질병관리본부유전체센터 학술연구용역사업 한국인질병위험도측정및 질병예측을위한바이오마커발굴 (I) 당뇨병위험도분석을위한형질마커발굴 결과보고서 (2006 년 4 월 28 일 ) 디엔에이링크 질병관리본부유전체센터
목 차 최종결과보고서 1. 사업명 1 2. 연구목적및필요성 1 가. 연구목적 1 나. 국내외연구동향 1 다. 연구의필요성 2 라. 연구용역사업의수행체계 3 3. 연구의내용및방법 4 가. 연구내용 4 나. 연구방법 4 1) sample set선정 5 2) DNA plating 5 3) Genotyping Assay Development 5 4) Genotyping Platform & Technology 6 5) 통계분석 8 4. 연구결과 10 가. 분석결과개요 10 1) sample 입고및분석현황 10 2) Genotyping 분석현황 10 3) Hardy-Weinberg Equilibrium test 15 4) SNP의발현빈도 27 5) Haplotype 빈도 27 나. 당뇨병발생과의관련성분석 40 다. 17개 SNP set분석 51 5. 검토사항 55 가. Minor allele frequency 차이 55 나. Novel SNP 여부 56 다. phenotype 자료 56 6. 연구결과에대한기여도, 기대효과및활용방안 57 가. 기대효과 57
나. 활용방안 57 7. 연구용역사업비 58 부록 1. Assay Information 59 부록 2. Gene blast Information 71 부록 3. Genotyping Technology 79
최종결과보고서 1. 사업명 당뇨병위험도분석을위한형질마커발굴 2. 연구목적및필요성 가. 연구목적 현재장기추적되고있는지역사회코호트전체를대상으로하여당뇨병발생위험도분 석및질병예측지표로서의활용도측정을위한형질지표의대규모분석 한국인에서중요한대표적생활습관질환인당뇨병의병인적요소에대한연구로확인된 유전체지표를지역사회코호트전체규모로분석 나. 국내외연구동향 인간의질병에관여하는유전체변이연구는해당질환의병리, 생리학적기작에기여한다고 예상되는적절한유전자 ( 질환후보유전자 ) 들을선정하고이들의변이형을발굴하고질환과 의연관성을통계적으로확인하는과정순으로진행되고있음. 가족을대상으로한유전연구에서당뇨병과관련한공복혈당및 HbA1c 의유전율은각각 72 85%, 55~67% 로나타나당뇨병발생에강한유전력이있음을규명하였음. Genome wide scanning 분석을통해당뇨병발생과관련한유전자의위치가보고되었으며 최근 Reynisdottir 등은 5q34.2-35.2 와당뇨병발생과의관련성을새롭게보고하였음. 제 2 형당뇨병은인슐린저항성과베타세포의기능부전을특징으로하며당뇨병발생과관 련한원인유전자는인슐린분비경로및작용경로에관여하는호르몬, 대사관여물질과관 여한유전자들및인슐린에의해발현이조절되는유전자들로알려져있음. - 1 -
다. 연구필요성 최근미국등의서구사회뿐아니라우리나라에서도사회경제적발전과식생활의서구화 및생활수준의향상으로당뇨병의유병률이급격히증가하고있고당뇨병으로인한합병 증및사망률증가로보건사회적, 경제적부담이급증하고있음. 당뇨병은유전적인성향이강한질병으로가족, 특히형제, 자매중에당뇨병을앓고있는분이있는경우에는현재본인이정상이라할지라도당뇨병에걸릴확률은일반인들에비해무려 40% 정도증가함. 그러나최근까지밝혀진당뇨병의원인유전자는전체의 5% 미만에불과함. 질환은유전적소인과환경적소인에의하여발생하며유전적소인의대부분은개인간유전적차이인 SNP에의해나타나고있어, 그러한유전적소인이밝혀진다면이를바탕으로질병발생위험도측정과발생예측이가능하게되며, 이로써발병전중재를통하여적극적인예방, 조기진단, 종합적예후향상을기대할수있음. 한국인에서중요한대표적생활습관질환인당뇨병에대한병인요소연구에의해서최근 밝혀진유전체지표를이용하여당뇨병발생의위험도를측정할수있을것으로판단됨. - 2 -
라. 연구용역사업의수행체계 Genotyping 대상 SNP 선정 21 개유전자 175 개 SNP Genotyping 대상 SNP 선정 6 개유전자 17 개 SNP 1,302 Sample Set 선정 6,653 Sample Set 선정 Assay development & allele frequency determination Autoprimer, ProbeITy, Primer3 High-throughtput genotyping SNPstream UHT, SNPstream 25K, SNaPshot, Sequencing SNP 및 haplotype 질환관련성분석 Allele Freq. HWE LD(linkage disequilibrium), Haplotype analysis, case-control analysis 결과정리및보고 - 3 -
3. 연구내용및방법 가. 연구내용 1) 유전자 - 유전자상호작용연구를위해 KSNP 발굴과정에서확인된인슐린신호전달및 Glucose Uptake 에관여하는유전자등 21 개유전자의 175 개 KSNP 를코호트내에서확인된 환자및정상인집단 ( 총 1302 명 ) 에서분석함 2) 당뇨병의병인요소연구에의해서확인된 6 개유전자의 17 개 KSNP 를질환군별유전체연구 센터와협력하여총 6,653 명환자, 내당장애및정상인집단에서분석 3) 분석결과는통계분석을통하여 SNP 및 haplotype 의질병관련성정보를확보 나. 연구방법 <Strategy> - 4 -
1) Sample set 선정 - 국립보건연구원유전체연구부와질환군별센터에서제공되는정상인 660명과당뇨환자 642 명총 1302명유전체시료를제공받음 - 정상인, 당뇨환자가포함된 6653명유전체시료를제공받음 - 선정된샘플의 genomic DNA 확보및 normalization 2) DNA plating - 96well plate 4개에분주되어있는 normalize된 sample DNA stock solution(1ng/ μl ) 을 Biomek FX를이용하여 384 plate에 2μl (2ng) 씩효율적으로배열. - 분주된 DNA는공기중에서건조시킨후보관. 384well plate 배열 3) Genotyping Assay Development - Assay design Genotyping primer를 design 하기위해서국립보건원유전체센터에서제공한 target SNP의 Flanking sequence를이용하여 blast를실시하였다. autoprimer, ProbeITy, Primer 3 software를이용하여 genotyping primer를 design하였다. 분석에사용된 primer sequence 와 blast 결과는부록 1,2 에나타내었음. - Multiplex Long PCR CYP계열의약물대사효소는유전자간의높은 homology을나타내는특성을보임. In Silico상에서 target SNP을포함한 500bp의 flanking sequence의 blast해보면 Pseudogene이나다른염색체상의유전자와 98% 이상일치하는결과를보여기존의 genotyping방법의분석결과의정확성에의문이제시됨. Multiplex Long PCR 방법은각유전자의특징적인 sequence를포함하도록 long PCR(1~3kb) 를시행한후다시 PCR product를 plate로이용하여 short PCR를실시하여각각의 SNP부위에대한유전형을분석함. - 5 -
2 1 3 4 5 8 9 7 6 10 11 1 2 3 4 5 67 8 9 1 0 11 M ultiplex long-pcr M ultiplex short-pcr Long PCR product를이용하여여러 primer쌍으로 2차 PCR을시행함으로써분석에필요한 DNA양및시간과비용을단축시킴. 이때 PCR primer를효율적으로 design 하는기술이중요. 본연구진은실질적으로 CYP11B2 유전자에대한 multiplex long PCR 방법을 genotyping 분석법과접목시켜성공적으로 SNP genoytping을실시하였음. 4) Genotyping Platform & Technology - Genotypting platform Single base extension 원리의 SNPstream Genotyping System 를이용하여소량의 DNA 를 이용하여 12 개의 SNP 를동시에분석할수있는 12multiplex UHT system 를주축으로 SNP IT, SNaPshot assay(abi3700), Sequencing 방법을이용하여정확한분석결과와 data missing 을최소화하였음. Platform 특징 SNPstream SNPstream SNaPshot Sequencing UHT 25K - SNP-IT assay (single-base primer extension assay) - Throughput : 800,000 genotyping/day - 12 plex reaction, 많은 SNP 많은샘플처리에용이 - SNP-IT assay (single-base primer extension assay) - Throughput : 25,000 genotyping/day - Full automation, 적은 SNP 많은샘플처리에용이 - SNaPshot assay (single-base primer extension assay) - 대규모시스템의보완용으로적합 - data confirm - 대규모시스템의보완용으로적합 - 6 -
자세한분석원리및분석과정은부록 3 에나타내었음. <High-throughput Liquid Handling> - 7 -
5) 통계분석 - SNP validation Hardy-Weinberg Equilibrium test, International hapmap Japanese panel data와 allele frequency 비교. ᄀ각 SNP의빈도분석 - 전체대상자및대조군, 당뇨군에서각각 MAF(minor allele frequency) 를구한다. ᄂ Marker의정보성 - 일반적으로 SNP의 minor allele의출현빈도가 10% 이상인경우를 common SNP라하며이러한 SNP은 marker로서의정보성을갖는다고함. - Heterozygosity Heterozygosity는단순하게자료내에서이형접합인개체의비율을나타내기도하나여기서는 HWE가만족될때이형접합체의기대비율을나타냄 - Association analysis ᄀ Haploview, SAS/Genetics, Phase 등유전통계분석이가능한 software를이용하여 LD map 및 haplotype을추정하고환자-대조군 SNP association분석을실시ᄂ SNP과 haplotype 등의정보에대해 dominant, recessive, codominant model등의다양한모형을적용하여특정 SNP 및 haplotype과질병연관성을분석. ᄃ표현형 (phenotype) 이질환자 / 정상인의이진 (binary) 변수이고, 유전정보가 genotype, haplotype 등의범주형 (categorical) 인경우에는카이제곱검정 (chi-square test), 피셔의정확검정 (Fisher exact test), 오즈비 (odds ratio), 상대위험도 (relative risk) 와신뢰구간 (confidence interval :CI) 을적용하여분석을실시함ᄅ범주형 (category) 변수간의연관성에관한유의성을검정하고자할때, 일반적으로카이제곱검정 (Chi-square test) 을많이이용함. 그러나이방법은카이제곱분포를가정한모수적 (parametric) 분석방법으로대표본가정이만족되는경우에만적용할수있음. 그러므로자료가대표본가정을만족하지않는경우에는 Fisher의정확검정 (Fisher's Exact test) 등의비모수적 (non-parametric) 분석방법을적용해야함. 위에서언급된두방법은범주형자료를명목형변수 (nominal variable) 로취급하여분석하는방법이지만, SNP 자료는 Minor allele의포함여부혹은포함개수를근거로순서형변수 (ordinal variable) 로취급할수있음. 순서형변수는순서정보를반영하여연관성분석을실시할수있으며, 모수적분석방법으로 Cochran-Armitage 추세검정 (Cochran-Armitage trend test), Cochran-Mantel-Haenszel 검정그리고비모수적방법으로 Jonckheere-Terpstra 검정 (Jonckheere-Terpstra test) 등을이용하여분석하였음. - 8 -
<Data Flow Chart> - 9 -
4. 연구결과 가. 분석결과개요 1) sample 입고및분석현황 - 최종결과제출시료는 175개 SNP분석 set는 1302개로 no153, 1957, 3465, 3502, 6923, 7973등 6개시료는중복제공되어하나씩만분석에포함되었고, 분석결과 70% 이상의 SNP에서 fail 된시료가 no7109, 7852, 7854, 7860, 7861, 7862등 6개였으며 no1687, 3987은 sample 분주과정의오류로분석에서제외되었음. - 17 개 SNP set 의최종결과제출시료는 6653 개로 no5039 는중복제공되어하나만분석에 포함되었고, 분석결과 70% 이상의 SNP 에서 fail 된시료가 no4312, 4838, 5800, 5826, 5827, 5869, 5902 등 7 개였으며 no1184, 1652 는 sample 분주과정의오류로분석에서제외되었음. 175개 SNP set 17개 SNP set 입고sample수 1316 6663 중복시료수 6 1 70% 이상 SNP결과 fail시료수 6 7 미분석시료수 2 2 최종분석시료수 1302 6653 2) Genotyping 분석현황 - 175개 set에대한분석은 6개의 SNP을제외한 169개의 SNP에대한분석이완료되었음. 분석이완료된 SNP 중 new36, new134, rs11779229의 frequency가 monomorphic, 0.002, 0.01 로나타나 setting시 monomorphic한결과를보인 SNP에대한대량분석은실시하지않음. 분석제외된 SNP은 sequencing으로빈도를재확인하였고 CYP11B2의 rs13276226, rs13268153, new30와 STXBP3의 new127 등 4개 SNP은 monomorphic한결과를보였음. assay가만들어지지않은 SNP은 FABP2의 rs1973598, STXBP3의 rs1149162 2개임. - 10 -
