원저 한국인에서제2 형당뇨병과 Kir6.2 및 Peroxisome Proliferator -activated Receptor-gamma (PPARγ) 유전자다형성의연관성 경북대학교의과대학내과학교실, 경북대학교의료전문서비스인력양성사업원 1, 계명대학교자연과학대학생물학과 2 이정은 김수원 1 서현애 전재한 문성수 김희경 도윤정 김보완 김정국 유민 2 이인규 Association of Kir6.2 and Peroxisome Proliferator-activated Receptor-gamma (PPARγ) Polymorphisms with Type 2 Diabetes in Koreans Jung Eun Lee, Su Won Kim 1, Hyun Ae Seo, Jae Han Jeon, Seong Su Moon, Hee Kyung Kim, Yun Jeong Doh, Bo Wan Kim, Jung Guk Kim, Min Yoo 2, In Kyu Lee Department of Internal Medicine, Kyungpook National University School of Medicine; Medical Education Program For Human Resources 1, Kyungpook National University; and Department of Biology 2, College of Natural Science, Keimyung University Abstract Background: The type 2 diabetes is a typical polygenic disease complex, for which several common risk alleles have been identified. Several variants may contribute significantly to the risk of type 2 diabetes conferring insulin resistance of liver, muscle and fat (Pro12Ala) and a relative insulin secretory deficiency (Glu23Lys). In this study, we evaluated the association of Pro12Ala variant of the peroxisome proliferatoractivated receptor-γ and the Glu23Lys variant of the ATP-sensitive potassium channel, Kir6.2 (KCNJ11) with the type 2 diabetes in Korean population. Method: This study included 331 subjects consisting of 172 patients with type 2 diabetes and 159 nondiabetic control subjects enrolled from the Kyungpook, Keimyung and Catholic university hospital in Daegu, Korea. We genotyped Kir6.2 (Glu23Lys) and PPARγ (Pro12Ala) polymorphism and examined their association with the type 2 diabetes. Result: In the separate analyses, the Kir6.2 Glu23Lys (P = 0.385) and the PPARγ Pro12Ala (P = 0.191) polymorphism showed no significant association with type 2 diabetes. In addition, the results of our study showed no evidence of a synergistic interaction between Kir6.2 and PPARγ gene in each group (P = 0.110, P = 0.276). Conclusion: In this study, no association was seen between the genetic polymorphisms of Kir6.2, PPARγ and type 2 diabetes. However, to clarify whether genetic polymorphisms of these genes contribute to the development of type 2 diabetes, further studies involving larger Korean populations may be needed. (J Kor Diabetes Assoc 31:455~464, 2007) Key Words: Kir6.2, Korean, Peroxisome Proliferator-activated Receptor-gamma (PPARγ), Type 2 Diabetes 서론제2형당뇨병은인슐린저항성과췌장베타세포의기능이상및이로인한혈당상승을특징으로하는대사성질환이며질환의발생은복합적인유전적경향을보인다 1-4). 제2형당뇨병이강한유전성을지닌다는근거로는제2형당뇨병에 서이란성쌍생아와비교하여일란성쌍생아간의당뇨병발병일치율이약 3.5배높다는사실과 1,2,5-9) 민족간의발병률에차이를보인다는점등을들수있다 7,8,10). 최근수년간새로운분자생물학적연구기법의발달과함께제2형당뇨병의발현과관련된원인유전자를발견하기위한많은연구결과가발표되었으며이분야에관심이고조되고있다 2,11,12). 그결 접수일자 : 2007 년 5 월 15 일, 통과일자 : 2007 년 10 월 2 일, 책임저자 : 이인규, 경북대학교의과대학내과학교실 455
과로제2 형당뇨병의발생에는많은유전자의다형성이관련되어있다는여러연구결과가보고되었다. 특히 Kir6.2 13-15), PPARγ (Peroxisome proliferator-activated receptor-gamma) 16-18), TCF7L2 (transcription factor 7-like 2) 19), CAPN10 (calpain -10) 20), PAI-1 (plasminogen activator inhibitor-1) 21), UCP (uncoupling protein)-1 22), APM1 (adiponectin) 23), AMPK (AMP-activated protein kinase) 24), MnSOD (manganese superoxide dismutase) 25) 등의유전자의다형성은제2형당뇨병의발생및합병증의악화와관련이있다. 그러나그결과는연구자마다다르며국가나각민족간에도상당히다른결과들이제시되고있다 26,27). 이중 Kir6.2와 PPARγ 유전자는제2형당뇨병발현과연관되어가장많은연구가이루어져있으며, 제2형당뇨병발생과관련이있다는결과들이많이발표되어있다. 베타세포의 ATP-sensitive 포타슘채널 (K ATP) 은인슐린분비조절에매우중요하다. 포도당이당수송체 (glucose transporter) 를통하여베타세포내로들어오면대사되어 ATP 가생성된다. 이로인하여베타세포내에 ATP/ADP 비가증가되면베타세포의 ATP-sensitive 포타슘채널 (K ATP) 이닫히게되고베타세포의세포막전위탈분극이일어난다 28,29). 세포막전위의탈분극은칼슘채널을활성화시켜칼슘을세포내로이동시키고인슐린분비를유발한다 30). K ATP 는 SUR1과 Kir6.2로구성된 heterooctamer로서 31,32), 이들유전자다형성과제2형당뇨병발생과의관련성에대한많은연구들이있다 1). 