.29~2.09) 0.84 (0.33~2.16) 0.77 (0.29~2.07) 3.99 (1.45~10.94)* 4.98 (1.57~15.82)* 4.01 (1.44~11.14)* 4.70 (1.44~15.29)* 4.37 (1.82~10.52)* 5.39 (2.02~14.39)* 4.37 (1.81~10.54)* 5.30 (1.94~14.48)* Model 1 was the odds ratio (95% confidence intervals) which was adjusted for age, model 2 was the OR adjusted for sex, model 3 was the OR adjusted for age+sex. *p<0.01. 동형접합성변이를동형접합성변이형이아닌모든경우, 즉정상형과이형접합성변이형을합한경우와비교하였을때에도같은결과가나왔다 (Table 5). 따라서전조성편두통환자군에서는 MTHFR 동형접합성변이유전자형이정상유전자형에대해서뿐만아니라, 동형접합성변이형이아닌모든경우에대해서도유의한차이를보인다는것을알수있었다. 그러므로 677TT 유전자형을가진사람은정상대조군에대해서뿐만아니라동형접합성변이를가지지않은모든사람들에비해서도전조성편두통을가질가능성이유의하게높다는것을의미한다. 저자들은편두통환자군을정상대조군이아닌긴장형두통환자군을대조군으로삼아같은분석을하는새로운시도를해보았다. 긴장형두통환자군은 MTHFR 유전자다형성및혈중호모시스테인농도에서정상대조군과비교할때유의한차이를보이지않았다. 또한전조성편두통환자군은 MTHFR 유전자다형성및호모시스테인농도에서긴장형두통환자군에비해서유의한차이를보였지만무전조성편두통환자군은유의한차이를보이지않았으며, 이는정상대조군을대상으로했을때의결과와일치하고있다. 그러므로긴장형두통환자군은 MTHFR C677T 유전자다형성및혈중호모시스테인농도검사에서는정상대조군과같은특징을보인다고생각된다. 편두통은높은유병률에도불구하고병태생리학적기전이아직도정확하게알려져있지않다. 뇌혈관의혈관확장이편두통의발생기전일것이라는혈관성이론 (vascular theory) 과삼차신경을자극하였을때뇌의경막에분포하는혈관에신경인성염증을유발하여편두통을발생한다는이론 (trigeminovascular theory) 등이알려져있다. 14-16 또한, 편두통은 70~80% 에서가족력을동반하는질환으로유전적요인에영향을받을것이라는가능성이오래전부터제시되어왔으며, 최근의유전자연구는이러한가능성을뒷받침해주고있다. 17,18 가족성편마비성편두통 (familial hemiplegic migraine; FHM) 의원인유전자로 19번염색체에위치하는 Ca 2+ Channel 유전자인 CACNL1A4 가밝혀져있는데, 이유전자가관여하는 P/Q type Ca 2+ Channel 은 serotonin (5-HT) 과같은신경전달물질의방출을조절하는것으 로알려져있다. 17,18 Storer 와 Goadby 19 는뇌혈관통증의모델로고양이를사용하여뇌혈관에서호모시스테인과유사한작용을하는 DL-homocysteic acid 가삼차신경원흥분을촉진시키고 ergometrine 과 5-HT 1B/1D 길항제에의해억제됨을발견하였으며, 이와같은결과는고호모시스테인혈증이경막이나혹은뇌혈관에민감하게작용하여편두통을일으키는소인일것을시사하고있다. 호모시스테인은필수아미노산인메티오닌 (methionine) 으로부터시스테인 (cysteine) 이합성되는과정에서형성되는중간대사물질이다. 20 호모시스테인농도의증가는호모시스테인의대사와관련된효소의유전적인결함이나비타민 B12 및엽산결핍등이원인으로알려져있다. 10,11 이중호모시스테인대사과정에관여하는효소의유전적인결함에는 cystathionine β-synthase, methionine synthase 및 MTHFR 등의결핍이나활성도저하가고호모시스테인혈증을유발하는데중요한역할을하는것으로알려져있다. 20,21 MTHFR 은호모시스테인이메티오닌으로재메틸화하는과정에서메틸기공여자인 N 5- methylenetetrahydrofolate 가 N 5,N 10 -methylenetetrahydrofolate 로부터환원하는데작용하는효소로 MTHFR 의활성도는이효소의유전자유형에따라차이가나며, 특히동형접합성변이형에서혈중호모시스테인의농도가유의하게높다고알려져있다. 21 저자들은전조성편두통과무전조성편두통이서로다른유전적및생화학적병인을가지는질환일가능성및편두통환자군에서 MTHFR 677TT 유전자변이가전조성편두통의유전적요인중의하나일가능성을제시하였다. 그런데본연구는전향적인연구 (cohort study) 가아니라 hospital based case control study 라는제한점을가지고있으므로, 앞으로보다광범위한지역을대상으로다수의환자들을대상으로한전향적인조사가필요할것으로생각된다. REFERENCES 1. Headache Classification Committee of the International Headache Society. Classification and diagnostic criteria for headache disorders, cranial neuralgias and facial pain. Cephalalgia 1988;8:1-96. 204 대한신경과학회지 22 권 3 호
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