각각의 target SNP 에대한분석 platform 및분석현황 Gene rs number platform Pass Fail CallRate XX XY YY X Y Hetero HWE zygosity p-value ACAS2 NEW1 UHT 1302 0 100 92 499 711 0.262 0.738 0.387 0.728 2295096 Snapshot 1292 10 99.2 136 537 619 0.313 0.687 0.430 0.226 4911163 UHT 1302 0 100 360 653 289 0.527 0.473 0.499 0.827 NEW4 UHT 1302 0 100 0 36 1266 0.014 0.986 0.027 0.613 3746450 UHT 1279 23 98.2 287 644 348 0.476 0.524 0.499 0.739 3818273 UHT 1289 13 99 353 650 286 0.526 0.474 0.499 0.686 2273683 UHT 1281 21 98.4 276 651 354 0.470 0.530 0.498 0.470 CD36 1049654 UHT 1300 2 99.8 107 561 632 0.298 0.702 0.418 0.260 3173798 UHT 1302 0 100 133 618 551 0.339 0.661 0.448 0.035 3765187 UHT 1302 0 100 6 175 1121 0.072 0.928 0.133 0.766 3211864 UHT 1301 1 99.9 7 137 1157 0.058 0.942 0.109 0.184 3211908 UHT 1298 3 99.8 20 311 967 0.135 0.865 0.234 0.376 1527483 Snapshot 1296 6 99.5 816 438 42 0.799 0.201 0.322 0.068 3211956 UHT 1295 7 99.5 794 455 46 0.789 0.211 0.333 0.050 CYP11B2 NEW15 25K 1302 0 100 601 590 111 0.688 0.312 0.429 0.044 11781082 Snapshot 1297 5 99.6 1047 233 17 0.897 0.103 0.185 0.327 10087214 UHT 1297 5 99.6 112 587 598 0.313 0.687 0.430 0.056 1799998 UHT 1290 12 99.1 597 584 109 0.689 0.311 0.428 0.042 NEW19 25K 1290 12 99.1 1217 57 16 0.966 0.034 0.067 0.000 13276226 sequencing 231 231 0 0 13268153 sequencing 42 42 0 0 11779229 25K 1242 60 95.4 1218 24 0 0.990 0.010 0.731 4539 25K 1287 15 98.8 268 471 548 0.391 0.609 0.476 0.000 6399 25K 1300 2 99.8 580 456 264 0.622 0.378 0.470 0.000 4536 25K 1299 3 99.8 538 474 287 0.597 0.403 0.481 0.000 4736320 25K 1302 0 100 291 653 358 0.474 0.526 0.499 0.836 4545 25K 1301 1 99.9 316 646 339 0.491 0.509 0.500 0.812 6431 25K 1276 26 98 0 394 882 0.154 0.846 0.261 0.000 3097 snapshot 1296 6 99.5 1141 149 6 0.938 0.062 0.117 0.634 NEW30 sequencing 42 42 0 0 3802230 25K 1287 15 98.8 573 602 112 0.679 0.321 0.436 0.009 NEW32 sequencing 1298 4 99.7 597 589 112 0.687 0.313 0.430 0.048 3802228 sequencing 1280 21 98.4 559 598 123 0.670 0.330 0.442 0.041 FABP1 2970902 Snapshot 1291 11 99.2 992 275 24 0.875 0.125 0.219 0.334 2970901 UHT 1302 0 100 834 412 56 0.799 0.201 0.321 0.572 NEW36 Mono 1302 0 100 1302 0 0 1.000 0.000 2919872 UHT 1293 9 99.3 827 410 56 0.798 0.202 0.322 0.567 2241883 25K 1301 1 99.9 50 450 801 0.211 0.789 0.333 0.176 1545224 UHT 1292 10 99.2 334 598 360 0.490 0.510 0.500 0.008 2919867 UHT 1285 17 98.7 360 599 326 0.513 0.487 0.500 0.016 FABP2 13306357 UHT 1298 4 99.7 1152 140 6 0.941 0.059 0.110 0.435 10034579 UHT 1291 11 99.2 237 620 434 0.424 0.576 0.488 0.551 2282688 UHT 1301 1 99.9 237 625 439 0.422 0.578 0.488 0.577 6857641 UHT 1293 9 99.3 438 624 231 0.580 0.420 0.487 0.735 1973598 assay fail 1397614 UHT 1296 6 99.5 1150 140 6 0.941 0.059 0.110 0.437 1397613 UHT 1301 1 99.9 238 624 439 0.423 0.577 0.488 0.533 1799883 UHT 1299 3 99.8 598 566 135 0.678 0.322 0.436 0.950 4834770 UHT 1298 4 99.7 238 623 437 0.423 0.577 0.488 0.541 11935130 UHT 1300 2 99.8 814 427 59 0.790 0.210 0.331 0.753 12510269 UHT 1301 1 99.9 519 606 176 0.632 0.368 0.465 0.966 2964 UHT 1300 2 99.8 176 606 518 0.368 0.632 0.465 0.953-11 -
rs number platform Pass Fail CallRate XX XY YY X Y Hetero HWE zygosity p-value FABP2 9999724 UHT 1300 2 99.8 518 606 176 0.632 0.368 0.465 0.953 11724758 UHT 1297 5 99.6 222 622 453 0.411 0.589 0.484 0.734 10212775 UHT 1293 8 99.4 174 604 515 0.368 0.632 0.465 0.883 10213267 UHT 1288 14 98.9 176 605 507 0.372 0.628 0.467 0.833 NEW57 UHT 1296 6 99.5 177 603 516 0.369 0.631 0.466 0.968 NEW58 25K 1279 23 98.2 153 540 586 0.331 0.669 0.443 0.098 9996644 UHT 1296 6 99.5 518 605 173 0.633 0.367 0.465 0.861 NEW60 UHT 1300 2 99.8 1189 109 2 0.957 0.043 0.083 0.761 FABP4 2290200 UHT 1300 2 99.8 1 88 1211 0.035 0.965 0.067 0.643 16909196 UHT 1300 2 99.8 6 162 1132 0.067 0.933 0.125 0.937 1054135 UHT 1278 24 98.2 144 598 536 0.347 0.653 0.453 0.238 SLC27A1 8109373 UHT 1293 9 99.3 13 232 1048 0.100 0.900 0.180 0.968 8110830 UHT 1287 15 98.8 215 644 428 0.417 0.583 0.486 0.299 NEW66 Snapshot 1294 8 99.4 1042 238 14 0.897 0.103 0.184 0.921 NEW67 25K 1290 12 99.1 1100 186 4 0.925 0.075 0.139 0.187 10418156 snapshot 1292 10 99.2 413 637 242 0.566 0.434 0.491 0.896 7251644 UHT 1292 10 99.2 412 640 240 0.567 0.433 0.491 0.758 3746318 25K 1296 6 99.5 833 415 48 0.803 0.197 0.317 0.677 11879407 UHT 1288 14 98.9 411 642 235 0.568 0.432 0.491 0.569 NEW72 Snapshot 1301 1 99.9 1248 50 3 0.978 0.022 0.042 0.002 4808657 UHT 1295 7 99.5 847 407 41 0.811 0.189 0.306 0.349 15401 UHT 1302 0 100 951 325 26 0.855 0.145 0.248 0.773 9137 UHT 1292 10 99.2 26 323 943 0.145 0.855 0.248 0.786 SLC27A4 7030121 25K 1301 1 99.9 660 526 115 0.709 0.291 0.412 0.487 NEW77 25K 1291 11 99.2 7 160 1124 0.067 0.933 0.126 0.615 NEW78 UHT 1295 7 99.5 807 423 65 0.786 0.214 0.336 0.324 2240953 Snapshot 1297 5 99.6 814 416 67 0.788 0.212 0.334 0.149 NEW80 UHT 1292 10 99.2 1162 125 5 0.948 0.052 0.099 0.408 NEW81 UHT 1298 4 99.7 813 419 66 0.788 0.212 0.334 0.212 7048106 25K 1284 18 98.6 0 657 627 0.256 0.744 0.381 0.000 ASPSCR1 NEW83 UHT 1296 6 99.5 355 645 296 0.523 0.477 0.499 0.927 NEW84 25K 1300 2 99.8 1 84 1215 0.033 0.967 0.064 0.714 NEW85 UHT 1301 1 99.9 1214 87 0 0.967 0.033 0.065 0.212 NEW86 UHT 1301 1 99.9 1150 149 2 0.941 0.059 0.111 0.211 8074498 25K 1301 1 99.9 57 443 801 0.214 0.786 0.336 0.666 NEW88 Snapshot 1302 0 100 1169 130 3 0.948 0.052 0.099 0.758 13087 25K 1300 2 99.8 117 545 638 0.300 0.700 0.420 0.968 CACNA1G 7501581 UHT 1293 9 99.3 964 302 27 0.862 0.138 0.237 0.559 NEW91 Snapshot 1299 3 99.8 1234 62 3 0.974 0.026 0.051 0.022 12946808 UHT 1279 23 98.2 5 156 1118 0.065 0.935 0.121 0.859 12449998 UHT 1299 3 99.8 1135 159 5 0.935 0.065 0.122 0.821 198551 25K 1276 26 98.0 1141 134 1 0.947 0.053 0.101 0.145 12936750 UHT 1282 20 98.5 11 215 1056 0.092 0.908 0.168 0.988 3744521 UHT 1300 2 99.8 20 275 1005 0.121 0.879 0.213 0.811 NEW97 UHT 1290 12 99.1 19 335 936 0.145 0.855 0.247 0.073 739925 UHT 1294 8 99.4 27 337 930 0.151 0.849 0.257 0.582 9893223 UHT 1302 0 100 1139 154 9 0.934 0.066 0.123 0.136 PPARD 9658117 UHT 1300 2 99.8 1128 167 5 0.932 0.068 0.127 0.654 2016520 UHT 1278 24 98.2 68 479 731 0.241 0.759 0.365 0.359 2076168 UHT 1297 5 99.6 70 488 739 0.242 0.758 0.367 0.362 2076167 UHT 1291 11 99.2 737 484 70 0.758 0.242 0.367 0.412 9658173 UHT 1293 9 99.3 0 58 1235 0.022 0.978 0.044 0.409 3734254 UHT 1298 4 99.7 744 488 66 0.761 0.239 0.364 0.220 1053049 UHT 1297 5 99.6 785 447 65 0.778 0.222 0.346 0.894-12 -