특히제11번염색제의 p15.1에위치한 KCNJ11에코딩된 Kir6.2 유전자의다형성중 E23K 변이는 cytosolic proximal N-terminal tail에위치한 glutamate (E) 가 lysine (K) 로변환된과오돌연변이 (missense mutation) 에의한유전적다형성으로 33), 최근에백인의제2형당뇨병환자에서가장높은비율로발견되어주목받고있다 1,26). 그러나 KCNJ11 E23K 유전자다형성과제2형당뇨병의연관성에대한결과가연구자마다다르며, 이는대상환자의선정이나인종간유전적차이와관련이있을것으로생각된다 27,33). Peroxisome proliferator-activated receptor (PPAR) 는여러호르몬에대한핵수용체군의하나로서지방세포의분화를조절하는전사요소로알려져있다 34,35). PPAR는 α, β, γ 세아형이있으며, 그중 PPARγ는지방세포에특이적이고, 지방세포의분화및지방산대사에관여하며당뇨병치료제인 thiazolidinediones가리간드로알려져있어제2형당뇨병및비만증과관련성이있는원인유전자일가능성이지적되어왔다 35-37). Yen 등 38) 은코카시안당뇨병환자를대상으로 PPARγ 유전자의 34번째염기배열에서 C G 치환되어그결과 12번째아미노산이 P A으로치환된과오돌연변이를찾아제2형당뇨병과유의한연관이있다고보고하였으나, 이결과도연구자 38-41) 나대상자들의인종에따라그결과가일치하지않았다 7,8,42,43). Kir6.2유전자는인슐 린분비에그관련성이중요한유전자이며, PPARγ 유전자는인슐린저항성과의관련성이중요하다고사료되는유전자이다. 제2형당뇨병발현은인슐린저항성및인슐린분비의상대적감소가그원인으로지적된다. 그러므로이두유전자는제2형당뇨병발생과관련이있다는결과들이많이발표되어있다. 그러나다수의여러인종에서제2형당뇨병의발생에있어 Kir6.2과 PPARγ와의관련성에관한논문은일치되지않은결과를보이고있다. 또한한국인을대상으로한연구결과는아직소수에불과하고, 결과또한일치하지는않았다 44,45). 이에본연구는한국인에서 KCNJ11 E23K 유전자다형성및 PPARγ P12A유전자다형성과제2형당뇨병환자에서 Kir6.2 및 PPARγ유전자다형성의빈도를알아보고자하였다. 대상및방법 1. 대상본연구는총 331명을대상으로하였고, 남자 112명, 여자 219명이였으며, 평균연령은제2형당뇨병환자군 55.0세, 대조군 47.1세였다. 제2형당뇨병환자군은 2006년 4월부터 2006년 8월까지경북대학병원과계명대학교동산의료원내분비내과를내원하였던환자중제2형당뇨병의과거력이있거나혈액검사상공복혈당이 126 mg/dl 이상인환자 172명을제2형당뇨병환자군으로정하였으며남자 76명, 여자 96명이였다. 대조군은 2005년 1월부터 2006년 1월까지경북대학교부설병원혹은대구가톨릭대학교부설병원건강검진센터에건강검진을목적으로내원하였던 1,427명중혈액검사상공복혈당이 100 mg/dl 미만이고, 뇌졸중, 협심증, 심근경색, 고지혈증, 결핵, 갑상선, 간질환, 고혈압, 천식, 관절염, 우울증, 파킨슨병, 골다공증, 전립선비대증, 암등의질병이없는 159명으로정하였으며, 남자 36명, 여자 123명이였다. 대상자에게시행된모든검사들은임상시험심사위원회 (Institutional Review Board, IRB) 의기준에맞게환자들의서면동의를받은후에이루어졌다. 2. 대상및연구방법 1) 설문조사및신체계측사회인구학적특성, 현병력, 과거력, 흡연력, 음주력은설문지를이용하여자기기입식방법으로작성되었다. 조사에사용된설문지는한국인만성병역학조사및유전체연구사업의설문지를참고하였다. 대상자는검사전날밤부터최소한 10시간이상금식한공복상태에서가벼운의복을착용하고신장과체중측정하였으며바른자세로시선은앞으로향하게한후신장과체중을동시에잴수있는자동신장체중계를이용하여측정하였다. 체질량지수 (body mass index, BMI = kg/m 2 ) 는측정한기본신체계측치로부터 456
이정은외 10 인 : 한국인에서제 2 형당뇨병과 Kir6.2 및 Peroxisome Proliferator-activated Receptor-gamma (PPARγ) 유전자다형성의연관성 산출하였다. 2) 임상검사제2형당뇨병의진단기준은세계보건기구 (World Health Organization, WHO) 진단기준 46) 을이용하였고, 혈액검사는전날밤부터최소한 10시간이상금식한공복상태에서대상자의상완정맥에서채혈하였다. 채혈한혈액을즉시분리하였고공복혈장포도당, 당화혈색소, 총콜레스테롤, 고밀도콜레스테롤, 저밀도콜레스테롤, 중성지방을측정하였다. 공복혈당은 Modular Analytics SWA (Roche Diagnostics GmbH, Mannheim, Germany) 를이용하여측정하였고, 당화혈색소, 총콜레스테롤, 고밀도콜레스테롤, 저밀도콜레스테롤, 중성지방은 Histachi Modular D2400 기계 (Roche, Tokyo, Japan) 를이용하였다. 3) 채혈및 DNA 분리채혈된혈액의일부는 EDTA (ethylenediamine tetra acetic acid) 처리된시험관에넣어냉장보관하였다가실험실로운반하여 DNA를분리하였다. DNA 분리는 Genomic DNA 분리키트 (Gentra) 를이용하였다. 혈액 2 ml에 6 ml 의적혈구용해용액을넣은후 5분간잘흔들어준다음 4 에서 3,500 rpm의속도로 5분간원심분리하고원심분리후상등액과분리하였다. Vortex로세포덩어리를잘풀어준후 2mL의세포용해용액을넣고 10초간 vortex로다시잘섞어준후실온에서 10분이상방치하였다. 0.7 ml의 protein precipitation 용액을넣고충분히 vortex한후 4 에서 3,500 rpm으로 10분간원심분리하여단백질을포함한모든불순물을가라앉힌후상등액을조심스럽게 2mL의 100% 이소프로필알코올용액에옮겨실모양의흰색 DNA 가보일때까지아래위로섞었다. 2,500 rpm으로 5분동안원심분리한후상등액은따라버리고, 가라앉은부분을 70% 에탄올로탈수시켰다. 150 μl DNA 수용액을넣어 6 5 에서 1~2시간배양시킨후사용할때까지 -20 에서보관하였다. 4) 유전자분석및유전자다형성검사유전자분석에사용된 DNA는말초혈액임파구에서분리하였고, real-time PCR (Light cycler (Roche Diagnostics, Germany) 을이용하였다. Kir6.2 및 PPARγ의유전적다형성을조사하기위해 real-time PCR (Light cycler (Roche, Germany) 을이용하였으며 PCR을위한 primer의염기서열은 Kir6.2는 sense primer, 5'-GGC ATC ATC CCC GAG GAA T-3', antisense primer, 5'-CCC TGC TCC CGG ATF TT-3' 였고, PPARγ는 sense primer, 5'-ATT TGG AAA CTG ATG TCT TGA CTC A-3', antisense primer, 5'-CAA TAG CCG TAT CTG GAA GGA AC-3' 였다. Kir6.2의 PCR 반응조건은배양전 (pre-incubation) 단계에서는 95 에서 10분, 증폭 (amplification) 단계에서는 95 에서 10초, 결합 (annealing) 온도에서 10초, 72 에서 8초를 55회반복, 융해 (melting curve) 단계는 95 에서 1분, 40 1분, 80 에서지속적인냉각 (continuous, cooling) 단계는 40 에서 30초로하였다. 또한, PPARγ의 PCR 반응조건은배양전 (pre-incubation) 단계에서는 95 에서 10분, 증폭 (amplification) 단계에서는 95 에서 10초, 결합 (annealing) 온도에서 10초, 72 에서 10초를 55회반복, 융해 (melting curve) 단계는 95 에서 1분, 42 1분, 80 에서지속적인냉각 (continuous, cooling) 단계는 40 에서 30초로하였다. 