대한진단검사의학회지 : 제 23 권제 4 호 2003 Korean J Lab Med 2003; 23: 234-41 임상화학 에스트로젠수용체유전자의다형성이골표지자와지질치에미치는영향 최현식 이난영 원동일 이정범 1 송정흡 2 송경은 경북대학교의과대학진단검사의학교실, 가정의학과교실 1, 경북대학교병원산업의학과 2 Effects of Estrogen Receptor Polymorphisms on Bone Markers and Serum Lipid Levels Hyun Sik Choi, M.D., Nan Young Lee, M.D., Dong Il Won, M.D., Jung Bum Lee, M.D. 1, Jung Hup Song M.D. 2, and Kyung Eun Song, M.D. Departments of Laboratory Medicine and Family Practice 1, Kyungpook National University School of Medicine; Department of Occupational Medicine 2, Kyungpook National University Hospital, Daegu, Korea Background : In post-menopausal women, osteoporosis and cardiovascular diseases which are partly due to estrogen deficiency, occur more common than in pre-menopause women. Estrogen action is supposed to be mediated by an estrogen receptor (ER) and two polymorphisms of the ER gene in particular, Pvu II and Xba I, have been described for several years for genetic association studies. Authors have investigated the frequencies and patterns of the ER gene polymorphisms and their association with bone markers and lipid levels. Methods : For 121 women who visited the health promotion center of Kyungpook National University Hospital, the ER gene polymorphisms were determined by the Pvu II and Xba I restriction enzymes following polymerase chain reaction. Results : The distributions of ER Pvu II and Xba I restriction fragment length polymorphisms were as follows: PP 15.7%, Pp 47.9%, pp 36.4% and XX 5.8%, Xx 31.4%, xx 62.8%, respectively. And in a combination of two polymorphisms, ppxx was the most common, followed by PpXx, Ppxx, PPXx, PPXX and PPxx in that order. No significant genotypic differences were found in bone mineral density, bone markers and menopausal status. LDL cholesterol and triglyceride levels were significantly different by genotypes in premenopausal women (P<0.05). Conclusions : The results suggest that ER polymorphisms might be associated with LDL cholesterol and triglyceride levels. Further evaluation in a larger population would be helpful to determine the effects of ER polymorphisms on lipid metabolism and therapeutic trial for cardiovascular diseases in women. (Korean J Lab Med 2003; 23: 234-41) Key Words : Osteoporosis, Estrogen Receptor gene polymorphism, Bone marker, Lipid 서 론 골다공증은골양이저하되고골조직의미세구조가약화되어 골절의위험이증가되므로오늘날과같은노령화사회에서는심각 접수 : 2002년 11월 11일접수번호 : KJCP1626 수정본접수 : 2003년 7월 29일교신저자 : 송경은우 700-721 대구광역시중구삼덕 2가 50 경북대학교의과대학진단검사의학교실전화 : 053-420-5294, Fax: 053-426-3367 E-mail: kesong@knu.ac.kr 한사회적질환이다 [1]. 특히폐경후의여성에서는에스트로젠이감소함에따라골흡수의증가와골소실이더욱가속화되므로에스트로젠대체요법이폐경후골소실의예방과치료에이용되고있다 [2, 3]. 에스트로젠수용체는인간의조골세포와파골세포에모두존재하며 [4, 5] 에스트로젠수용체유전자는골다공증발병기전의유전적인자중의하나로인식되어있다 [6-8]. 또한폐경전여성에비해폐경후여성에서는관상동맥질환이더욱빈번히일어나며 [9] 호르몬대체요법을받은폐경후여성에서관상동맥질환에대한위험이감소되는데 [10], 이는에스트로 234