Gene rs number platform Pass Fail CallRate XX XY YY X Y Hetero HWE zygosity p-value PPARD 760783 UHT 1295 7 99.5 49 461 785 0.216 0.784 0.338 0.063 PPARG 12486170 Snapshot 1297 5 99.6 859 399 39 0.816 0.184 0.300 0.369 13306747 Snapshot 1301 1 99.9 1156 141 4 0.943 0.057 0.108 0.891 SNAP23 9302112 UHT 1297 5 99.6 714 509 74 0.747 0.253 0.378 0.177 SNTG1 3812426 UHT 1293 9 99.3 834 404 55 0.801 0.199 0.319 0.494 2247368 UHT 1288 14 98.9 875 374 39 0.825 0.175 0.289 0.900 4873415 UHT 1278 24 98.2 851 391 36 0.819 0.181 0.297 0.263 4873417 UHT 1298 4 99.7 837 417 44 0.805 0.195 0.313 0.365 4367560 UHT 1294 8 99.4 962 302 30 0.860 0.140 0.241 0.279 1481475 UHT 1278 24 98.2 32 334 912 0.156 0.844 0.263 0.829 7817955 UHT 1287 15 98.8 32 335 920 0.155 0.845 0.262 0.819 12542191 UHT 1299 3 99.8 817 427 55 0.793 0.207 0.328 0.933 1508620 UHT 1300 2 99.8 591 589 120 0.681 0.319 0.434 0.120 1911830 UHT 1301 1 99.9 119 593 589 0.319 0.681 0.435 0.081 6473337 UHT 1299 3 99.8 645 564 90 0.714 0.286 0.409 0.025 7837449 UHT 1301 1 99.9 1096 201 4 0.920 0.080 0.148 0.099 STXBP3 1124426 UHT 1300 2 99.8 753 477 70 0.763 0.237 0.362 0.623 1124427 snapshot 1301 1 99.9 755 464 82 0.759 0.241 0.366 0.347 6604120 UHT 1292 10 99.2 83 464 745 0.244 0.756 0.369 0.349 1277044 UHT 1297 5 99.6 753 459 85 0.758 0.242 0.367 0.186 NEW127 sequencing 42 42 0 0 1277042 UHT 1293 9 99.3 85 457 751 0.242 0.758 0.367 0.174 1690734 UHT 1289 13 99 83 454 752 0.240 0.760 0.365 0.198 1298092 UHT 1297 5 99.6 751 462 84 1 0 0.368 0.258 1149162 assay fail 12132867 snapshot 1298 4 99.7 785 440 73 0.774 0.226 0.350 0.276 1277239 UHT 1301 1 99.9 757 459 85 0.758 0.242 0.367 0.175 NEW134 UHT 1295 7 99.5 1289 6 0 0.998 0.002 0.005 0.933 1149154 UHT 1300 2 99.8 85 457 758 0.241 0.759 0.366 0.154 1149153 UHT 1299 3 99.8 95 469 735 0.254 0.746 0.379 0.094 STXBP4 9902718 UHT 1300 2 99.8 1212 85 3 0.965 0.035 0.068 0.248 17817382 UHT 1283 19 98.5 440 606 237 0.579 0.421 0.487 0.266 9303362 UHT 1298 4 99.7 437 614 247 0.573 0.427 0.489 0.231 NEW140 UHT 1290 12 99.1 943 326 21 0.857 0.143 0.245 0.232 17818028 UHT 1302 0 100 1213 85 4 0.964 0.036 0.069 0.060 10468513 UHT 1299 3 99.8 1211 84 4 0.965 0.035 0.068 0.054 1156287 UHT 1300 1 99.9 4 86 1210 0.036 0.964 0.070 0.067 11658717 UHT 1300 2 99.8 1210 86 4 0.964 0.036 0.070 0.067 1894936 UHT 1295 7 99.5 37 331 927 0.156 0.844 0.264 0.261 17818142 UHT 1299 3 99.8 820 422 57 0.794 0.206 0.327 0.772 UCP1 3811790 UHT 1287 15 98.8 166 587 534 0.357 0.643 0.459 0.813 3811789 UHT 1302 0 100 11 208 1083 0.088 0.912 0.161 0.772 NEW149 UHT 1286 15 98.8 8 183 1095 0.077 0.923 0.143 0.906 2071415 UHT 1302 0 100 20 257 1025 0.114 0.886 0.202 0.401 NEW151 UHT 1297 5 99.6 10 191 1096 0.081 0.919 0.149 0.598 7688743 UHT 1292 10 99.2 783 439 70 0.776 0.224 0.348 0.412 2270565 UHT 1301 1 99.9 1039 247 15 0.894 0.106 0.190 0.941 UCP2 659366 25K 1296 6 99.5 390 604 302 0.534 0.466 0.498 0.022 17174145 25K 1300 2 99.8 1194 99 7 0.957 0.043 0.083 0.002 NEW156 Snapshot 1294 8 99.4 1079 205 10 0.913 0.087 0.159 0.939 660339 25K 1278 24 98.2 335 665 278 0.522 0.478 0.499 0.126 UCP3 1800849 UHT 1293 9 99.3 100 538 655 0.285 0.715 0.408 0.469 NEW159 UHT 1299 3 99.8 649 546 104 0.710 0.290 0.412 0.466 2075576 UHT 1286 16 98.8 98 541 647 0.287 0.713 0.409 0.300-13 -
Gene rs number platform Pass Fail CallRate XX XY YY X Y Hetero HWE zygosity p-value UCP3 1726747 UHT 1299 3 99.8 320 663 316 0.502 0.498 0.500 0.454 3781907 UHT 1288 13 99 192 620 476 0.390 0.610 0.476 0.668 2734828 25K 1302 0 100 817 484 1 0.813 0.187 0.304 0.000 2075577 UHT 1295 7 99.5 324 665 306 0.507 0.493 0.500 0.327 647126 UHT 1299 3 99.8 307 670 322 0.494 0.506 0.500 0.253 7930460 UHT 1298 4 99.7 633 562 103 0.704 0.296 0.417 0.158 VAMP2 NEW167 UHT 1298 3 99.8 1229 67 2 0.973 0.027 0.053 0.283 1061032 UHT 1288 14 98.9 378 619 291 0.534 0.466 0.498 0.217 NEW169 UHT 1299 3 99.8 10 184 1105 0.079 0.921 0.145 0.445 11548690 UHT 1297 5 99.6 1110 175 12 0.923 0.077 0.142 0.087 1150 snapshot 1290 12 99.1 339 622 329 0.504 0.496 0.500 0.201 VAMP3 707457 UHT 1300 2 99.8 1187 109 4 0.955 0.045 0.086 0.377 NEW173 snapshot 1293 9 99.3 731 486 76 0.753 0.247 0.372 0.686 2301489 25K 1301 1 99.9 258 622 421 0.437 0.563 0.492 0.303 NEW175 UHT 1297 5 99.6 4 109 1184 0.045 0.955 0.086 0.380 전체 SNP 에대한 call rate 의평균은다음표와같음. 175 개 set Range 17 개 set Range 완료 169(96.6%) 17/17(100%) call rate 99.46% 98~100% 99.24% 98.1~99.7% 각 sample 에대한 genotyping 결과는 CD file 에 excel 형태로첨부하였음. - 14 -
3) Hardy-Weinberg Equilibrium test - 전체대상자에대한 HWE test 결과 7 개 SNP 이 p<0.001 에서 HWE 가깨진결과 (Hardy weinberg disequilibrium, HWD) 를보였음. 17 개 SNP set 에서도높은 Homology 를보인 PIK3CA 의 21142T/C 가 HWD 결과를보였음. HWE test p value 175개 SNP 17개 SNP p<0.001 7 0 p<0.01 11 0 p<0.05 20 2 Total 20 2-15 -
당뇨군과대조군의유전형빈도및 HWE test 결과 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p ACAS2 UP NEW1 GG GT TT N DM 354 240 48 642 0.262 0.386 0.839 Control 357 259 44 660 0.263 0.388 0.410 INTRON 2295096 AA AG GG DM 304 260 70 634 0.315 0.432 0.640 Control 315 277 66 658 0.311 0.428 0.204 EXON2 Phe91Phe 4911163 CC CT TT DM 181 314 147 642 0.474 0.499 0.484 Control 179 339 142 660 0.472 0.498 0.629 INTRON NEW4 GG AG AA DM 625 17 0 642 0.013 0.026 0.708 Control 641 19 0 660 0.014 0.028 1.000 INTRON 3746450 GG GT TT DM 177 310 147 634 0.476 0.499 0.384 Control 171 334 140 645 0.476 0.499 0.617 INTRON 3818273 GG AG AA DM 179 313 145 637 0.473 0.498 0.389 Control 174 337 141 652 0.475 0.499 0.715 INTRON 2273683 CC CT TT DM 178 312 141 631 0.471 0.498 0.233 Control 176 339 135 650 0.468 0.498 0.847 CD36 UP 1049654 AA AC CC DM 311 278 52 641 0.298 0.418 0.580 Control 321 283 55 659 0.298 0.419 0.354 INTRON 3173798 TT CT CC DM 287 296 59 642 0.322 0.437 0.109 Control 264 322 74 660 0.356 0.459 0.162 EXON3 Pro89Ser 3765187 CC CT TT DM 548 91 3 642 0.076 0.140 0.967 Control 573 84 3 660 0.068 0.127 1.000 INTRON 3211864 AA AG GG DM 572 64 6 642 0.059 0.111 0.411 Control 585 73 1 659 0.057 0.107 0.016 INTRON 3211908 CC CT TT DM 476 153 10 639 0.135 0.234 0.613 Control 491 158 10 659 0.135 0.234 0.563 INTRON 1527483 GG AG AA DM 404 213 22 639 0.201 0.321 0.114 Control 412 225 20 657 0.202 0.322 0.344 DOWN 3211956 TT GT GG DM 393 221 25 639 0.212 0.334 0.072 Control 401 234 21 656 0.210 0.332 0.377 CYP11B2 UP NEW15 AA AT TT DM 291 298 53 642 0.315 0.431 0.417 Control 310 292 58 660 0.309 0.427 0.053-16 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p CYP11B2 UP 11781082 CC CT TT DM 515 116 7 638 0.102 0.183 0.215 Control 532 117 10 659 0.104 0.186 0.870 UP 10087214 CC CT TT DM 289 297 53 639 0.315 0.432 0.457 Control 309 290 59 658 0.310 0.428 0.053 UP 1799998 TT CT CC DM 290 296 51 637 0.312 0.430 0.458 Control 307 288 58 653 0.309 0.427 0.039 EXON 1 Pro36Pro NEW19 GG AG AA DM 603 24 9 636 0.033 0.064 <.0001 Control 614 33 7 654 0.036 0.069 <.0001 EXON 3 Lys172Arg 4539 AA AG GG DM 272 234 130 636 0.388 0.475 <.0001 Control 276 237 138 651 0.394 0.478 <.0001 INTRON 6399 CC CT TT DM 284 227 131 642 0.381 0.472 <.0001 Control 296 229 133 658 0.376 0.469 <.0001 EXON 5 Ala290Ala 4536 GG AG AA DM 269 229 144 642 0.403 0.481 <.0001 Control 269 245 143 657 0.404 0.482 <.0001 INTRON 4736320 TT AT AA DM 169 339 134 642 0.473 0.499 0.245 Control 189 314 157 660 0.476 