2 μm의 probe, 5 μm의 forward primer와 reverse primers, 1X PCR Master Mix Light Cycler 480 Genotyping Master, Roche), 10 ng DNA을포함하여총 5 μl씩반응시켰다. 3. 통계학적분석제2형당뇨병환자군과대조군사이의대립형질 (allele) 의빈도와유전자형 (genotype) 빈도의차이에대한통계분석은 SPSS version 15.0를사용하였다. 변수는평균 ± 표준편차로나타내었고각각의단일염기다형성이하디-바인베르크 (Hardy-Weinberg) 평형을따르는지를알아보기위해 chisquare 검정을이용하였으며 (P < 0.05), 다변량로지스틱회귀분석을이용하여각염기변이와제2형당뇨병과의연관성을알아보았고교차비 (Odds Ratio : OR) 과신뢰구간 (95% Confidence Interval: CI) 을계산하였다. 교차비와신뢰구간도대상자의특성이다르므로나이, 성별, BMI (kg/m 2 ), 흡연상태 ( 비흡연, 과거흡연, 현재흡연 ) 와음주상태 ( 비음주, 과거음주, 현재음주 ) 를보정하여계산하였다. 통계적유의수준은 P값 0.05 이하로판단하였다. 결과 1. 대상자의일반적특성및건강행태대조군과제2형당뇨병환자군대상자의평균연령은각각 47.1과 55.0세로제2형당뇨병환자군에서유의하게높게나타났으며남녀비는대조군에서여성이, 제2형당뇨병환자군에서는남성이유의하게많았다. 흡연상태는제2형당뇨병환자군이대조군에비해현재흡연자가유의하게많았고, 음주상태는제2형당뇨병환자군이대조군에비해과거음주자가많았고현재음주자는유의하게더적은것으로나타났다. 체질량지수 (BMI), 중성지방, 저밀도콜레스테롤은제2형당뇨병환자군에서의미있게높았으며, 고밀도콜레스테롤은대조군에서의미있게높았다. 총콜레스테롤은대조군과제2형당뇨병환자군사이에유의한차이가없었다 (Table 1). 457
Table 1. Dermographic and health behavior variables among study subjects Control (n = 159) Type 2 DM (n = 172) P Age (year) * 47.14 ± 5.14 55.02 ± 13.46 0.000 BMI (kg/m 2 ) * 22.38 ± 2.33 23.17 ± 3.54 0.017 Sex, n (%) 0.000 Male 36 (22.6) 76 (44.2) Female 123 (77.4) 96 (55.8) Smoking Status, n (%) 0.018 Nonsmoker 119 (74.8) 108 (62.8) Ex-smoker 24 (15.1) 28 (16.3) Smoker 16 (10.1) 36 (20.9) Drinking Status, n (%) 0.000 Nondrinker 79 (49.7) 103 (59.9) Ex-drinker 12 (7.5) 34 (19.8) Drinker 68 (42.8) 35 (20.3) Total cholesterol (mg/dl) * 182.64 ± 24.87 178.54 ± 53.16 0.378 High-density lipoprotein (mg/dl) * 60.23 ± 10.77 52.52 ± 23.98 0.000 Low-density lipoprotein (mg/dl) * 105.75 ± 22.47 113.83 ± 46.01 0.045 Triglyceride (mg/dl) * 83.45 ± 37.50 144.53 ± 101.90 0.000 * expressed as mean ± SD. P < 0.05. Table 2. Genotype and allele frequencies of Kir 6.2 gene in both group Polymorphism Amino acid Contol, n (%) T2D, n (%) P Crude OR Adjusted OR * (95% CI) (95% CI) G/G Glu/Glu 55 (34.6) 62 (36.0) 0.385 1 1 G/A Glu/Lys 75 (47.2) 88 (51.2) 1.041 (0.647-1.676) 0.811 (0.382-1.723) A/A Lys/Lys 29 (18.2) 22 (12.8) 0.673 (0.347-1.305) 0.527 (0.171-1.618) GG 55 (34.6) 62 (36.0) 0.782 1 1 GA + AA 104 (65.4) 110 (64.0) 0.938 (0.597-1.473) 0.742 (0.360-1.529) G allele 0.582 0.616 0.383 A allele 0.418 0.384 * odds ratio adjusted for age, sex, BMI, smoking status, drinking status. OR, odds ratio; T2D, type 2 diabetes mellitus. Table 3. Genotype frequencies of Kir 6.2 gene by gender in both group Sex Polymorphism Amino acid Control, n (%) T2D, n (%) P Crude OR Adjusted OR * (95 % CI) (95 % CI) Male G/G Glu/Glu 10 (27.8) 25 (32.9) 0.816 1 1 G/A Glu/Lys 19 (52.8) 39 (51.3) 0.821 (0.329-2.051) 0.756 (0.195-2.937) A/A Lys/Lys 7 (19.4) 12 (15.8) 0.686 (0.209-2.245) 0.141 (0.018-1.089) Female G/G Glu/Glu 45 (36.6) 37 (38.5) 0.293 1 1 G/A Glu/Lys 56 (45.5) 49 (51.0) 1.064 (0.596-1.900) 1.085 (0.394-2.990) A/A Lys/Lys 22 (17.9) 10 (10.4) 0.553 (0.233-1.313) 0.882 (0.183-4.259) * odds ratio adjusted for age, sex, BMI, smoking status, drinking status. OR, odds ratio; T2D, type 2 diabetes mellitus. 2. Kir6.2 유전자다형성과제 2 형당뇨병의연관성 E23K Kir6.2 유전자다형성의경우대조군과제 2 형당 뇨병환자군사이에서 GG, GA, AA유전자형 (genotype) 의분포는유의한차이를보이지않았고 (P = 0.385), 각군에서성별에따른유전자형의분포도유의한차이를보이지않았고 (P 458