에스트로젠수용체유전자다형성과골표지자및지질치 235 젠이심장에대해보호역할을하기때문이며에스트로젠수용체 1, 2를통해매개된다고보고하였다 [11]. 이에따라폐경후여성에서에스트로젠수용체유전자가심혈관질환이나지질에미치는효과에대한연구들 [12-15] 이새로운관심사로대두되었다. 에스트로젠수용체는와, 두개의아형을가지고있다. 에스트로젠수용체 1으로도알려진에스트로젠수용체는염색체 6q25.1에위치해있으며보고된유전자의여러다형성 [15] 중에서특히Pvu II와 Xba I에의한다형성은가장많이연구되고있다. Pvu II 다형성은 exon 2로부터약 0.4 kb 지점에위치해있고 intron내의 T/C 전위에의해생긴것이며 [16], Xba I 다형성은 intron내에 Pvu II로부터 50 bp 떨어진곳에위치하고 G/A 전위에의한것으로알려져있다 [17]. 이수용체들에대해유방암 [18], 자궁내막암 [19], 초경 [20] 등뿐만아니라골다공증이나심혈관질환과의관련성에대한보고가많은데국내에서는에스트로젠수용체유전자의다형성과골다공증에대한연구는있으나 [21, 22] 지질과의관련성에대해서는아직보고된바가없다. 이에저자들은경북대학교병원건강증진센터를방문한여성들을대상으로에스트로젠수용체의유전자다형성의양상과빈도를조사하여보았고, 이들과골다공증을반영하는골표지자들과의관련성및혈청지질치에미치는영향을살펴보았다. 재료및방법 1. 대상이번연구는경북대학교병원건강증진센터를방문한건강한여성 200명을대상으로하였다. 병력조사상자궁절제술혹은양측성난소절제술을받았거나경구피임약, 지질강하제, 또는골대사에영향을미치는에스트로젠, 비타민 D, 칼슘제등을복용중인환자, 그리고간질환, 신장질환, 당뇨병등다른질병이있는환자들을제외한 121명을연구대상으로선정하였다. 이들을생리상태를기준으로폐경전, 후로나누었을때폐경전여성은 59명으로평균나이는 42.03 ±6.53세이었고폐경후는 62명으로 60.0 ± 6.63세이었다. 공복시에채혈을하였는데항응고제 EDTA를처리한혈액은 DNA 추출에사용하였고항응고제를처리하지않은혈액은응고후혈청을분리하여검사를시행할때까지 -80 에보관하였다. 2. 에스트로젠수용체유전자다형성분석에스트로젠수용체유전자다형성검사는 EDTA를처리한혈액을 Wizard genomic DNA purification kit (Promega, Madison, WI, USA) 을사용하여 DNA를추출한후 Lau 등 [23] 과 Vandevyver 등 [24] 의방법을변형한제한효소분절길이다형성 (RFLP, Restriction Fragment Length Polymorphism) 방법을 1 2 3 4 5 6 7 8 9 10 Fig. 1. Genotype analysis of ER Pvu II and Xba I restriction fragment length polymporphims (RFLPs). Lane 1 and 6, size markers; 2 and 3, XX; 4, Xx; 5, xx; 7 and 8, PP; 9, Pp; 10, pp. 시행하였다. 시발체는 sense 시발체 5-CTGCCACCCTATCTGTATCTT- TTCCTATTCTCC-3, antisense 시발체 5-TCTTTCTCTGC- CACCCTGGCGTCGATTATCTGA-3 를사용하였는데국내 Bioneer사에의뢰하여제조하였다. 중합효소연쇄반응은 Accupower PCR premix (Bioneer사, 한국 ) 를사용하여증류수 17 L에 20 pmol/ L의농도로맞춘 sense와 antisense 시발체각각 1 L씩과 DNA 1 L를혼합하였다. GeneAmp 9600 thermal cycler (Perkin Elmer, Norwalk, CT, USA) 를이용하여 94 에서 3분간반응시킨후 94 에서 30초간변성 (denaturation), 62 에서 20초동안소환 (annealing), 72 에서 1분 30초간연장 (extension) 하는조건으로총 35회의반응을반복하였으며이후 72 에서 6 분간연장반응을시켰다. 먼저이반응물 5 L로 2% 한천젤에서전기영동하여 1.3 kb의증폭산물을확인하였다. 제한효소분절길이다형성은중합효소연쇄반응산물중 10 L씩을취하여각각제한효소 Pvu II와 Xba I (BioLabs Inc., Beverly, MA, USA) 으로 37 에서 3시간반응시킨후 2% 한천겔에서전기영동하였다. Pvu II나 Xba I에의해잘리지않는경우에는 1.3 kb에 band가보이게되며이때각각 P, X로표기하고, 잘리는경우각각 p, x로표기하였다 (Fig. 1). 3. 골대사의생화학적표지자검사 혈청 osteocalcin과제 1형콜라젠카르복시텔로펩티드의골기원분해산물 (bone-derived degradation products of type I collagen carboxy-telopeptide, CTX) 은 Elecsys 2010 (Hitachi Boehringer Mannheim, Tokyo, Japan) 을이용하여측정하였다. 4. 혈청지질검사 총콜레스테롤과중성지방은효소법으로, LDL- 콜레스테롤과