0.499 0.134 EXON 8 Ser434Gly 4545 AA AG GG DM 170 316 148 634 0.483 0.499 0.871 Control 169 330 155 654 0.489 0.500 0.961 INTRON 6431 TT CT CC DM 438 192 0 630 0.152 0.258 <.0001 Control 444 202 0 646 0.156 0.264 <.0001 EXON 9 3097 GG AG AA DM 567 71 3 641 0.060 0.113 0.842 Control 574 78 3 655 0.064 0.120 0.491 EXON 9 3802230 GG GT TT DM 282 301 52 635 0.319 0.434 0.182 Control 291 301 60 652 0.323 0.437 0.022 EXON 9 NEW32 CC CT TT DM 288 298 53 639 0.316 0.432 0.462 Control 309 291 59 659 0.310 0.428 0.047 EXON 9 3802228 AA AG GG DM 271 303 57 631 0.330 0.442 0.475 Control 288 295 66 649 0.329 0.441 0.032 FABP1 UP 2970902 CC CG GG DM 488 139 9 636 0.123 0.216 0.112 Control 504 136 15 655 0.127 0.221 0.801 UP 2970901 CC AC AA DM 421 195 26 642 0.192 0.311 0.813 Control 413 217 30 660 0.210 0.332 0.569-17 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p FABP1 UP 2919872 GG AG AA DM 418 197 26 641 0.194 0.313 0.726 Control 409 213 30 652 0.209 0.331 0.646 EXON 3 Thr39Ala 2241883 AA AG GG DM 380 235 26 641 0.224 0.348 0.713 Control 421 215 24 660 0.199 0.319 0.164 INTRON 1545224 CC CT TT DM 160 308 170 638 0.508 0.500 0.005 Control 200 290 164 654 0.472 0.498 0.387 DOWN 2919867 TT CT CC DM 158 313 161 632 0.502 0.500 0.002 Control 202 286 165 653 0.472 0.498 0.812 FABP2 UP 13306357 TT CT CC DM 573 65 2 640 0.059 0.102 0.362 Control 579 75 4 658 0.063 0.118 0.715 UP 10034579 GG GT TT DM 225 307 106 638 0.407 0.483 0.485 Control 209 313 131 653 0.440 0.493 0.941 UP 2282688 GG AG AA DM 227 309 106 642 0.406 0.482 0.533 Control 212 316 131 659 0.439 0.492 0.961 UP 6857641 GG AG AA DM 227 309 103 639 0.403 0.481 0.632 Control 211 315 128 654 0.437 0.492 0.900 UP 1397614 GG AG AA DM 571 65 2 638 0.054 0.102 0.362 Control 579 75 4 658 0.063 0.118 0.708 INTRON 1397613 AA AC CC DM 227 309 106 642 0.406 0.482 0.482 Control 212 315 132 659 0.439 0.493 0.961 EXON 2 Ala54Thr 1799883 GG AG AA DM 303 276 63 642 0.313 0.430 1.000 Control 295 290 72 657 0.330 0.442 0.990 EXON 2 Asn71Asn 4834770 CC CT TT DM 225 307 106 638 0.407 0.483 0.483 Control 212 316 132 660 0.439 0.493 0.941 INTRON 11935130 GG GT TT DM 396 217 27 640 0.212 0.334 0.406 Control 418 210 32 660 0.208 0.329 0.689 INTRON 12510269 TT CT CC DM 242 306 93 641 0.384 0.473 0.931 Control 277 300 83 660 0.353 0.457 0.814 EXON 4 2964 AA AG GG DM 241 306 93 640 0.384 0.473 0.934 Control 277 300 83 660 0.353 0.457 0.795 EXON 4 9999724 GG AG AA DM 241 306 93 640 0.384 0.473 0.934 Control 277 300 83 660 0.353 0.457 0.795-18 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p FABP2 EXON 4 11724758 CC CT TT DM 213 312 115 640 0.423 0.488 0.684 Control 240 310 107 657 0.399 0.480 0.968 EXON 4 10212775 CC CG GG DM 241 306 93 640 0.384 0.473 1.000 Control 274 298 81 653 0.352 0.456 0.795 EXON 4 10213267 TT CT CC DM 235 305 93 633 0.388 0.475 1.000 Control 272 300 83 655 0.356 0.458 0.711 EXON 4 NEW57 CC CT TT DM 240 305 92 637 0.384 0.473 0.742 Control 276 298 85 659 0.355 0.458 0.757 EXON 4 NEW58 AA AG GG DM 278 271 85 634 0.348 0.454 0.418 Control 308 269 68 645 0.314 0.431 0.146 EXON 4 9996644 CC AC AA DM 242 306 92 640 0.383 0.473 1.000 Control 276 299 81 656 0.351 0.456 0.765 DOWN NEW60 AA AG GG DM 590 49 1 640 0.040 0.077 0.692 Control 599 60 1 660 0.047 0.090 1.000 FABP4 INTRON 2290200 AA AC CC DM 599 42 0 641 0.033 0.063 0.889 Control 612 46 1 659 0.036 0.070 1.000 DOWN 16909196 AA AT TT DM 566 73 2 641 0.060 0.113 0.806 Control 566 89 4 659 0.074 0.136 1.000 DOWN 1054135 AA AG GG DM 252 295 82 629 0.365 0.463 0.152 Control 284 303 62 649 0.329 0.441 0.765 SLC27A1 UP 8109373 GG GT TT DM 517 115 7 639 0.101 0.182 1.000 Control 531 117 6 654 0.099 0.178 0.831 UP 8110830 AA AG GG DM 199 325 109 633 0.429 0.490 0.811 Control 229 319 106 654 0.406 0.482 0.227 INTRON NEW66 GG AG AA DM 512 117 8 637 0.104 0.187 1.000 Control 530 121 6 657 0.101 0.182 0.654 INTRON NEW67 GG AG AA DM 547 86 3 636 0.072 0.134 0.105 Control 553 100 1 654 0.078 0.144 1.000 INTRON 10418156 TT CT CC DM 190 323 125 638 0.449 0.495 0.751 Control 223 314 117 654 0.419 0.487 0.559 INTRON 7251644 CC CG GG DM 189 322 126 637 0.451 0.495 1.000 Control 223 318 114 655 0.417 0.486 0.597-19 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p SLC27A1 INTRON 3746318 GG AG AA DM 391 219 28 638 0.216 0.338 0.900 Control 442 196 20 658 0.179 0.294 0.702 INTRON 11879407 TT GT GG DM 191 323 121 635 0.445 0.494 1.000 Control 220 319 114 653 0.419 0.487 0.452 EXON 12 NEW72 GG GT TT DM 616 24 2 642 0.022 0.043 0.188 Control 632 26 1 659 0.021 0.042 0.035 EXON 12 4808657 GG AG AA DM 406 213 22 641 0.200 0.321 0.789 Control 441 194 19 654 0.177 0.292 0.354 EXON 12 15401 GG AG AA DM 484 148 10 642 0.131 0.227 1.000 Control 467 177 16 660 0.158 0.267 0.731 EXON 12 9137 TT CT CC DM 479 147 11 637 0.133 0.230 0.882 Control 464 176 15 655 0.157 0.265 0.943 SLC27A4 UP 7030121 GG AG AA DM 317 262 63 642 0.302 0.422 0.927 Control 343 264 52 659 0.279 0.403 0.413 UP NEW77 GG CG CC DM 552 82 2 636 0.068 0.126 0.202 Control 572 78 5 655 0.067 0.125 0.760 INTRON NEW78 CC CT TT DM 389 216 32 637 0.220 0.343 0.286 Control 418 207 33 658 0.207 0.329 0.776 EXON 4 Gly208Ser 2240953 GG AG AA DM 393 212 35 640 0.220 0.344 0.275 Control 421 204 32 657 0.204 0.325 0.365 INTRON NEW80 GG AG AA DM 573 63 1 637 0.051 0.097 0.100 Control 589 62 4 655 0.053 0.101 1.000 INTRON NEW81 TT CT CC DM 392 214 34 640 0.220 0.344 0.278 Control 421 205 32 658 0.204 0.325 0.499 DOWN 7048106 AA AG GG DM 296 339 0 635 0.267 0.391 <.0001 Control 331 318 0 649 0.245 0.370 <.0001 ASPSCR1 UP NEW83 CC AC AA DM 168 323 148 639 0.484 0.500 0.694 Control 187 322 148 657 0.470 0.498 0.763 UP NEW84 GG CG CC DM 598 44 642 0.034 0.066 0.677 Control 617 40 1 658 0.032 0.062 1.000 INTRON NEW85 GG GT TT DM 602 39 0 641 0.030 0.059 0.332 Control 612 48 0 660 0.036 0.070 1.000-20 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p ASPSCR1 INTRON NEW86 CC AC AA DM 566 75 1 642 0.060 0.113 0.393 Control 584 74 1 659 0.058 0.109 0.718 EXON 7 Leu251Gln 8074498 TT AT AA DM 400 216 25 641 0.207 0.329 1.000 Control 401 227 32 660 0.220 0.344 0.533 INTRON NEW88 GG GT TT 0 DM 578 64 0 642 0.050 0.095 0.434 Control 591 66 3 660 0.055 0.103 0.394 EXON 14 Asp486Asp 13087 TT CT CC DM 316 268 57 641 0.298 0.418 1.000 Control 322 277 60 659 0.301 0.421 0.987 CACNA1G UP 7501581 GG CG CC DM 480 148 8 636 0.129 0.225 0.122 Control 484 154 19 657 0.146 0.250 0.364 UP NEW91 AA AG GG DM 607 32 1 640 0.027 0.052 0.016 Control 627 30 2 659 0.026 0.050 0.362 INTRON 12946808 GG GT TT DM 557 75 2 634 0.062 0.117 0.967 Control 561 81 3 645 0.067 0.126 1.000 EXON 8 Arg530Arg 12449998 AA AC CC DM 575 82 3 660 0.063 0.119 0.967 Control 575 82 3 660 0.067 0.124 1.000 INTRON 198551 GG AG AA DM 569 66 0 635 0.052 0.099 0.486 Control 572 68 1 641 0.055 0.103 0.403 INTRON 12936750 GG AG AA DM 521 105 5 631 0.091 0.166 