이정은외 10 인 : 한국인에서제 2 형당뇨병과 Kir6.2 및 Peroxisome Proliferator-activated Receptor-gamma (PPARγ) 유전자다형성의연관성 Table 4. Genotype and allele frequencies of PPARγ gene in both group Polymorphism Amino acid Control, n (%) T2D, n (%) P Crude OR Adjusted OR * (95% CI) (95% CI) CC Pro/Pro 148 (93.1) 153 (89.0) 0.191 1 1 CG Pro/Ala 11 (6.9) 19 (11.0) 1.671 (0.769-3.631) 1.193 (0.390-3.648) GG Ala/Ala 0 0 C allele 0.965 0.945 0.262 G allele 0.035 0.055 * odds ratio adjusted for age, sex, BMI, smoking status, drinking status. OR, odds ratio; T2D, type 2 diabetes mellitus. Table 5. Genotype frequencies of PPARγ gene by gender in both group Sex Polymorphism Amino acid Control, n (%) T2D, n (%) P Crude OR Adjusted OR * (95% CI) (95% CI) Male CC Pro/Pro 33 (91.7) 67 (88.2) 0.575 1 1 CG Pro/Ala 3 (8.3) 9 (11.8) 1.478 (0.375-5.824) 0.922 (0.137-6.223) Female CC Pro/Pro 115 (93.5) 86 (89.6) 0.296 1 1 CG Pro/Ala 8 (6.5) 10 (10.4) 1.672 (0.633-4.413) 0.894 (0.202-3.953) * odds ratio adjusted for age, sex, BMI, smoking status, drinking status. OR, odds ratio; T2D, type 2 diabetes mellitus. Table 6. Distribution of the genotype combination of Kir6.2 and PPARγ genes in both group PPARγ Kir6.2 GG GA AA Control, n (%) CC 48 (30.19) 72 (45.28) 28 (17.61) 0.110 CG 7 (4.40) 3 (1.89) 1 (0.63) T2D, n (%) CC 52 (30.23) 81 (47.09) 20 (11.63) 0.276 CG 109 (5.81) 7 (4.07) 2 (1.17) * odds ratio adjusted for age, sex, BMI, smoking status, drinking status. OR, odds ratio; T2D, type 2 diabetes mellitus. P = 0.816, P = 0.293), 이는나이, 성별, 비만도 (BMI, kg/m 2 ), 흡연상태 ( 비흡연, 과거흡연, 현재흡연 ) 와음주상태 ( 비음주, 과거음주, 현재음주 ) 를보정한상태에서도유의한차이를보이지않았다 (Adjusted OR = 0.811, 0.527). 대조군과제2형당뇨병환자군사이에서 Wild type GG형과 Mutation type인 GA + AA형의분포역시의미있는차이를나타내지않았다 (P = 0.782). 제2형당뇨병환자군의 K 대립유전자빈도 (allele frequency) 는 0.384으로대조군의 K 대립유전자빈도인 0.418에비해의미있는차이를보이지않았다 (P = 0.383) (Table 2, 3). 3. PPARγ 유전자다형성과제2 형당뇨병의연관성대조군과제 2형당뇨병환자군모두에서 GG 유전자형 (genotype) 은나타나지않았고, CC, CG 유전자형 (genotype) 의분포에있어서도두군사이에유의한차이를보이지는않았고 (P = 0.191), 각군에서성별에따른유전자형의분포도유의한차이를보이지않았으며 (P = 0.575, P = 0.296), 나이, 성 별, 비만도, 흡연상태 ( 비흡연, 과거흡연, 현재흡연 ) 와음주상태 ( 비음주, 과거음주, 현재음주 ) 에따라보정한상태에서도통계적으로유의한차이를나타내지않았다 (Adjusted OR = 1.193). 제2형당뇨병환자군의 Ala 대립유전자빈도는 0.055 으로대조군의 Ala 대립유전자빈도인 0.035에비해높지만, 의미있는차이를보이지않았다 (P = 0.262) (Table 4, 5). 4. Kir6.2 및 PPARγ 유전자다형성간의상호작용과제 2 형당뇨병의연관성 E23K Kir6.2 유전자다형성 GG, GA, AA 유전자형 (genotype) 과 PPARγ CC, CG 유전자형 (genotype) 간의상호작용분포에있어서도대조군과제2형당뇨병환자군에서각각유의한차이를보이지는않았다 (P = 0.110, P = 0.276) (Table 6). 고 찰 췌장베타세포의포도당대사에의해생성된 ATP 에의 459
해차단되어베타세포의세포막전위를탈분극시키는역할을하는베타세포의 ATP-sensitive 포타슘채널 (K ATP) 은 Kir6.2와 SUR1으로구성되어있다 1,3). Kir6.2 변이는 K ATP 의과발현을일으키고인슐린분비를감소시킴으로써제2형당뇨병발생의위험성을증가시키게된다 1). Kir6.2 다형성에대한연구는제2형당뇨병발생과의관련성을연구하는과정에서시도되기시작하였고, 백인에있어시행되었던연구결과들이많이보고되고있다 13-15,47,48). Kir6.2 다형성과제2 형당뇨병의발병과의관련성에대한연구는영국인, 덴마크인, 미국인, 일본인등여러민족에서시도되었고, 각민족에서상이한결과를나타내었다. 2001년 Yamada Y 등에의해일본인을대상으로한연구 (n = 176, K 대립유전자빈도 (allele frequency) : 대조군 0.342 vs 당뇨군 0.388) 49), 2003년 Eva-Maria D. Nielsen 등에의한덴마크인을대상으로한연구 (n = 1665, K 대립유전자빈도 : 대조군 0.381 vs 당뇨병군 0.405, P = 0.36) 15), 2006년 Norihde Yokoi 등이시행한일본인에서의연구결과 (n = 2834, K 대립유전자빈도 : 대조군 0.37 vs 당뇨병군 0.39, P = 0.15) 50) 에서는 E23K Kir6.2 다형성과제2형당뇨병의발생률사이에관련성이없는것으로나타났었다. 그러나통계학적의미는없었지만 K 대립유전자빈도가제2형당뇨병환자군에서높은점을살펴볼수있었다. 이전에발표된영국인, 덴마크인, 미국인등백인을대상으로한연구 13,14,47) 를 meta-analysis 한 2003년 Eva-Maria D. Nielsen 등의연구에의하면 E23K Kir6.2 다형성은 K23K 유전자형에서의미있게제2 형당뇨병의위험성을증가시키고 (n = 2824, OR = 1.49, P = 0.00022), 체질량지수를증가시키는결과를보여주었다 15). Peroxisome proliferator-activated receptor (PPAR) 는여러호르몬에대한핵수용체군의하나로써 PPARγ는지방세포에특이적이고지방세포의분화및지방산대사에관여하는것으로알려져왔으며, 이유전자의다형성과제2형당뇨병의발현이관련되어있다는여러보고가있었다 35). 지금까지보고된 PPARγ 다형성 (P12A) 에대한연구결과도 Kir6.2와마찬가지로민족에따라다른결과를보였다. 2000 년 Francesco P. Mancini는이탈리아인을대상으로연구하여 PPARγ와제2형당뇨병이관련성이없음을발표하였고 51), 2000년한국인을대상으로 PPARγ 다형성 (P12A) 에대한연구에서도 P12A 변이는제2형당뇨병또는비만과관련성이없다는결과가있었다 45). 하지만, 2001년및 2004년일본에서일본인을대상으로 PPARγ 다형성 (P12A) 과제2형당뇨병을연관성에대해시행한연구에서는모두 Ala 대립유전자빈도 (allele frequency) 가제2형당뇨병의위험성을감소시킨다는결과를보고하고있다 18,52). 2004년스코틀랜드인을대상으로이루어진 PPARγ 다형성 (P12A) 에대한연구에서는 Ala 대립유전자빈도는대조군 0.143보다제2형당뇨병환자군 0.111 (n = 3058, P = 0.0003) 에서의미있게 낮았고 16), 2005년한국인을대상으로 PPARγ 다형성 (Pro12Ala) 에대한연구결과에서도, Ala 대립유전자빈도가대조군 0.053, 제2형당뇨병환자군 0.036 (n = 975, P = 0.024) 으로 Ala 대립유전자빈도가제2형당뇨병환자군에서유의하게낮았다 44). 