236 최현식 이난영 원동일외 3 인 HDL-콜레스테롤은직접측정법으로 Hitachi 7060 (Hitachi Ltd., Tokyo, Japan) 을사용하여측정하였다. 5. 골밀도검사와 BMI (body mass index) L2에서 L4까지요추의골밀도검사는 Lunar Dual Energy X-ray absorptiometry (Lunar Corp., Madison, WI, USA) 로시행하였고 BMI는체중을키의제곱으로나누어계산하였다 (kg/m 2 ). 6. 통계처리모든검사치는평균 ± 표준편차로나타내었으며폐경전, 후군에서의에스트로젠수용체유전자다형성빈도의비교는 Chi-square test를사용하였고유전자형에따른차이는 Kruskal-Wallis test와 ANOVA, multirange test를사용하였다. 각분석은 SPSS 프로그램 (version 10.0, SPSS Inc., Chicago, IL, USA) 을이용하였으며통계적유의성은 p값 0.05 미만을기준으로검정하였다. 결 과 1. 에스트로젠수용체유전자다형성빈도 121명의전체대상자에서각대립유전자의빈도는 P가 39.7%, p가 60.3% 이었고 X가 21.5%, x가 78.5% 이었다. Pvu II 다형성은 PP가 19예 (15.7%), Pp가 58예 (47.9%), pp가 44예 (36.4%) 로 Pp가가장많았고, Xba I에의한다형성은 XX가 7예 (5.8%), Xx 가 38예 (31.4%), xx가 76예 (62.8%) 로 xx가가장많았다. 또한이들을조합한경우는 PPXX가 7예, PPXx가 10예, PPxx가 2예, PpXx가 28예, Ppxx가 30예그리고 ppxx가 44예였고 PpXX, ppxx와 ppxx는한예도없었다. Chi-square test로폐경전, 후군을비교해보았을때각대립유전자의빈도나유전자의다형성빈도에서는유의한차이가없었다 (Table 1). 2. 폐경전군에서에스트로젠수용체유전자의다형성에따른일반적특성, 골표지자및지질치의비교 폐경전군에서 Pvu II 와 Xba I 제한효소에의한각각의에스 Table 1. Distribution of estrogen receptor gene polymorphism n Pvu II Xba I Combination PP Pp pp XX Xx xx PPXX PPXx PPxx PpXx Ppxx ppxx Premenopausal 59 10 (16.9)* 32 (54.2) 17 (28.2) 2 (3.4) 22 (37.3) 35 (59.3) 2 (3.4) 6 (10.2) 2 (3.4) 16 (27.1) 16 (27.1) 17 (28.8) Postmenopausal 62 9 (14.5) 26 (41.9) 27 (43.5) 5 (8.1) 16 (25.8) 41 (66.1) 5 (8.1) 4 (6.5) 0 (0) 12 (19.4) 14 (22.6) 27 (43.5) Total 121 19 (15.7) 58 (47.9) 44 (36.4) 7 (5.8) 38 (31.4) 76 (62.8) 7 (5.8) 10 (8.3) 2 (1.7) 28 (23.1) 30 (24.8) 44 (36.4) *( ), % Table 2. The general characteristics, bone marker and lipid profiles for the Pvu II and Xba I restriction fragment length polymorphims (RFLPs) in premenopausal woemen Pvu II genotype PP (n=10) Pp (n=32) pp (n=17) Xba I genotype XX (n=2) Xx (n=22) xx (n=35) Age (years) 38.4±6.8* 42.8±6.3 42.7±6.5 40.0±1.4 41.2±7.2 42.7±6.5 Height (cm) 159.4±4.5 157.9±5.4 157.6±3.7 161.3±6.1 158.0±4.9 157.8±4.7 Weight (kg) 58.2±5.2 57.0±6.7 55.5±4.5 57.8±6.1 57.7±6.4 56.0±5.2 BMI (kg/m 2 ) 23.0±2.8 22.9±2.5 22.4±1.8 22.2±0.7 23.1±2.6 22.5±2.2 OC (ng/ml) 16.1±1.2 13.0±4.1 20.8±14.0 16.1±0.1 13.4±3.8 17.0±10.7 CTX (ng/ml) 0.26±0.03 0.17±0.13 0.26±0.27 0.24±0.01 0.17±0.07 0.23±0.23 BMD spine (g/m 2 ) 1.22±0.08 1.16±0.13 1.18±0.12 1.22±0.05 1.18±0.14 1.17±0.11 T-score -0.84±0.74-0.36±1.06-0.46±0.97-0.75±0.49-0.49±1.13-0.44±0.93 T-cholesterol (mg/dl) 178.9±23.7 194.3±34.8 179.1±28.6 165.1±5.7 181.1±30.7 190.8±33.3 LDL-cholesterol (mg/dl) 115.9±20.1 111.2±27.6 102.8±27.0 84.5±9.2 107.2±24.1 112.6±27.9 HDL-cholesterol (mg/dl) 53.1±11.1 61.0±14.4 61.2±9.2 65.6±21.2 56.5±12.9 61.5±12.2 Triglyceride (mg/dl) 88.0±42.5 106.2±103.1 65.4±21.3 63.5±43.1 115.3±116.6 76.8±38.4 *values are presented as the mean±sd, P<0.05 by Kruskal-Wallis test. Abbreviations: BMI, body mass index; OC, osteocalcin; CTX, degradation products of type I collagen C-telopeptide; BMD, bone mineral density; LDL, low density lipoprotein; HDL, high density lipoprotein.