0.812 Control 535 110 6 651 0.094 0.170 0.908 INTRON 3744521 CC CG GG DM 504 126 11 641 0.115 0.204 0.720 Control 501 149 9 659 0.127 0.221 0.342 INTRON NEW97 AA AG GG DM 458 171 7 636 0.145 0.249 0.750 Control 478 164 12 654 0.144 0.246 0.040 EXON 36 Pro2223Pro 739925 TT CT CC DM 462 166 11 639 0.147 0.251 0.880 Control 468 171 16 655 0.155 0.262 0.373 EXON 36 Pro2301Pro 9893223 GG AG AA DM 567 71 4 642 0.062 0.115 0.306 Control 572 83 5 660 0.070 0.131 0.293 PPARD INTRON 9658117 GG GT TT DM 560 78 4 642 0.067 0.125 0.194 Control 568 89 1 658 0.069 0.129 0.516 EXON 4 2016520 TT CT CC DM 360 243 31 634 0.241 0.365 1.000 Control 371 236 37 644 0.241 0.366 0.217-21 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p PPARD INTRON 2076168 TT GT GG DM 364 244 31 639 0.239 0.364 1.000 Control 375 244 39 658 0.245 0.370 0.221 EXON 7 Asn162Asn 2076167 TT CT CC DM 364 241 31 636 0.238 0.363 1.000 Control 373 243 39 655 0.245 0.370 0.266 INTRON 9658173 GG AG AA DM 601 36 0 637 0.028 0.055 0.662 Control 634 22 0 656 0.017 0.033 1.000 EXON 11 3734254 TT CT CC DM 368 246 27 641 0.234 0.358 1.000 Control 376 242 39 657 0.244 0.368 0.074 EXON 11 1053049 TT CT CC DM 388 226 26 640 0.217 0.340 0.265 Control 397 221 39 657 0.228 0.352 0.330 DOWN 760783 GG GT TT DM 387 231 21 639 0.214 0.336 0.564 Control 398 230 28 656 0.218 0.341 0.055 PPARG UP 12486170 AA AG GG DM 417 202 19 638 0.188 0.305 0.791 Control 442 197 20 659 0.180 0.295 0.355 EXON 6 Pro296Pro 13306747 CC CG GG DM 559 79 3 641 0.066 0.124 0.642 Control 597 62 1 660 0.048 0.092 0.755 SNAP23 INTRON 9302112 TT CT CC DM 343 259 38 640 0.262 0.386 0.525 Control 371 250 36 657 0.245 0.370 0.232 SNTG1 UP 3812426 AA AG GG DM 422 189 27 638 0.190 0.308 1.000 Control 412 215 28 655 0.207 0.328 0.321 UP 2247368 CC CT TT DM 447 171 19 637 0.164 0.274 0.610 Control 428 203 20 651 0.187 0.304 0.592 INTRON 4873415 GG AG AA DM 430 184 18 632 0.174 0.288 0.249 Control 421 207 18 646 0.188 0.305 0.751 INTRON 4873417 TT CT CC DM 418 201 23 642 0.192 0.311 0.319 Control 419 216 21 656 0.197 0.316 0.847 INTRON 4367560 TT CT CC DM 475 147 16 638 0.140 0.241 0.629 Control 487 155 14 656 0.139 0.240 0.258 INTRON 1481475 GG AG AA DM 449 165 13 627 0.152 0.258 0.463 Control 463 169 19 651 0.159 0.267 0.633 INTRON 7817955 TT CT CC DM 457 166 13 636 0.151 0.256 0.464 Control 463 169 19 651 0.159 0.267 0.645-22 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p SNTG1 INTRON 12542191 TT AT AA DM 409 205 26 640 0.201 0.321 1.000 Control 408 222 29 659 0.212 0.335 0.961 INTRON 1508620 AA AG GG DM 292 288 61 641 0.320 0.435 0.208 Control 299 301 59 659 0.318 0.434 0.408 EXON 15 Thr329Thr 1911830 AA AG GG DM 291 290 61 642 0.321 0.436 0.145 Control 298 303 58 659 0.318 0.434 0.356 INTRON 6473337 CC AC AA DM 320 276 45 641 0.285 0.408 0.087 Control 325 288 45 658 0.287 0.409 0.161 EXON 19 7837449 TT GT GG DM 553 85 3 641 0.071 0.132 0.051 Control 543 116 1 660 0.089 0.163 1.000 STXBP3 UP 1124426 CC AC AA DM 386 225 31 642 0.224 0.347 0.673 Control 367 252 39 658 0.251 0.376 0.807 EXON 1 1124427 CC CG GG DM 386 216 40 642 0.231 0.355 1.000 Control 369 248 42 659 0.252 0.377 0.191 INTRON 6604120 CC CT TT DM 381 215 40 636 0.232 0.356 1.000 Control 364 249 43 656 0.255 0.380 0.197 INTRON 1277044 CC CG GG DM 384 216 40 640 0.231 0.356 0.535 Control 369 243 45 657 0.253 0.378 0.199 INTRON 1277042 GG CG CC DM 384 216 40 640 0.231 0.356 0.539 Control 367 241 45 653 0.253 0.378 0.199 INTRON 1690734 GG GT TT DM 383 215 38 636 0.229 0.353 0.470 Control 369 239 45 653 0.252 0.377 0.290 INTRON 1298092 TT CT CC DM 382 219 39 640 0.232 0.356 0.535 Control 369 243 45 657 0.253 0.378 0.314 EXON 14 Leu371Leu 12132867 GG CG CC DM 397 210 33 640 0.216 0.338 0.447 Control 388 230 40 658 0.236 0.360 0.448 INTRON 1277239 GG AG AA DM 386 216 40 642 0.231 0.355 0.536 Control 371 243 45 659 0.253 0.378 0.191 INTRON NEW134 GG AG AA DM 638 3 0 641 0.002 0.005 0.953 Control 651 3 0 654 0.002 0.005 1.000 EXON 19 1149154 GG AG AA DM 386 215 40 641 0.230 0.354 0.530 Control 372 242 45 659 0.252 0.377 0.177-23 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p STXBP3 DOWN 1149153 GG CG CC DM 374 224 43 641 0.242 0.367 0.280 Control 361 245 52 658 0.265 0.390 0.234 STXBP4 UP 9902718 TT CT CC DM 592 47 2 641 0.040 0.076 0.603 Control 620 38 1 659 0.030 0.059 0.268 UP 17817382 GG AG AA DM 217 290 125 632 0.427 0.489 1.000 Control 223 316 112 651 0.415 0.485 0.117 UP 9303362 GG AG AA DM 220 294 125 639 0.426 0.489 0.877 Control 217 320 122 659 0.428 0.490 0.136 UP NEW140 AA AG GG DM 473 155 9 637 0.136 0.235 0.537 Control 470 171 12 653 0.149 0.254 0.354 UP 17818028 CC CG GG DM 593 47 2 642 0.040 0.076 0.092 Control 620 38 2 660 0.032 0.062 0.261 UP 10468513 CC AC AA DM 592 47 2 641 0.040 0.076 0.079 Control 619 37 2 658 0.031 0.060 0.268 EXON 3 Arg14Gly 1156287 AA AG GG DM 591 48 2 641 0.041 0.078 0.093 Control 619 38 2 659 0.032 0.062 0.282 INTRON 11658717 AA AG GG DM 592 48 2 642 0.040 0.078 0.093 Control 618 38 2 658 0.032 0.062 0.275 INTRON 1894936 AA AG GG DM 461 161 16 638 0.151 0.257 0.255 Control 466 170 21 657 0.161 0.271 0.665 EXON 10 17818142 AA AG GG DM 408 198 34 640 0.208 0.329 0.337 Control 412 224 23 659 0.205 0.326 0.127 UP 3811790 GG GT TT DM 272 292 73 637 0.344 0.451 0.502 Control 262 295 93 650 0.370 0.466 0.687 UCP1 UP 3811789 AA AT TT DM 519 116 7 642 0.101 0.182 0.906 Control 564 92 4 660 0.076 0.140 0.856 UP NEW149 GG AG AA DM 541 90 4 635 0.077 0.142 0.964 Control 554 93 4 651 0.078 0.143 0.786 INTRON 2071415 TT CT CC DM 491 138 13 642 0.128 0.223 0.824 Control 534 119 7 660 0.101 0.181 0.371 EXON 2 Ala63Thr NEW151 GG AG AA DM 527 105 7 639 0.093 0.169 0.897 Control 569 86 3 658 0.070 0.130 0.494-24 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p UCP1 INTRON 7688743 CC CT TT DM 381 218 36 635 0.228 0.352 0.640 Control 402 221 34 657 0.220 0.343 0.515 EXON 5 Met228Leu 2270565 AA AT TT DM 500 132 10 642 0.118 0.209 0.820 Control 539 115 5 659 0.095 0.172 0.704 UCP2 UP 659366 GG AG AA DM 208 284 147 639 0.452 0.495 0.533 Control 182 320 155 657 0.479 0.499 0.009 UP 17174145 CC CT TT DM 580 55 5 640 0.051 0.096 0.211 Control 614 44 2 660 0.036 0.070 0.018 EXON 1 NEW156 CC CT TT DM 534 97 7 638 0.087 0.159 0.337 Control 545 108 3 656 0.087 0.159 0.307 EXON 4 Ala54val 660339 CC CT TT DM 173 326 134 633 0.469 0.498 0.208 Control 162 339 144 645 0.486 0.500 0.393 UCP3 UP 1800849 CC CT TT DM 333 256 50 639 0.279 0.402 0.302 Control 322 282 50 654 0.292 0.414 0.935 EXON 3 NEW159 TT CT CC DM 328 262 50 640 0.283 0.406 0.459 Control 321 284 54 659 0.297 0.418 0.817 INTRON 2075576 GG AG AA DM 329 258 48 635 0.279 0.402 0.261 Control 318 283 50 651 0.294 0.415 0.792 INTRON 1726747 AA AT TT DM 144 334 161 639 0.513 0.500 1.000 Control 176 329 155 660 0.484 0.499 0.244 INTRON 3781907 TT CT CC DM 233 301 100 634 0.395 0.478 0.451 Control 243 319 92 654 0.385 0.473 0.865 INTRON 2734828 CC CT TT DM 407 235 0 642 0.183 0.299 <.0001 Control 410 249 1 660 0.190 0.308 <.0001 EXON 5 Tyr209Tyr 2075577 TT CT CC DM 146 337 154 637 0.506 0.500 1.000 Control 178 328 152 658 0.480 0.499 0.142 EXON 7 647126 CC CT TT DM 145 340 155 640 0.508 0.500 1.000 Control 177 330 152 659 0.481 0.499 0.112 DOWN 7930460 TT CT CC DM 324 263 52 639 0.287 0.409 0.082 Control 309 299 51 659 0.304 0.423 0.893 VAMP2 INTRON NEW167 GG CG CC DM 603 38 0 641 0.030 0.058 0.012 Control 626 29 2 657 0.025 0.131 1.000-25 -
Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p VAMP2 EXON 5 1061032 CC AC AA DM 175 309 150 634 0.480 0.499 0.279 Control 203 310 141 654 0.453 0.496 0.550 EXON 5 NEW169 CC CT TT DM 531 105 3 639 0.087 0.159 0.027 Control 574 79 7 660 0.070 0.131 0.610 EXON 5 11548690 CC CT TT DM 549 85 5 639 0.074 0.138 0.122 Control 561 90 7 658 0.079 0.146 0.383 EXON 5 1150 CC CT TT DM 158 303 175 636 0.513 0.500 0.586 Control 181 319 154 654 0.479 0.499 0.241 VAMP3 UP 707457 CC AC AA DM 581 59 2 642 0.049 0.093 0.377 Control 606 50 2 658 0.041 0.079 0.658 EXON 1 NEW173 CC AC AA DM 366 237 37 640 0.243 0.368 0.757 Control 365 249 39 653 0.250 0.375 0.867 INTRON 2301489 CC CG GG DM 209 315 118 642 0.429 0.490 0.155 Control 212 307 140 659 0.445 0.494 0.971 EXON 5 NEW175 AA AG GG DM 579 59 2 640 0.049 0.094 0.378 Control 605 50 2 657 0.041 0.079 0.665-26 -
4) SNP 의발현빈도 - 분석이완료된 SNP의발현빈도는 monomorphic인것이전체의 2.6% 이며 Minor allele frequency가 10% 미만인것이 28.4% 임. 175개 SNP set 17개 SNP set Monomorphic 5-0<MAF<0.05 20 2 0.05 MAF<0.1 25 2 0.1 MAF<0.2 27 6 0.2 MAF<0.3 41 5 0.3 MAF<0.4 25 1 0.4 MAF<0.5 30 1 Total 173 17 5) Haplotype 빈도 - 20 개유전자에서분석된 SNP 의수와 Haplotype block 및 5% 이상발현되는 Haplotype 의 수는다음과같음. SNP수 Block수 Haplotype 수 ACAS2 7 1 4 ASPSCR1 7 2 6 CACNA1G 10 1 2 CD36 7 2 6 CYP11B2 15 4 12 FABP1 6 2 6 FABP2 19 1 4 FABP4 3 1 3 PPARD 8 1 2 SLC27A1 12 3 10 SLC27A4 7 1 3 SNTG1 12 5 12 STXBP3 12 1 2 STXBP4 10 1 5 UCP1 7 1 3 UCP2 4 1 3 UCP3 9 2 6 VAMP2 5 2 6 VAMP3 4 1 3-27 -
- FABP4 의 LD 및 Haplotype frequency Haplotype Frequency A-A 0.65260 A-G 0.28051 T-G 0.06689 SNP1 SNP2 D' r^2 rs2290200 rs16909196 1 0.003 rs2290200 rs1054135 1 0.019 rs16909196 rs1054135 1 0.133 - SCL27A4 의 Haplotype frequency Haplotype Frequency G-G-C-G-G-T 0.70307 A-G-T-A-G-C 0.20885 A-C-C-G-G-T 0.05187 A-C-C-G-G-T 0.01475 A-G-C-G-G-T 0.01407 G-G-T-A-G-C 0.00313 A-G-T-G-G-C 0.00154 G-G-T-G-G-T 0.00116 - G-C-C-G-A-T 0.00078 G-G-C-G-A-T 0.00039 G-C-C-G-G-T 0.00039-28 -
- SCL27A4의 LD SNP1 SNP2 D' r^2 rs7030121 NEW77 0.968 0.165 rs7030121 NEW78 0.969 0.624 rs7030121 rs2240953 0.978 0.627 rs7030121 NEW80 0.958 0.124 rs7030121 NEW81 0.978 0.632 rs7030121 rs7048106 0.881 0.661 NEW77 NEW78 1 0.02 NEW77 rs2240953 1 0.019 NEW77 NEW80 0.992 0.758 NEW77 NEW81 1 0.019 NEW77 rs7048106 0.851 0.15 NEW78 rs2240953 1 0.984 NEW78 NEW80 1 0.015 NEW78 NEW81 1 0.993 NEW78 rs7048106 0.778 0.473 rs2240953 NEW80 1 0.015 rs2240953 NEW81 1 0.991 rs2240953 rs7048106 0.779 0.471 NEW80 NEW81 1 0.015 NEW80 rs7048106 0.931 0.138 NEW81 rs7048106 0.785 0.479-29 -
- UCP1 의 Haplotype block 과 haplotype frequency Haplotype Frequency G-C 0.77469 G-T 0.14387 A-T 0.08097 A-C 0.00047 - UCP1 의 LD SNP1 SNP2 D' r^2 rs3811790 rs3811789 0.704 0.027 rs3811790 NEW149 0.554 0.014 rs3811790 rs2071415 0.98 0.068 rs3811790 NEW151 1 0.049 rs3811790 rs7688743 0.01 0 rs3811790 rs2270565 0.674 0.03 rs3811789 NEW149 1 0.008 rs3811789 rs2071415 0.898 0.608 rs3811789 NEW151 0.99 0.895 rs3811789 rs7688743 0.993 0.326 rs3811789 rs2270565 0.89 0.645 NEW149 rs2071415 1 0.011 NEW149 NEW151 1 0.007 NEW149 rs7688743 0.947 0.262 NEW149 rs2270565 1 0.01 rs2071415 NEW151 1 0.685 rs2071415 rs7688743 0.594 0.156 rs2071415 rs2270565 0.875 0.709 NEW151 rs7688743 0.993 0.299 NEW151 rs2270565 0.984 0.717 rs7688743 rs2270565 0.679 0.189-30 -
- VAMP2의 LD SNP1 SNP2 D' r^2 NEW167 rs1061032 1 0.023 NEW167 NEW169 0.637 0.001 NEW167 rs11548690 0.399 0 NEW167 rs1150 1 0.028 rs1061032 NEW169 1 0.096 rs1061032 rs11548690 1 0.073 rs1061032 rs1150 0.998 0.894 NEW169 rs11548690 1 0.007 NEW169 rs1150 1 0.087 rs11548690 rs1150 1 0.082 - VAMP2 의 Haplotype block 과 haplotype frequency - 31 -
1 ACAS2 유전자 Haplotype Frequency GACGGGC 0.51309 TGTGTAT 0.26188 GATGTAT 0.15674 GGTGTAT 0.05367 GACAGGC 0.01379 TATGTAT 0.00041 GGTATAT 0.00002 GATATAT 0.00001 2 CD36 유전자 - 32 -
3 CYP11B2 유전자 - 33 -
4 FABP1 유전자 5 FABP2 유전자 Haplotype Frequency TGGGGAGCGCGATGCTGA 0.3409 TTAAGCATGTAGCCTCAC 0.32138 TGGGGAGCTCGATGCTGA 0.20891 CTAAACGTGTAGCCTCAC 0.05837 TTAAGCGTGTAGTCTCAC 0.04267 TGGGGAGCGCGATGCTAA 0.027 TGGGGAGCTTAGCCTTAC 0.00039 TTAAGCATGTAGCCTTAC 0.00039-34 -
6 SCL27A1 유전자 - 35 -
7 ASPSCR1 유전자 8 CACNA1C 유전자 Haplotype Frequency GAG 0.93314 TCA 0.05331 TCG 0.01003 TAG 0.00157 GCA 0.00079 GCG 0.00077 TAA 0.00038-36 -
9 PPARD 유전자 Haplotype Frequency TTTGTTG 0.73512 CGCGCCT 0.21272 TTTATTG 0.02099 CGCGCTG 0.01565 CGCGCCG 0.00625 CGCGTTG 0.00392 TGCGCCT 0.00121 CTTGTTG 0.00118 CGCACCT 0.00072 TTTGCCT 0.00042 TGCGCCG 0.00041 TTCGCCG 0.00038 TTTGCTG 0.00036 TGCACCT 0.00035 CGCACTG 0.00033 10 SNTG1 유전자 - 37 -
11 STXBP3 유전자 Haplotype Freq CCCCGGTGGGGG 0.74113 AGTGCTCCAGAC 0.21587 AGTGCTCGAGAC 0.01448 CCCCGGTGGGGC 0.01232 CGTGCTCCAGAC 0.00375 AGTCGGTGGGGG 0.00231 AGTGCTCCAAAC 0.00197 CCCCGGCGGGGG 0.00193 CCCGCTCCAGAC 0.00192 CGTGCTCGAGAC 0.00090 ACTGCTCCAGAC 0.00077 CCCCGGTGGAAC 0.00077 CCCGGTCCAGAC 0.00038 ACTGCTCGAGAC 0.00038 AGTGGGTGGGGG 0.00038 AGTGCGTGGGGG 0.00038 CGTGCTCCAAAC 0.00035 12 STXBP4 유전자 Haplotype Frequency GACCAAAA 0.36645 GACCAAAG 0.20639 AACCAAGA 0.15609 AGCCAAAA 0.14283 AACCAAAA 0.09176 AAGAGGAA 0.03533 GACCGGAA 0.00063 AAGAAAAA 0.00033 AGCCGGAA 0.00014 AGGAAAAA 0.00006-38 -
13 UCP2 유전자 Haplotype Frequency GCC 0.48855 ACC 0.38118 ACT 0.08410 GTC 0.04247 GCT 0.00271 ATC 0.00068 GTT 0.00030 ATT 0.00001 14 UCP3 유전자 각각의유전자의 LD test 결과및 haplotype block 와 haplotype frequency 는 CD 의 excel 파일 의 haplotype 기본정보 sheet 에나타내었음. SNP MAF >5% 이상일때추정된 haplotype 결과도 함께나타내었음 - 39 -
나. 당뇨병발생과의관련성 - 175개 SNP set의당뇨-대조군의 phenotype 자료를이용하여각 SNP과당뇨병발생과의관련성에대한분석을진행하였음. Monomorphic하거나 MAF 0.01미만인 rs13276226, 13268153, new30, new36, new127, new134, re11779229와 assay fail된 rs1973598, rs1149162를제외한 166개 SNP에대한 association 분석을진행하였음. - FABP1, SLC27A1, UCP1, VAMP2 유전자와당뇨병발생위험증가와유의한관련성을보였고 CACNA1G 유전자는당뇨병발생위험감소와유의한관련성을나타냄. 각모델별로유전자별당뇨발생에대한 OR 의 p value 를나타낸그림임. rs2919867 1.344(1.052-1.717) rs1545224 1.316(1.031-1.680) Dominant model rs3746318 1.293(1.029-1.623) rs3811789 1.392(1.039-1.865) p=0.05 -Log 10 (p-value) - 40 -
Co-dominant model rs3746318 1.261(1.037-1.534) rs3811789 1.372(1.044-1.802) New51,rs2071415 New169 p=0.05 -Log 10 (p-value) Recessive model rs7501581 0.428(0.186-0.984) p=0.05 -Log 10 (p-value) - 41 -