이는 Ala 대립유전자 (allele) 가제2형당뇨병의위험성을감소시키는것과관련되어있음을보여주는것이다. 2006년미국당뇨병학회 (American Diabetes Association) 의발표에따르면 PPARγ 다형성 (P12A) 과제 2형당뇨병의연관성연구에서남아시아인종군에서는관련성이없는것으로결과가나온반면 (n = 976, 대조군.23 vs. 당뇨병군 0.2, P = 0.5), 백인군에서는당뇨병환자에서 PPAR γ 12Ala의발견빈도가의미있게낮았다 (n = 457, 대조군 0.2 vs 당뇨병군 0.09, P = 0.006) 40). 2006년 Steno 당뇨병센터 (Diabetes Center) 와 Hagedorn 연구소 (Research Institute) 의 Sara K. Hansen 등이발표한 Kir6.2(KCNJ11) 유전자및 PPARγ 유전자다형성과제2형당뇨병발생과의관련성에대한연구에서 Kir6.2 (KCNJ11) Glu23Lys의 K 대립유전자는제2형당뇨병유발과관련성이있었으나 (n = 5978, 대조군 0.363 vs. 당뇨병군 0.404, P = 0.0002), PPARγ P12A의 Ala 대립유전자는제2형당뇨병과연관성이없었고 (n = 6447, 대조군 0.139 vs 당뇨병군 0.130, P = 0.25) KCNJ11 Glu23Lys과 PPARγ P12A 유전자다형성을복합분석한결과도제2형당뇨병발병과연관성이없는것으로나타났다 53). 이처럼다수의여러인종에서제2형당뇨병의발생에있어 Kir6.2과 PPARγ와의관련성을밝히고자시도하였으나한국인을대상으로한연구결과가아직소수에불과하고, 결과또한일치하지는않았다 44,45). 본연구에서는 K 대립유전자빈도가대조군에서 0.418인반면제2형당뇨병환자군에서는 0.384으로낮았으나유의한차이는없었으며, Ala 대립유전자빈도는대조군에서 0.035인반면제2형당뇨병환자군에서는 0.055으로의미밌는차이는없었다. 이는이미보고된다른국내외논문들과는상의한결과이나, 그원인으로는연구대상환자수가기존연구에비하여적어서이런결과를보일수있다고사료된다. 또한통계적으로의미있는결과들은대상수가많은연구들인점을고려할때앞으로더많은대상으로한추시가필요하다. 끝으로이연구에이용된 real time PCR 방법을이용한유전자다형성분석법은, 과거의전기영동에의존하는수동적인유전자분석법에비하여관련유전자에대한연구를더욱용이하게만들고, 이부분의연구속도를더욱향상시킬것으로사료된다. 본논문의결과를요약하면, 한국인에서 Kir6.2 및 PPARγ 유전자다형성이제2형당뇨병의위험성에영향을미치는지에대해각각의유전자를분석을시도하였고, 또두유전자가연합하여제2형당뇨병의위험성에영향을미칠수있 460
이정은외 10 인 : 한국인에서제 2 형당뇨병과 Kir6.2 및 Peroxisome Proliferator-activated Receptor-gamma (PPARγ) 유전자다형성의연관성 는지에대해서도살펴보았다. 예상과는다르게제2형당뇨병에서 Kir6.2와 PPARγ 유전자다형성에유의한차이를볼수없었다. 이는이미보고된논문의결과와는상이한결과이다. 그러나본연구의대조군과실험군의대상수는연령을고려할때다소미흡한부분이있다. 또한, 대구지방에주로분포한환자및대조군을대상으로한본연구의결과를한국인전체를대표한결과로받아들이기에는한계가있어서, 더확실한결론에도달하기위하여서는타지방의환자를포함한좀더많은한국인을대상으로하여추가적인연구가시도되어야할것으로생각된다. 요약배경 : 인슐린작용및분비에는많은유전자들이관여하므로, 제2형당뇨병발병에는이러한유전자의변화가관여할것으로생각된다. Kir6.2는포타슘채널 (K ATP) 의구성단위이며, 포타슘채널의유전변이는베타세포의전기적활성도및포도당항상성을변화시켜제2형당뇨병을일으키는데관여하는것으로알려져있다. PPARγ는여러호르몬에대한핵수용체군의하나로서, 지방세포의분화및포도당항상성에관여한다. 따라서본연구는 Kir6.2와 PPARγ 유전자다형성과제2형당뇨병의연관성을알아보고자하였다. 방법 : 대구경북대학교병원, 대구계명대학교병원및대구카톨릭대학교병원을방문한제2형당뇨병환자군 172명및대조군 159명을대상으로하였다. Kir6.2 (Glu23Lys) 와 PPARγ (Pro12Ala) 의유전자다형성이제2형당뇨병과관련성이있는지알아보았다. 결과 : Kir6.2 Glu23Lys (P = 0.385) 와 PPARγ Pro12Ala (P = 0.191) 유전자다형성은제2형당뇨병환자군과대조군에서유의한차이를보이지않았다. 또한 Kir6.2와 PPARγ 유전자상호작용도각군에서유의한영향을보이지않았다 (P = 110, P = 0.276). 결론 : 본연구에서는 Kir6.2와 PPARγ 유전자다형성은제 2형당뇨병과연관성을보이지않았지만, 좀더확실한결과를얻기위하여서는타지방의환자를포함한대규모의한국인환자를대상으로이러한유전자다형성이제2 형당뇨병발병에영향을미치는지에대한연구가시행되어야할것이다. 감사의글이논문은 2007년도정부 ( 과학기술부 ) 의재원으로한국과학재단의국가지정연구실사업으로수행된연구이며 (No. ROA-2006-000-10271-0 (2007)), 산업자원부지역혁신인력사업연구비의지원으로수행되었음. 참고문헌 1. Riedel MJ, Steckley DC, Light PE: Current status of the e23k Kir6.2 polymorphism: Implications for type 2 diabetes. Hum Genet 116:133-45, 2005 2. Duggirala R, Blangero J, Almasy L, Dyer TD, Williams KL, Leach RJ, O'Connell P, Stern MP: Linkage of type 2 diabetes mellitus and of age at onset to a genetic location on chromosome 10q in Mexican Americans. Am J Hum Genet 64:1127-40, 1999 3. Rewers M, Norris JM, Eisenbarth GS, Erlich HA, Beaty B, Klingensmith G, Hoffman M, Yu L, Bugawan TL, Blair A, Hamman RF, Groshek M, McDuffie RS, Jr.: Beta-cell autoantibodies in infants and toddlers without IDDM relatives: Diabetes autoimmunity study in the young (DAISY). J Autoimmun 9:405-10, 1996 4. Polonsky KS, Sturis J, Bell GI: Seminars in medicine of the Beth Israel Hospital, Boston. Non-insulindependent diabetes mellitus-a genetically programmed failure of the beta cell to compensate for insulin resistance. N Engl J Med 334:777-83, 1996 5. Barnett AH, Eff C, Leslie RD, Pyke DA: Diabetes in identical twins. A study of 200 pairs. Diabetologia 20:87-93, 1981 6. Newman B, Selby JV, King MC, Slemenda C, Fabsitz