에스트로젠수용체유전자다형성과골표지자및지질치 237 트로젠수용체유전자의다형성에따른일반적특성을살펴보면나이, 키, 체중등에서유의한차이가없었고, 골표지자 osteocalcin, CTX 및골밀도검사결과역시유의한차이가없었다. 지질치중에서는중성지방이 Pp가 106.2±103.1 mg/dl, Xx가 115.3± 116.6 mg/dl로이형접합체가동형접합체에비해유의하게높은치를보였다 (P<0.05, Kruskal-Wallis test)(table 2). Pvu II와 Xba I 제한효소에의한다형성의유형을조합하여보았을때는일반적특성이나골표지자는각군사이에유의한차이가없었다. 지질치중에서 LDL-콜레스테롤치가 PpXx가 101.9 ±26.2 mg/dl, Ppxx가 120.6±26.6 mg/dl, ppxx가 102.8±27.0 mg/dl로 PpXx와 Ppxx 사이에, Ppxx와 ppxx 사이에유의한 차이를보였으며 (P<0.05, ANOVA&multirange test)(table 3), Pp와 Xx에서유의하게높았던중성지방은이들을조합한 PpXx 에서다른다형성에비해높은치를보였으나통계학적으로유의한차이가없었다. 3. 폐경후군에서에스트로젠수용체유전자의다형성에따른일반적특성, 골표지자및지질치의비교 폐경후군에서 Pvu II와 Xba I 제한효소에의한각각의에스트로젠수용체유전자의다형성에따른일반적특성은나이, 체중등은차이가없었으나키는 Pp가 pp에비해, Xx가 xx에비해유 Table 3. The general characteristics, bone marker and lipid profiles for the combination of Pvu II and Xba I restriction fragment length polymporphims (RFLPs) in premenopausal woemen PPXX (n=2) PPXx (n=6) PPxx (n=2) PpXx (n=16) Ppxx (n=16) ppxx (n=17) Age (years) 40.0±1.4* 37.8±8.3 38.5±7.8 42.3±6.8 43.9±5.9 42.6±6.5 Height (cm) 161.3±6.1 157.7±4.6 162.4±1.9 158.0±5.2 157.8±5.7 157.6±3.7 Weight (kg) 57.8±6.1 59.0±6.1 56.3±2.1 56.9±6.6 57.0±7.0 55.5±4.5 BMI (kg/m 2 ) 22.2±0.7 23.8±3.4 21.4±1.3 22.8±2.4 22.9±2.7 22.4±1.8 OC (ng/ml) 16.1±1.0 16.2±1.7 16.1±0.1 12.9±3.9 13.3±4.8 20.8±14.0 CTX (ng/ml) 0.24±0.03 0.27±0.05 0.26±0.07 0.15±0.07 0.19±0.20 0.26±0.27 BMD spine (g/m 2 ) 1.22±0.05 1.23±0.11 1.21±0.04 1.15±0.13 1.18±0.12 1.18±0.12 T-score -0.75±0.50-0.90±0.94-0.75±0.35-0.23±1.11-0.49±1.03-0.46±0.97 T-cholesterol (mg/dl) 165.0±5.7 177.8±28.4 196.0±5.7 185.9±33.0 202.7±35.5 179.1±28.6 LDL-cholesterol (mg/dl) 84.5±9.2 121.5±13.1 130.5±10.6 101.9±26.2 120.6±26.6 102.8±27.0 HDL-cholesterol (mg/dl) 65.6±21.2 49.5±7.1 51.7±2.7 58.3±13.8 63.7±14.9 61.2±9.2 Triglyceride (mg/dl) 63.5±43.1 102.3±45.9 69.5±27.6 121.3±138.2 91.1±48.7 65.4±21.3 *values are presented as the mean±sd, P<0.05 by ANOVA and multirange test. Abbreviations: BMI, body mass index; OC, osteocalcin; CTX, degradation products of type I collagen C-telopeptide; BMD, bone mineral density; LDL, low density lipoprotein; HDL, high density lipoprotein. Table 4. The general characteristics, bone marker and lipid profiles for the Pvu II and Xba I restriction fragment length polymorphims (RFLPs) in postmenopausal woemen Pvu II genotype PP (n=9) Pp (n=26) pp (n=27) Xba I genotype XX (n=5) Xx (n=16) xx (n=41) Age (years) 62.1±6.6* 59.3±6.3 60.0±7.0 59.4±5.7 59.8±7.3 60.2±6.6 Height (cm 2 ) 153.5±4.6 155.8±3.9 152.5±4.4 154.6±4.2 156.2±4.5 153.2±4.2 Weight (kg) 58.0±11.7 57.4±5.3 53.7±7.2 58.8±13.6 58.9±6.7 54.3±6.5 BMI (kg/m 2 ) 24.5±4.3 23.6±1.9 23.1±2.7 24.6±5.7 24.1±2.2 23.1±2.4 OC (ng/ml) 28.6±10.4 22.4±8.7 21.0±9.0 22.3±8.8 25.0±9.2 21.8±9.4 CTX (ng/ml) 0.52±0.29 0.39±0.20 0.37±0.21 0.46±0.17 0.40±0.24 0.40±0.22 BMD spine (g/m 2 ) 0.96±0.27 0.95±0.15 0.90±0.14 1.05±0.30 0.97±0.20 0.90±0.13 T-score -1.3±2.3-1.45±1.29-1.84±1.41-0.54±2.5-1.25±1.64-1.87±1.08 Years since menopause 12.9±7.9 10.0±9.6 11.7±9.0 10.0±7.0 9.8±7.0 11.8±9.9 T-cholesterol (mg/dl) 214.9±58.1 203.7±21.1 207.6±31.2 214.6±81.1 200.4±24.1 208.6±26.6 LDL-cholesterol (mg/dl) 127.0±42.6 125.3±25.1 127.9±23.7 127.2±9.2 119.9±24.2 129.2±22.9 HDL-cholesterol (mg/dl) 49.4±11.1 50.7±9.3 52.1±14.3 43.0±7.9 51.9±8.9 51.8±12.9 Triglyceride (mg/dl) 145.7±73.6 132.7±78.7 123.3±62.5 165.0±84.6 136.8±82.2 123.9±64.2 *values are presented as the mean±sd, P<0.05 by ANOVA and multirange test. Abbreviations: BMI, body mass index, OC; osteocalcin; CTX, degradation products of type I collagen C-telopeptide; BMD, bone mineral density; LDL, low density lipoprotein; HDL, high density lipoprotein.