SNP 및 Haplotype 분석결과요약 Gene SNP Position DM Control DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p FABP1 1545224 INTRON 0.508 0.472 1.3161 0.027 1.141 0.086 1.0853 0.520 (1.0308,1.6804) (0.983,1.324) (0.846,1.3924) 2919867 DOWN 0.502 0.472 1.3437 0.018 1.122 0.043 1.011 0.932 (1.0519,1.7165) (0.966,1.304) (0.7863,1.2999) h1(act) 0.494 0.526 0.9647 0.779 0.888 0.066 0.7529 0.024 (0.7504,1.2403) (0.764,1.032) (0.5886,0.9631) h2(atc) 0.285 0.276 1.0857 0.462 1.043 0.677 0.9501 0.806 (0.8718,1.3521) (0.877,1.239) (0.6314,1.4296) h3(gtc) 0.212 0.192 1.1574 0.208 1.142 0.416 1.2398 0.475 (0.9219,1.453) (0.938,1.389) (0.6867,2.2383) FABP4 1054135 DOWN 0.365 0.329 1.164 0.181 1.178 0.106 1.4193 0.049 (0.9319,1.454) (0.998,1.392) (1.0003,2.0137) 16909196 DOWN 0.060 0.074 0.8065 0.195 0.805 0.190 0.5125 0.687 (0.5823,1.117) (0.59,1.097) J (0.0935,2.808) F h1(a-a) 0.635 0.671 0.7045 1 0.851 0.333 0.8644 0.598 (0.4965,0.9996) (0.721,1.005) (0.6919,1.0799) h2(a-g) 0.306 0.267 1.3165 0.043 1.281 0.063 1.5288 0.146 (1.0564,1.6405) (1.075,1.528) (1.0001,2.337) h3(t-g) 0.059 0.073 0.8029 0.581 0.801 0.567 0.5144 1 (0.5764,1.1183) (0.585,1.098) J (0.0939,2.8183) F SCL27A1 3746318 INTRON 0.216 0.179 1.2927 0.027 1.261 0.020 1.4643 0.199 (1.0294,1.6232) (1.037,1.534) CA (0.8162,2.6269) 11879407 INTRON 0.445 0.419 1.1811 0.164 1.114 0.363 1.113 0.458 (0.934,1.4936) (0.952,1.304) (0.8388,1.477) h1(gt) 0.555 0.582 0.8853 0.400 0.892 0.339 0.8441 0.158 (0.6667,1.1755) (0.762,1.045) (0.6672,1.0678) h2(gg) 0.231 0.237 1.0153 0.894 0.971 0.461 0.7418 0.248 (0.8132,1.2676) (0.806,1.169) (0.4461,1.2337) h3(ag) 0.214 0.181 1.2613 0.047 1.234 0.036 1.4103 0.250 (1.0034,1.5855) (1.013,1.502) CA (0.7827,2.5414) 15401 EXON 12 0.131 0.158 0.7899 0.060 0.799 0.045 0.6369 0.264 (0.6178,1.0099) (0.64,0.996) CA (0.2868,1.4141) CACNA1G 7501581 UP 0.129 0.146 0.9092 0.457 0.867 0.118 0.4278 0.040 (0.7077,1.1681) (0.694,1.083) (0.1859,0.9843) PPARD 9658173 INTRON 0.028 0.017 1.7262 0.046 (1.0039,2.9681) UCP1 3811789 UP 0.101 0.076 1.3923 0.026 1.372) 0.023 1.8079 0.340 (1.0394,1.8651) (1.044,1.802 CA (0.5267,6.2056) 2071415 INTRON 0.128 0.101 1.3034 0.051 1.3 0.032 1.928 0.157 (0.9986,1.7012) (1.022,1.653) CA (0.7642,4.8639) NEW151 EXON 2 0.093 0.070 1.3587 0.047 1.361 0.032 2.4182 0.219 (1.0041,1.8387) (1.026,1.805) CA (0.6226,9.3929) F - 42 -
Gene SNP Position DM Control DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p UCP1 7688743 INTRON 0.228 0.22 1.05 0.662 1.048 0.875 1.1012 0.695 1(0.8407,1.3138) (0.873,1.259) (0.6801,1.7832) h1(g-c) 0.771 0.781 0.8783 0.600 0.943 0.811 0.9426 0.605 (0.5405,1.4273) (0.785,1.133) (0.7538,1.1788) h2(g-t) 0.138 0.149 0.9154 0.486 0.922 0.762 0.8603 0.671 (0.7138,1.174) (0.743,1.145) (0.4298,1.7223) h3(a-t) 0.09 0.07 1.3107 0.081 1.31 0.077 2.0831 0.334 (0.9665,1.7777) (0.984,1.743) J (0.5187,8.3652) F VAMP2 1061032 EXON 5 0.480 0.453 1.1806 0.176 1.114 0.360 1.1276 0.368 (0.9284,1.5013) (0.956,1.297) (0.8683,1.4643) NEW169 EXON 5 0.087 0.070 1.3575 0.050 1.249 0.037 0.44 0.343 (0.9988,1.8451) (0.94,1.66) (0.1133,1.7091) F h1(c-c) 0.521 0.547 0.8891 0.379 0.901 0.387 0.8526 0.194 (0.6844,1.1551) (0.774,1.05) (0.6704,1.0844) h2(a-c) 0.393 0.384 1.0979 0.416 1.038 0.638 0.9736 0.860 (0.8766,1.3749) (0.888,1.213) (0.7226,1.3118) h3(a-t) 0.086 0.069 1.389 0.038 1.272 0.027 0.4415 0.343 (1.018,1.8952) (0.954,1.696) (0.1137,1.7151) F CA: Cochran-Armitage Trend Test, J: Jonckheere-Terpstra Test, F: Fisher's Exact test - 43 -
Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p ACAS2 NEW1 UP 0.9586 0.704 0.994 0.719 1.1313 0.569 (0.7706,1.1923) (0.835,1.183) (0.7401,1.7293) 2295096 INTRON 0.9969 0.978 1.021 0.816 1.1133 0.554 (0.8014,1.2402) (0.867,1.203) (0.7802,1.5885) 4911163 EXON2 0.9478 0.666 1.006 0.668 1.0833 0.549 (0.7434,1.2084) (0.862,1.174) (0.8341,1.407) NEW4 INTRON 0.9176 0.800 0.918 0.800 (0.4726,1.7817) (0.473,1.782) 3746450 INTRON 0.9315 0.572 1.002 0.585 1.0888 0.526 (0.7281,1.1916) (0.857,1.17) (0.8372,1.4161) 3818273 INTRON 0.9314 0.569 0.994 0.658 1.0681 0.623 (0.7291,1.1898) (0.851,1.162) (0.8212,1.3891) 2273683 INTRON 0.945 0.651 1.009 0.613 1.0977 0.493 (0.7397,1.2072) (0.863,1.18) (0.841,1.4329) CD36 1049654 UP 1.0077 0.945 0.999 0.981 0.9695 0.878 (0.8107,1.2526) (0.842,1.185) (0.6527,1.4402) 3173798 INTRON 0.8246 0.086 0.852 0.174 0.8014 0.228 (0.6617,1.0277) (0.721,1.008) (0.5587,1.1496) 3765187 EXON3 1.1298 0.447 1.118 0.448 1.0282 1.000 (0.8251,1.5468) (0.829,1.507) J (0.2068,5.113) F 3211864 INTRON 0.9674 0.852 1.041 0.892 6.2075 0.066 (0.6841,1.3681) (0.754,1.438) J (0.7452,51.7059) F 3211908 INTRON 1.0008 0.995 1.003 0.998 1.0318 0.945 (0.7797,1.2846) (0.798,1.259) (0.4265,2.4959) 1527483 INTRON 0.9782 0.848 0.996 0.881 1.1357 0.685 (0.7807,1.2256) (0.818,1.213) (0.6136,2.1018) 3211956 DOWN 0.9843 0.890 1.011 0.750 1.2312 0.489 (0.787,1.2311) (0.832,1.227) (0.682,2.2225) CYP11B2 NEW15 UP 1.0683 0.552 1.028 0.727 0.934 0.731 (0.8591,1.3285) (0.867,1.219) (0.6327,1.3786) 11781082 UP 1.0005 0.997 0.978 0.791 0.72 0.506 (0.7592,1.3184) (0.762,1.256) (0.2724,1.9032) 10087214 UP 1.0723 0.531 1.026 0.672 0.9182 0.667 (0.8619,1.334) (0.865,1.217) (0.6229,1.3535) 1799998 UP 1.0617 0.592 1.015 0.655 0.8928 0.572 (0.8529,1.3215) (0.855,1.205) (0.6026,1.3228) NEW19 EXON 1 0.84 0.471 0.937 0.468 1.3267 0.576 (0.5227,1.35) (0.649,1.351) (0.4911,3.5842) 4539 EXON 3 0.9849 0.893 0.981 0.945 0.9551 0.738 (0.7896,1.2285) (0.851,1.131) (0.7297,1.2501) 6399 INTRON 1.0307 0.786 1.016 0.963 1.0119 0.931 (0.8282,1.2828) (0.882,1.171) (0.7723,1.326) 4536 EXON 5 0.9613 0.726 0.995 0.831 1.0393 0.773 (0.7709,1.1989) (0.865,1.145) (0.7997,1.3509) 4736320 INTRON 1.1231 0.350 0.988 0.162 0.8451 0.207 (0.8803,1.4329) (0.847,1.153) (0.6507,1.0976) J: Jonckheere-Terpstra Test, F: Fisher's Exact test - 44 -
Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p CYP11B2 4545 EXON 8 0.9511 0.692 0.974 0.924 0.9804 0.880 (0.7421,1.2188 (0.834,1.137) (0.7578,1.2684) 6431 INTRON 0.9635 0.759 0.964 0.759 (0.7598,1.2219) (0.76,1.222) 3097 EXON 9 0.9249 0.648 0.934 0.651 1.0219 1.000 (0.661,1.294) (0.68,1.282) J (0.2055,5.0821) F 3802230 EXON 9 1.009 0.936 0.981 0.783 0.88 0.519 (0.8099,1.2572) (0.826,1.165) (0.5967,1.2979) NEW32 EXON 9 1.076 0.511 1.029 0.659 0.9198 0.673 (0.8649,1.3386) (0.868,1.22) (0.624,1.3558) 3802228 EXON 9 1.0598 0.607 1.007 0.598 0.8772 0.491 (0.8497,1.3219) (0.85,1.193) (0.6043,1.2733) FABP1 2970902 UP 1.0123 0.926 0.971 0.470 0.6124 0.245 (0.7816,1.311) (0.771,1.223) (0.2661,1.4097) 2970901 UP 0.8777 0.259 0.898 0.525 0.8864 0.659 (0.6997,1.101) (0.743,1.087) (0.5182,1.5161) 2919872 UP 0.8979 0.353 0.912 0.633 0.8765 0.630 (0.7154,1.127) (0.753,1.103) (0.5124,1.4994) 2241883 EXON 3 1.2099 0.095 1.166 0.248 1.1203 0.694 (0.9674,1.5131) (0.962,1.413) (0.6363,1.9727) FABP2 13306357 UP 0.857 0.381 0.849 0.373 0.5125 0.687 (0.6067,1.2105) (0.613,1.176) J (0.0936,2.8081) F 10034579 UP 0.864 0.215 0.873 0.211 0.794 0.110 (0.6858,1.0887) (0.748,1.02) (0.5982,1.0538) 