R, Friedman GD: Concordance for type 2 (non-insulin-dependent) diabetes mellitus in male twins. Diabetologia 30:763-8, 1987 7. Brosseau JD, Eelkema RC, Crawford AC, Abe TA: Diabetes among the three affiliated tribes: Correlation with degree of Indian inheritance. Am J Public Health 69:1277-8, 1979 8. Chakraborty R, Ferrell RE, Stern MP, Haffner SM, Hazuda HP, Rosenthal M: Relationship of prevalence of non-insulin-dependent diabetes mellitus to Amerindian admixture in the Mexican Americans of San Antonio, texas. Genet Epidemiol 3:435-54, 1986 9. Rich SS: Mapping genes in diabetes. Genetic epidemiological perspective. Diabetes 39:1315-9, 1990 10. Knowler WC, Williams RC, Pettitt DJ, Steinberg AG: Gm3;5,13,14 and type 2 diabetes mellitus: An association in American indians with genetic admixture. Am J Hum Genet 43:520-6, 1988 11. Kahn CR, Vicent D, Doria A: Genetics of non-insulindependent (type-ii) diabetes mellitus. Annu Rev Med 47:509-31, 1996 461
12. Pillay TS, Langlois WJ, Olefsky JM: The genetics of non-insulin-dependent diabetes mellitus. Adv Genet 32:51-98, 1995 13. Gloyn AL, Hashim Y, Ashcroft SJ, Ashfield R, Wiltshire S, Turner RC: Association studies of variants in promoter and coding regions of beta-cell ATP-sensitive K-channel genes sur1 and Kir6.2 with type 2 diabetes mellitus (UKPDS 53). Diabet Med 18: 206-12, 2001 14. Hani EH, Boutin P, Durand E, Inoue H, Permutt MA, Velho G, Froguel P: Missense mutations in the pancreatic islet beta cell inwardly rectifying K+ channel gene (Kir6.2/bir): A meta-analysis suggests a role in the polygenic basis of type II diabetes mellitus in Caucasians. Diabetologia 41:1511-5, 1998 15. Nielsen EM, Hansen L, Carstensen B, Echwald SM, Drivsholm T, Glumer C, Thorsteinsson B, Borch- Johnsen K, Hansen T, Pedersen O: The e23k variant of Kir6.2 associates with impaired post-ogtt serum insulin response and increased risk of type 2 diabetes. Diabetes 52:573-7, 2003 16. Doney AS, Fischer B, Cecil JE, Boylan K, McGuigan FE, Ralston SH, Morris AD, Palmer CN: Association of the pro12ala and c1431t variants of PPARg and their haplotypes with susceptibility to type 2 diabetes. Diabetologia 47:555-8, 2004 17. Ghoussaini M, Meyre D, Lobbens S, Charpentier G, Clement K, Charles MA, Tauber M, Weill J, Froguel P: Implication of the pro12ala polymorphism of the PPAR-gamma 2 gene in type 2 diabetes and obesity in the French population. BMC Med Genet 6:11, 2005 18. Mori H, Ikegami H, Kawaguchi Y, Seino S, Yokoi N, Takeda J, Inoue I, Seino Y, Yasuda K, Hanafusa T, Yamagata K, Awata T, Kadowaki T, Hara K, Yamada N, Gotoda T, Iwasaki N, Iwamoto Y, Sanke T, Nanjo K, Oka Y, Matsutani A, Maeda E, Kasuga M: The pro12 ALA substitution in PPAR-gamma is associated with resistance to development of diabetes in the general population: Possible involvement in impairment of insulin secretion in individuals with type 2 diabetes. Diabetes 50:891-4, 2001 19. Zhang C, Qi L, Hunter DJ, Meigs JB, Manson JE, van Dam RM, Hu FB: Variant of transcription factor 7-like 2 (tcf7l2) gene and the risk of type 2 diabetes in large cohorts of U.S. Women and men. Diabetes 55:2645-8, 2006 20. Horikawa Y: Calpain-10 (NIDDM1) as a susceptibility gene for common type 2 diabetes. Endocr J 53:567-76, 2006 21. Meigs JB, Dupuis J, Liu C, O'Donnell CJ, Fox CS, Kathiresan S, Gabriel SB, Larson MG, Yang Q, Herbert AG, Wilson PW, Feng D, Tofler GH, Cupples LA: PAI-1 gene 4G/5G polymorphism and risk of type 2 diabetes in a population-based sample. Obesity (Silver Spring) 14:753-8, 2006 22. Fukuyama K, Ohara T, Hirota Y, Maeda K, Kuno S, Zenibayashi M, Teranishi T, Kouyama K, Maeda E, Sakamoto N, Kasuga M: Association of the -112a>c