238 최현식 이난영 원동일외 3 인 Table 5. The general characteristics, bone marker and lipid profiles for the combination of Pvu II and Xba I restriction fragment length polymorphims (RFLPs) in postmenopausal women PPXX (n=5) PPXx (n=4) PpXx (n=12) Ppxx (n=14) ppxx (n=27) Age (years) 59.4±5.7* 66.0±5.8 57.0±6.3 60.4±5.9 60.0±7.0 Height (cm) 154.6±4.2 153.6±5.4 157.4±3.7 154.4±3.7 152.5±4.4 Weight (kg) 58.8±13.6 57.4±9.3 59.6±5.5 55.6±4.9 53.7±7.2 BMI (kg/m 2 ) 24.6±5.7 24.2±2.3 24.1±2.3 23.3±1.7 23.1±2.7 OC (ng/ml) 22.3±8.8 33.3±8.0 21.2±7.0 23.1±10.2 21.0±9.0 CTX (ng/ml) 0.45±0.17 0.54±0.35 0.34±0.16 0.44±0.23 0.37±0.21 BMD spine (g/m 2 ) 1.05±0.30 0.88±0.21 1.01±0.19 0.89±0.11 0.90±0.14 T-score -0.54±2.48-2.04±1.71-0.89±1.55-1.91±0.90-1.84±1.17 Years since menopoause 10.0±7.0 15.2±7.7 7.4±5.3 12.1±11.9 11.7±8.9 T-cholesterol (mg/dl) 214.6±81.1 211.6±15.4 195.4±26.2 210.6±14.6 207.6±31.2 LDL-cholesterol (mg/dl) 127.2±59.5 129.0±10.5 115.7±27.9 131.9±21.8 127.9±23.7 HDL-cholesterol (mg/dl) 43.0±7.9 54.5±10.4 50.7±8.4 51.2±10.3 52.1±14.3 Triglyceride (mg/dl) 165.0±84.6 117.0±52.1 145.7±93.6 124.9±69.9 123.3±62.5 *values are presented as the mean±sd, P<0.05 by ANOVA and multirange test. Abbreviations: BMI, body mass index; OC; osteocalcin; CTX, degradation products of type I collagen C-telopeptide; BMD, bone mineral density; LDL, low density lipoprotein; HDL, high density lipoprotein. 의하게컸다 (P<0.05, ANOVA & multirange test). 골표지자중 osteocalcin과 CTX 및골밀도검사결과에서통계학적인유의성은없었으며지질치에서도각군사이에유의한차이를보이지않았다 (Table 4). Pvu II와 Xba I 제한효소에의한다형성의유형을조합하여보았을때에도일반적특성중에키는 PpXx가 ppxx에비해유의한차이를보였으나 (P<0.05, ANOVA & multirange test), 골표지자및지질치가각군사이에유의한차이가없었다 (Table 5). 고찰성스테로이드호르몬중의하나인에스트로젠은생식계외에도골격계, 심혈관계등을포함한많은생리적과정에서중요한영향을미치고있으며 [7, 9] 특히폐경후의여성에서는에스트로젠치가감소함에따라골다공증이나관상동맥질환의발병이증가하게된다 [8, 11]. 저자들은 Pvu II와 Xba I 에스트로젠수용체유전자다형성이골밀도와지질치에미치는영향을조사하여보았다. 우선다형성의빈도를보면, Pvu II는저자의결과에서 Pp가 47.9%, pp가 36.4%, PP가 15.7% 의순이었는데같은한국인을대상으로한 Kim 등 [22] 과는일치하는소견이었으나 Han 등 [21] 은 pp를 14.2% 로가장낮게보고하였으며일본인 [25] 이나중국인 [26] 등의보고에서도 Pp가가장많고 PP가가장적었다. Xba I 에서는저자들의연구결과 xx가 62.8% 로가장많고 Xx가 31.4%, XX가 5.8% 순이었다. 다른연구자들의보고 [21, 27] 에서는 xx가가장많아저자들의결과와일치하는소견이었으나 Kim 등 [22] 은 Xx를가장많이보고하였다. Pvu II와 Xba I을조합하였을경우저자들의결과에서는 ppxx가가장많고다음이 PpXx였으며 PpXX, ppxx와 ppxx는한예도없었는데이는 Han 등 [21], Ushiroyama 등 [25] 과일치하는소견이었으나 Kim 등 [22] 은 PpXx를가장많다고하였다. 다형성의빈도가부분적으로조금씩차이가나는것은한등 [21] 과김등 [22] 이각각 598명, 229명으로저자들과는수적인차이를배제할수없으므로더연구할필요가있다고사료되었다. 폐경후군의일반적특성에서 Pp가 pp에비해, Xx가 xx에비해유의하게키가컸고 Pvu II와 Xba I을조합하였을경우 PpXx 가 ppxx에비해신장이유의하게차이가있음을알수있었는데다른연구에서도대체로 Pp나 Xx와같은이형접합체가 PP, pp나 XX, xx 등동형접합체에비해신장이크다고보고하였다 [26]. 에스트로젠수용체유전자다형성과골밀도및골표지자와의관계에서는저자의결과로는각다형성사이에유의한차이를발견할수없었는데이는여러연구자들 [21-26] 과일치되는소견이었다. 그러나에스트로젠수용체유전자의다형성과골밀도와의관계는보고자에따라상이한결과를보였는데 Kim 등 [22] 은 pp가 PP에비해요추골밀도가 7.5% 낮고 Xx가 xx에비해대퇴골경부의골밀도가 4.8% 낮아서에스트로젠수용체내에서도다른유전형이신체부위에따라다르게영향을미친다고주장하였고, Kobayashi 등 [17] 은폐경후여성에서 Px 일배체형 (haplotype) 이유의하게골밀도가낮아에스트로젠수용체유전자의변이가폐경후골다공증의원인중하나라고하였다. 그외에스트로젠수용체유전자가비타민 D 수용체유전자나 COLIA 1 유전자와상호작용하여골밀도에영향을미친다고주장한것처럼 [6], 각유전자의개별적인영향은상대적으로미약해도이들여러유전자들이복합적으로골밀도에영향을미치는지에대해서는앞으로동질의인구를대상으로한연구가필요하리라생각된다. 생화학적골표지자와의관련성에대해서도가로단면연구 (cross section study) 보다는코호트연구등세로연구 (longitudinal study) 를통한골소실연구가필요할것이다.
에스트로젠수용체유전자다형성과골표지자및지질치 239 에스트로젠이심혈관에대해보호작용을하는기전은혈관벽에직접작용하는것과지단백대사에영향을미치는것으로알려져있다. 에스트로젠은혈관평활근세포와혈관내피세포의증식을억제하고 [28] 세포벽의이온투과성을조절하거나내피세포에서유래되는물질들에작용하여혈관확장을유도하는데 [29], 이러한효과는에스트로젠수용체의변이에따라영향을받는다는주장이다 [30]. 지질에대해서는에스트로젠이간에있는 LDL 수용체의수를증가시키고혈중 LDL-콜레스테롤치를감소시키는데 [10] 쥐실험에서순수한항에스트로젠을투여했을때에스트로젠의 LDL 저하효과가억제되었다고하였다 [31]. 또한에스트로젠은간세포에서아포지단백 A-I의생산을증가시켜혈중 HDL-콜레스테롤이증가하게된다 [10, 32]. 실제로적극적인에스트로젠의투여가중성지방치의증가를방지한다거나 [33], 에스트로젠대체요법을시작한여성에서 LDL-콜레스테롤과아포지단백 B가유의하게감소하였고 HDL-콜레스테롤은증가하였다는보고가있다 [34]. 저자들의연구에서폐경전군에서 Pvu II와 Xba I을조합하였을경우각군사이에 LDL-콜레스테롤치가유의한차이를보였으나폐경후군에서는의의가없었고총콜레스테롤과 HDL-콜레스테롤은모든군에서차이가없었다. 에스트로젠수용체유전자다형성과지질의관계에대한연구는매우드문데 Kikuchi 등 [27] 이소아를대상으로조사한것을보면총콜레스테롤과 HDL-콜레스테롤은차이가없으나 LDL-콜레스테롤이 XX가 Xx나 xx에유의하게높아저자들과는상이한결과를보였다. 또한 Lu 등 [15] 은 PP와 XX가다른다형성군에비해 HDL-콜레스테롤이유의하게감소되었다고하였는데저자의결과에서도통계적인유의성은없었으나폐경후군에서 PP, XX, PPXX에서다른유전형에비해가장낮은치를보였다. 이번저자들의연구결과중에서특이한것은폐경전여성군에서 Xba I 다형성과 Pvu II 다형성모두중성지방치가 Pp와 Xx가각각다른군에비해유의하게높았는데다른보고에서는건강대조군과관상동맥질환자 [30], 소아 [27] 모두총콜레스테롤, HDL-콜레스테롤및중성지방치가다형성군사이에차이가없다고하였다. Herrington 등 [35] 이비록저자들과다른방법으로에스트로젠수용체유전자의다형성을분석하였지만, 에스트로젠대체요법을시행한후추적관찰하였을때에스트로젠수용체유전자의다형성에따라 HDL-콜레스테롤및성호르몬결합글로불린치의변화가유의한차이가있었다고보고한것은매우흥미로운것이다. 앞으로에스트로젠대체요법을시행하였을때에스트로젠수용체유전자의다형성에따라치료의반응에차이가있는지, 특히지질치의변화에차이가있는지에대한연구가필요할것이다. 또한지질과의관련성에대한기전이명확하게밝혀지고에스트로젠을치료적으로투여할때조직에서의에스트로젠수용체를연구하여이에선택적인수용체작용제를개발한다면에스트로젠대체요법시유전자다형성에따라치료를조절하는데도움이되리라생각된다. 결론적으로저자의결과에서는 121명의여성에서에스트로젠수용체유전자의다형성의빈도는 Pp와 xx가가장많았고, 폐경 전군에서중성지방과 LDL-콜레스테롤이유전자다형성에따라유의한차이를보였는데, 앞으로대상을더확대하고, 또한각다형성에따라경과를추적하여치료효과의차이를비교연구하여 LDL-콜레스테롤의대사에에스트로젠수용체유전자다형성의역할을명확하게밝힌다면, 지질강하제나에스트로젠대체요법을시행할때에스트로젠수용체유전자다형성검사결과가치료를조절하거나치료효과를판단하는데도움이되는좋은자료가될수있으리라생각한다. 요약배경 : 폐경후의여성에서는에스트로젠이감소함에따라골흡수의증가와골소실이더욱가속화되며또한관상동맥질환도폐경전의여성에비해더빈번히일어난다. 국내에서는에스트로젠수용체유전자의다형성과골다공증과의관련성에대한연구는있으나지질에대해서는아직보고된바가없다. 이에저자는경북대학교병원건강증진센터를방문한여성들을대상으로에스트로젠수용체의유전자의다형성양상과빈도를조사하였고이들이골표지자들과지질치에미치는영향을살펴보았다. 방법 : 에스트로젠수용체유전자다형성검사는 EDTA를처리한혈액으로 DNA를추출한다음중합효소연쇄반응을시행한후제한효소 Pvu II와 Xba I으로처리하는제한효소분절길이다형성 (RFLP, Restriction Fragment Length Polymorphism) 방법을사용하였다. Pvu II나 Xba I에의해잘리지않는경우에는 1.3 kb에 band가보이게되며이때각각p, X로표기하고, 잘리는경우각각 p, x로표기하였다. 결과 : 전체대상자 121 명중에서 Pvu II에의한다형성을보면 PP가 19예 (15.7%), Pp가 58예 (47.9%), pp가 44예 (36.4%) 였고, Xba I에의한다형성은 XX가 7예 (5.8%), Xx가 38예 (31.4%), xx가 76예 (62.8%) 였다. 또한이들을조합한경우는 PPXX가 7예, PPXx가 10예, PPxx가 2예, PpXx가 28예, Ppxx가 30예그리고 ppxx가 44예였고 PpXX, ppxx와 ppxx는한예도없었다. 일반적특정중폐경후군에서키가각군에따라유의한차이를보였으나골표지자는모든군사이에유의한차이가없었다. 지질치는총콜레스테롤치는에스트로젠수용체유전자다형성의각유형별로유의한차이를볼수없었으며, 폐경전군에서 LDL-콜레스테롤치과중성지방치가차이를보였다. 결론 : 에스트로젠수용체유전자의다형성에따라폐경전군에서는 LDL-콜레스테롤및중성지방치가, 폐경후군에서는키에차이가있음을알수있었으며앞으로우리한국인여성들을대상으로광범위한연구가이루어져에스트로젠수용체유전자의다형성과지질과의관련성을밝혀진다면에스트로젠대체요법시유전자다형성에따라치료를조절하는데도움이되리라생각된다. 참고문헌
240 최현식 이난영 원동일외 3 인 1. Gambert SR, Schultz BM, Hamdy RC. Osteoporosis. Clinical features, prevention, and treatment. Endocrinol Metab Clin North Am 1995; 24: 317-71. 2. Lindsay R, Hart DM, Forrest C, Baird C. Prevention of spinal osteoporosis in oophorectomised women. Lancet 1980; 2: 1151-4. 3. Stevenson JC, Cust MP, Gangar KF, Hillard TC, Lees B, Whitehead MI. Effects of transdermal versus oral hormone replacement therapy on bone density in spine and proximal femur in postmenopausal women. Lancet 1990; 336: 265-9. 