polymorphism of the uncoupling protein 1 gene with insulin resistance in Japanese individuals with type 2 diabetes. Biochem Biophys Res Commun 339:1212-6, 2006 23. Wang H, Zhang H, Jia Y, Zhang Z, Craig R, Wang X, Elbein SC: Adiponectin receptor 1 gene (Adipor1) as a candidate for type 2 diabetes and insulin resistance. Diabetes 53:2132-6, 2004 24. Musi N: AMP-activated protein kinase and type 2 diabetes. Curr Med Chem 13:583-9, 2006 25. Cui Y, Xu X, Bi H, Zhu Q, Wu J, Xia X, Qiushi R, Ho PC: Expression modification of uncoupling proteins and MNsod in retinal endothelial cells and pericytes induced by high glucose: The role of reactive oxygen species in diabetic retinopathy. Exp Eye Res 83:807-16, 2006 26. Hansen L, Pedersen O: Genetics of type 2 diabetes mellitus: Status and perspectives. Diabetes Obes Metab 7:122-35, 2005 27. Zietz B, Leonhardt K, Schaffler A: [candidate genes and polymorphism analysis in type 2 diabetes mellitus.]. Med Klin (Munich) 101:605-16, 2006 28. Clement K, Lahlou N, Ruiz J, Hager J, Bougneres P, Basdevant A, Guy-Grand B, Froguel P: Association of poorly controlled diabetes with low serum leptin in morbid obesity. Int J Obes Relat Metab Disord 21:556-61, 1997 29. Gribble FM, Reimann F: Sulphonylurea action revisited: The post-cloning era. Diabetologia 46:875-91, 2003 30. Haider S, Antcliff JF, Proks P, Sansom MS, Ashcroft FM: Focus on Kir6.2: A key component of the ATP-sensitive potassium channel. J Mol Cell Cardiol 38:927-36, 2005 462
이정은외 10 인 : 한국인에서제 2 형당뇨병과 Kir6.2 및 Peroxisome Proliferator-activated Receptor-gamma (PPARγ) 유전자다형성의연관성 31. Inagaki N, Gonoi T, Clement JPt, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, Bryan J: Reconstitution of IKATP: An inward rectifier subunit plus the sulfonylurea receptor. Science 270:1166-70, 1995 32. Clement JPt, Kunjilwar K, Gonzalez G, Schwanstecher M, Panten U, Aguilar-Bryan L, Bryan J: Association and stoichiometry of k(atp) channel subunits. Neuron 18:827-38, 1997 33.Li L, Shi Y, Wang X, Shi W, Jiang C: Single nucleotide polymorphisms in K(ATP) channels: Muscular impact on type 2 diabetes. Diabetes 54:1592-7, 2005 34. Chawla A, Schwarz EJ, Dimaculangan DD, Lazar MA: Peroxisome proliferator-activated receptor (PPAR) gamma: Adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology 135: 798-800, 1994 35. Guo L, Tabrizchi R: Peroxisome proliferator-activated receptor gamma as a drug target in the pathogenesis of insulin resistance. Pharmacol Ther 111:145-73, 2006 36. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA: An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem 270:12953-6, 1995 37. Stumvoll M, Haring H: The peroxisome proliferatoractivated receptor-gamma2 pro12ala polymorphism. Diabetes 51:2341-7, 2002 38. Yen CJ, Beamer BA, Negri C, Silver K, Brown KA, Yarnall DP, Burns DK, Roth J, Shuldiner AR: Molecular scanning of the human peroxisome proliferator activated receptor gamma (HPPAR gamma) gene in diabetic Caucasians: Identification of a pro12ala PPAR gamma 2 missense mutation. Biochem Biophys Res Commun 241:270-4, 1997 39. Ek J, Andersen G, Urhammer SA, Hansen L, Carstensen B, Borch-Johnsen K, Drivsholm T, Berglund L, Hansen T, Lithell H, Pedersen O: Studies of the pro12ala polymorphism of the peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2) gene in relation to insulin sensitivity among glucose tolerant caucasians. Diabetologia 44:1170-6, 2001 40. Radha V, Vimaleswaran KS, Babu HN, Abate N, Chandalia M, Satija P, Grundy SM, Ghosh S, Majumder PP, Deepa R, Rao SM, Mohan V: Role of genetic polymorphism peroxisome proliferator-activated receptor-gamma2 pro12ala on ethnic susceptibility to diabetes in South-Asian