4. Eriksen EF, Colvard DS, Berg NJ, Graham ML, Mann KG, Spelsberg TC, et al. Evidence of estrogen receptors in normal human osteoblastlike cells. Science 1988; 241: 84-6. 5. Pensler JM, Radosevich JA, Higbee R, Langman CB. Osteoclasts isolated from membranous bone in children exhibited nuclear estrogen and progesterone receptors. J Bone Miner Res 1990; 5: 797-802. 6. Brown MA, Haughton MA, Grant SF, Gunnell AS, Henderson NK, Eisman JA. Genetic control of bone mineral density and turnover: role of the collagen 1 1, estrogen receptor, and vitamin D receptor genes. J Bone Miner Res 2001; 16: 758-64. 7. Ralston SH. The genetics of osteoporosis. QJM 1997; 90: 247-51. 8. Zmuda JM, Cauley JA, Ferrell RE. Recent progress in understanding the genetic susceptibility to osteoporosis. Genet Epidemiol 1999; 16: 356-67. 9. Castelli WP. Epidemiology of coronary heart disease: The Framingham Study. Am J Med 1984; 76: 4-12. 10. Nasr A and Breckwoldt M. Estrogen replacement therapy and cardiovascular protection: lipid mechanisms are the tip of an iceberg. Gynecol Endocrinol 1998; 12: 43-59. 11. Barrett-Conor E and Bush TL. Estrogen and coronary heart disease in women. JAMA 1991; 265: 1861-7. 12. Binder EF, Williams DB, Schechtman KB, Jeffe DB, Kohrt WM. Effects of hormone replacement therapy on serum lipids in elderly women. a randomized, placebo-controlles trial. Ann Intern Med 2001; 134: 754-60. 13. Hong MK, Romm PA, Reagan K, Green CE, Rackley CE. Effects of estrogen replacement therapy on serum lipid values and angiographically defined coronary artery disease in postmenopausal women. Am J Cardiol 1992; 15: 176-8. 14. Windler E. Modification of serum lipids and cardiovascular risk by estrogenic active compounds. Gynecol Endocrinol 1999; 13(S6): 21-8. 15. Lu H, Higashikata T, Inazu A, Nohara A, Yu W, Shimizu M, et al. Association of estrogen receptor- gene polymorphisms with coronary artery disease in patients with familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 2002; 22: 821-7. 16. Yaich L, Dupont WD, Cavener DR, Parl FF. Analysis of the PvuII restriction fragment-length polymorphism and exon structure of the estrogen receptor gene in breast cancer and peripheral blood. Cancer Res 1992; 52: 77-83. 17. Kobayashi S, Inoue S, Hosoi T, Ouchi Y, Shiraki M, Orimo H. Association of bone mineral density with polymorphism of the estrogen receptor gene. J Bone Miner Res 1996; 11: 306-11. 18. Andersen TI, Heimdal KR, Skrede M, Tveit K, Berg K, Borresen AL. Oestrogen receptor (ESR) polymorphisms and breast cancer susceptibility. Hum Genet 1994; 94: 665-70. 19. Weiderpass E, Persson I, Melhus H, Wedren S, Kindmark A, Baron JA. Estrogen receptor alpha gene polymorphisms and endometrial cancer risk. Carcinogenesis 2000; 21: 623-7. 20. Stavrou I, Zois C, Ioannidis JP, Tsatsoulis A. Association of polymorphisms of the oestrogen receptor gene with the age of menarche. Hum Reprod 2002; 17: 1101-5. 21. Han K, Choi J, Moon I, Yoon H, Han I, Min H, et al. Non-association of estrogen receptor genotypes with bone mineral density and bone turnover in Korean pre-, peri-, and postmenopausal women. Osteoporos Int 1999; 9: 290-5. 22. Kim JG, Lim KS, Kim EK, Choi YM, Lee JY. Association of vitamin D receptor and estrogen receptor gene polymorphisms with bone mass in postmenopausal Korean women. Menopause 2001; 8: 222-8. 23. Lau EM, Young RP, Lam V, Li M, Woo J. Estrogen receptor gene polymorphism and bone mineral density in postmenopausal Chinese women. Bone 2001; 29: 96-8. 24. Vandevyver C, Vanhoof J, Declerck K, Stinissen P, Vandervorst C, Michiels L, et al. Lack of association between estrogen receptor genotypes and bone mineral density, fracture history, or muscle strength in elderly women. J Bone Miner Res 1999; 14: 1576-82. 25. Ushiroyama T, Heishi M, Higashio S, Ikeda A, Ueki M. The association between postmenopausal vertebral bone mineral density and estrogen receptor gene alleles in ethnic Japanese living in western Japan. Res Commun Mol Pathol Pharmacol 2001; 109: 15-24. 26. Bagger YZ, Jorgensen HL, Heegaard AM, Bayer L, Hansen L, Hassager C. No major effect of estrogen receptor gene polymorphisms on bone mineral density or bone loss in postmenopausal Danish women. Bone 2000; 26: 111-6. 27. Kikuchi T, Hashimoto N, Kawasaki T, Uchiyama M. Association of serum low-density lipoprotein metabolism with oestrogen receptor gene polymorphism in healthy children. Acta Paediatr 2000; 89: 42-5. 28. Vargas R, Wroblewska B, Rego A, Hatch J, Ramwell PW. Oestradiol inhibits smooth muscle cell proliferation of pig coronary artery. Br J Pharmacol 1993; 109: 612-7. 29. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects of estrogen. FASEB J 1996; 10: 615-24. 30. Matsubara Y, Murata M, Kawano K, Zama T, Aoki N, Yoshino H,
에스트로젠수용체유전자다형성과골표지자및지질치 241 et al. Genotype distribution of estrogen receptor polymorphisms in men and postmenopausal women from healthy and coronary populations and its relation to serum lipid levels. Arterioscer Thromb Vasc Biol 1997; 17: 3006-12. 31. Campos H, Walsh BW, Judge H, Sacks FM. Effect of estrogen on very low density lipoprotein and low density lipoprotein subclass metabolism in postmenopausal women. J Clin Endocrinol Metab 1997; 82: 3955-63. 32. Lamon-Fava S, Ordovas JM, Schaefer EJ. Estrogen increases apolipoprotein (apo) A-I secretion in hep G2 cells by modulating transcription of the apo A-I gene promoter. Arterioscler Thromb Vasc Biol 1999; 19: 2960-5. 33. Clarke SC, Schofield PM, Grace AA, Metcalfe JC, Kirschenlohr HL. Tamoxifen effects on endothelial function and cardiovascular risk factors in men with advanced atherosclerosis. Circulation 2001; 103: 1497-502. 34. Folsom AR, McGovern PG, Nabulsi AA, Shahar E, Kahn ES, Winkhart SP, et al. Changes in plasma lipids and lipoproteins associated with starting or stopping postmenopausal hormone replacement therapy. Atherosclerosis Risk in Communities Study. Am Heart J 1996; 132: 952-8. 35. Herrington DM, Howard TD, Hawkins GA, Reboussin DM, Xu J, Zheng SL, et al. Estrogen-receptor polymorphisms and effects of estrogen replacement on high-density lipoprotein cholesterol in women with coronary disease. N Eng J Med 2002; 346: 967-74.