and Caucasian subjects: Evidence for heterogeneity. Diabetes Care 29:1046-51, 2006 41. Tonjes A, Scholz M, Loeffler M, Stumvoll M: Association of pro12ala polymorphism in peroxisome proliferator-activated receptor {gamma} with pre-diabetic phenotypes: Meta-analysis of 57 studies on nondiabetic individuals. Diabetes Care 29:2489-97, 2006 42. Jaziri R, Lobbens S, Aubert R, Pean F, Lahmidi S, Vaxillaire M, Porchay I, Bellili N, Tichet J, Balkau B, Froguel P, Marre M, Fumeron F: The PPARg pro12ala polymorphism is associated with a decreased risk of developing hyperglycemia over 6 years and combines with the effect of the apm1 g-11391a single nucleotide polymorphism: The data from an epidemiological study on the insulin resistance syndrome (desir) study. Diabetes 55:1157-62, 2006 43. McKeigue PM, Shah B, Marmot MG: Relation of central obesity and insulin resistance with high diabetes prevalence and cardiovascular risk in South Asians. Lancet 337:382-6, 1991 44. Moon MK, Cho YM, Jung HS, Park YJ, Yoon KH, Sung YA, Park BL, Lee HK, Park KS, Shin HD: Genetic polymorphisms in peroxisome proliferatoractivated receptor gamma are associated with type 2 diabetes mellitus and obesity in the Korean population. Diabet Med 22:1161-6, 2005 45. Oh EY, Min KM, Chung JH, Min YK, Lee MS, Kim KW, Lee MK: Significance of pro12ala mutation in peroxisome proliferator-activated receptor-gamma2 in Korean diabetic and obese subjects. J Clin Endocrinol Metab 85:1801-4, 2000 46. World health organization: Definition, diagnosis and classification of diabetes mellitus and its complications. Report of a who consultation. Part 1, Diagnosis and classification of diabetes mellitus. Geneva: World Health Organization, 1999 47. Inoue H, Ferrer J, Warren-Perry M, Zhang Y, Millns H, Turner RC, Elbein SC, Hampe CL, Suarez BK, Inagaki N, Seino S, Permutt MA: Sequence variants in the pancreatic islet beta-cell inwardly rectifying K+ channel Kir6.2 (bir) gene: Identification and lack 463
of role in Caucasian patients with NIDDM. Diabetes 46:502-7, 1997 48. Sakura H, Wat N, Horton V, Millns H, Turner RC, Ashcroft FM: Sequence variations in the human Kir6.2 gene, a subunit of the beta-cell ATP-sensitive K-channel: No association with niddm in while caucasian subjects or evidence of abnormal function when expressed in vitro. Diabetologia 39:1233-6, 1996 49. Yamada Y, Kuroe A, Li Q, Someya Y, Kubota A, Ihara Y, Tsuura Y, Seino Y: Genomic variation in pancreatic ion channel genes in Japanese type 2 diabetic patients. Diabetes Metab Res Rev 17:213-6, 2001 50. Yokoi N, Kanamori M, Horikawa Y, Takeda J, Sanke T, Furuta H, Nanjo K, Mori H, Kasuga M, Hara K, Kadowaki T, Tanizawa Y, Oka Y, Iwami Y, Ohgawara H, Yamada Y, Seino Y, Yano H, Cox NJ, Seino S: Association studies of variants in the genes involved in pancreatic beta-cell function in type 2 diabetes in Japanese subjects. Diabetes 55:2379-86, 2006 51. Mancini FP, Vaccaro O, Sabatino L, Tufano A, Rivellese AA, Riccardi G, Colantuoni V: Pro12ala substitution in the peroxisome proliferator-activated receptor-gamma2 is not associated with type 2 diabetes. Diabetes 48:1466-8, 1999 52. Horiki M, Ikegami H, Fujisawa T, Kawabata Y, Ono M, Nishino M, Shimamoto K, Ogihara T: Association of pro12ala polymorphism of PPARgamma gene with insulin resistance and related diseases. Diabetes Res Clin Pract 66 Suppl 1:S63-7, 2004 53. Hansen SK, Nielsen EM, Ek J, Andersen G, Glumer C, Carstensen B, Mouritzen P, Drivsholm T, Borch- Johnsen K, Jorgensen T, Hansen T, Pedersen O: Analysis of separate and combined effects of common variation in KCNJ11 and PPARg on risk of type 2 diabetes. J Clin Endocrinol Metab 90:3629-37, 2005 464