2282688 UP 0.8671 0.224 0.876 0.222 0.7971 0.116 (0.6889,1.0913) (0.6007,1.0577) 6857641 UP 0.8645 0.215 0.872 0.205 0.7897 0.105 (0.6865,1.0886) (0.746,1.019) (0.5933,1.0511) 1397614 UP 0.86 0.392 0.852 0.384 0.5142 0.687 (0.6088,1.2148) (0.615,1.18) J (0.0938,2.8169) F 1397613 INTRON 0.8671 0.224 0.873 0.203 0.7895 0.101 (0.6889,1.0913) (0.748,1.019) (0.5952,1.0473) 1799883 EXON 2 0.9117 0.407 0.924 0.644 0.8841 0.499 (0.733,1.1341) (0.784,1.09) (0.6186,1.2635) 4834770 EXON 2 0.8686 0.231 0.877 0.225 0.797 0.115 (0.6899,1.0936) (0.751,1.023) (0.6008,1.0572) 11935130 INTRON 1.0643 0.587 1.025 0.662 0.8644 0.586 (0.8501,1.3325) (0.849,1.237) (0.5118,1.4599) 12510269 INTRON 1.1924 0.121 1.141 0.258 1.1798 0.308 (0.9548,1.4892) (0.973,1.339) (0.8582,1.6218) 2964 EXON 4 1.1974 0.112 1.144 0.244 1.1819 0.303 (0.9586,1.4956) (0.975,1.343) (0.8598,1.6248) 9999724 EXON 4 1.1974 0.112 1.144 0.244 1.1819 0.303 (0.9586,1.4956) (0.975,1.343) (0.8598,1.6248) 11724758 EXON 4 1.1538 0.220 1.106 0.431 1.1259 0.421 (0.918,1.4501) (0.946,1.293) (0.8432,1.5035) J: Jonckheere-Terpstra Test, F: Fisher's Exact test - 45 -
Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p FABP2 10212775 EXON 4 1.1969 0.114 1.149 0.233 1.2006 0.263 (0.9577,1.4959) (0.979,1.349) (0.8717,1.6536) 10213267 EXON 4 1.2028 0.106 1.148 0.231 1.1869 0.291 (0.9615,1.5047) (0.978,1.349) (0.8632,1.632) NEW57 EXON 4 1.192 0.122 1.131 0.287 1.1399 0.418 (0.954,1.4895) (0.964,1.327) (0.83,1.5656) NEW58 EXON 4 1.1704 0.161 1.157 0.189 1.3138 0.115 (0.9391,1.4586) (0.985,1.36) (0.9354,1.845) 9996644 EXON 4 1.1945 0.118 1.146 0.245 1.1918 0.283 (0.9561,1.4923) (0.976,1.345) (0.8648,1.6424) NEW60 DOWN 0.8322 0.356 0.841 0.357 1.0313 1.000 (0.563,1.2301) (0.575,1.23) J (0.0644,16.5231) F FABP4 2290200 INTRON 0.913 0.679 0.895 0.674 1.000 (0.5932,1.4051) (0.586,1.368) J F SLC27A1 8109373 UP 1.0187 0.896 1.026 0.948 1.1962 0.748 (0.7713,1.3455) (0.793,1.327) (0.3998,3.5789) 8110830 UP 1.1751 0.173 1.102 0.395 1.0754 0.627 (0.9315,1.4824) (0.94,1.292) (0.8023,1.4415) NEW66 INTRON 1.0189 0.894 1.035 0.837 1.38 0.551 (0.7737,1.3416) (0.803,1.333) (0.4761,3.9997) NEW67 INTRON 0.8909 0.463 0.919 0.479 3.0948 0.368 (0.6544,1.2128) (0.682,1.239) J (0.3211,29.8304) F 10418156 INTRON 1.22 0.096 1.131 0.243 1.1184 0.433 (0.9651,1.5422) (0.967,1.322) (0.8455,1.4792) 7251644 INTRON 1.2236 0.092 1.149 0.204 1.1702 0.272 (0.9678,1.5471) (0.982,1.344) (0.8837,1.5494) NEW72 EXON 12 0.988 0.966 1.025 0.970 2.0563 0.620 (0.5701,1.7122) (0.617,1.703) J (0.186,22.7327) F 4808657 EXON 12 1.1984 0.122 1.167 0.298 1.1878 0.588 (0.9529,1.5072) (0.956,1.426) (0.6366,2.2163) 9137 EXON 12 0.8013 0.078 0.818 0.201 0.7497 0.471 (0.6263,1.0252) (0.656,1.021) (0.3417,1.645) SLC27A4 7030121 UP 1.1128 0.335 1.116 0.394 1.2701 0.222 (0.8953,1.3832) (0.943,1.319) (0.8647,1.8656) NEW77 UP 1.0487 0.774 1.007 0.800 0.4101 0.452 (0.7577,1.4516) (0.742,1.367) J (0.0793,2.1215) F NEW78 INTRON 1.1104 0.361 1.074 0.635 1.0018 0.995 (0.8868,1.3903) (0.892,1.293) (0.6082,1.6499) 2240953 EXON 4 1.1212 0.320 1.099 0.598 1.1299 0.627 (0.8951,1.4044) (0.913,1.322) (0.6907,1.8485) NEW80 INTRON 0.9968 0.986 0.953 0.964 0.2559 0.374 (0.6936,1.4324) (0.677,1.343) J (0.0285,2.2957) F NEW81 INTRON 1.1238 0.309 1.096 0.595 1.0976 0.713 (0.8974,1.4074) (0.911,1.319) (0.6687,1.8014) 7048106 DOWN 1.1921 0.116 1.192 0.116 (0.9576,1.4841) (0.957,1.484) - 46 -
Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p ASPSCR1 NEW83 UP 1.1155 0.381 1.058 0.681 1.0367 0.786 (0.8736,1.4243) (0.907,1.234) (0.7998,1.3437) NEW84 UP 1.1073 0.650 1.077 0.655 1.000 (0.7131,1.7194) (0.699,1.66) J F NEW85 INTRON 0.826 0.391 0.826 0.391 (0.5334,1.279) (0.533,1.279) NEW86 INTRON 1.0456 0.797 1.044 0.797 1.0265 1.000 (0.7447,1.4679) (0.749,1.456) J (0.0641,16.4466) F 8074498 EXON 7 0.9328 0.542 0.925 0.652 0.7965 0.403 (0.746,1.1665) (0.766,1.117) (0.4665,1.3597) NEW88 INTRON 0.9484 0.772 0.909 0.752 0.2496F (0.6624,1.3579) (0.642,1.286) J 13087 EXON 14 0.9827 0.875 0.985 0.984 0.9744 0.894 (0.7906,1.2215) (0.833,1.165) (0.6663,1.4249) CACNA1G NEW91 UP 1.0652 0.804 1.029 0.809 0.5141 1.000 (0.6468,1.7543) (0.645,1.641) J (0.0465,5.6834) F 12946808 INTRON 0.9232 0.636 0.918 0.630 0.6772 1.000 (0.6633,1.2851) (0.67,1.259) J (0.1128,4.0666) F 12449998 EXON 8 0.9543 0.780 0.947 0.773 0.6876 1.000 (0.6877,1.3243) (0.692,1.295) J (0.1145,4.1287) F 198551 INTRON 0.9616 0.830 0.947 0.822 1.000 (0.673,1.3739) (0.665,1.348) J F 12936750 INTRON 0.9738 0.856 0.97 0.960 0.8586 0.802 (0.7305,1.298) (0.742,1.267) (0.2607,2.8277) 3744521 INTRON 0.8619 0.263 0.9 0.390 1.261 0.608 (0.6645,1.118) (0.711,1.139) (0.519,3.0638) NEW97 INTRON 1.0555 0.665 1.015 0.441 0.5954 0.274 (0.8265,1.348) (0.81,1.271) (0.2329,1.5221) 739925 EXON 36 0.9588 0.734 0.94 0.657 0.6995 0.364 (0.7524,1.2219) (0.756,1.167) (0.3221,1.5192) 9893223 EXON 36 0.8598 0.368 0.87 0.367 0.8213 1.000 (0.6186,1.195) (0.642,1.18) J (0.2196,3.0724) F PPARD 9658117 INTRON 0.9241 0.630 0.966 0.655 4.1191 0.212 (0.6703,1.2741) (0.71,1.313) J (0.4592,36.9532) F 2016520 EXON 4 1.0343 0.765 0.999 0.698 0.8434 0.496 (0.8287,1.2909) (0.831,1.201) (0.5165,1.3772) 2076168 INTRON 1.0011 0.992 0.971 0.670 0.8093 0.391 (0.8035,1.2473) (0.809,1.165) (0.4984,1.314) 2076167 EXON 7 0.9884 0.917 0.963 0.686 0.8093 0.392 (0.7928,1.2322) (0.802,1.155) (0.4984,1.3142) 3734254 EXON 11 0.9927 0.948 0.947 0.349 0.6968 0.158 (0.7966,1.2369) (0.788,1.138) (0.4213,1.1526) 1053049 EXON 11 0.9917 0.942 0.942 0.281 0.671 0.122 (0.7937,1.2391) (0.783,1.133) (0.4035,1.1159) 760783 DOWN 1.0045 0.969 0.973 0.627 0.7621 0.355 (0.8038,1.2554) (0.803,1.179) (0.4282,1.3566) - 47 -
Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p PPARG 12486170 UP 1.0795 0.515 1.058 0.788 0.9807 0.952 (0.8575,1.3589) (0.865,1.294) (0.5184,1.8553) 13306747 EXON 6 1.3901 0.063 1.396 0.061 3.0987 0.367 (0.9815,1.9687) (0.998,1.952) J (0.3215,29.8679) F SNAP23 9302112 INTRON 1.1232 0.298 1.096 0.580 1.0889 0.722 (0.9024,1.3981) (0.915,1.313) (0.6809,1.7412) SNTG1 3812426 UP 0.8678 0.223 0.904 0.452 0.9895 0.970 (0.6908,1.0901) (0.746,1.095) (0.5765,1.6985) 2247368 UP 0.8158 0.089 0.855 0.220 0.97 0.925 (0.6452,1.0315) (0.697,1.048) (0.5127,1.8352) 4873415 INTRON 0.879 0.277 0.907 0.523 1.0228 0.947 (0.6965,1.1093) (0.739,1.113) (0.5272,1.9843) 4873417 INTRON 0.9474 0.642 0.972 0.786 1.1235 0.704 (0.7547,1.1893) (0.799,1.184) (0.6155,2.051) 4367560 INTRON 0.9889 0.930 1.006 0.885 1.1796 0.655 (0.7705,1.2691) (0.809,1.253) (0.5709,2.4373) 1481475 INTRON 0.9763 0.847 0.951 0.626 0.7043 0.334 (0.766,1.2444) (0.768,1.177) (0.3448,1.4384) 7817955 INTRON 0.9646 0.771 0.941 0.602 0.6941 0.314 (0.7572,1.2288) (0.76,1.164) (0.3399,1.4176) 12542191 INTRON 0.9181 0.457 0.931 0.754 0.9199 0.762 (0.7329,1.15) (0.77,1.126) (0.5356,1.58) 1508620 INTRON 0.9927 0.948 1.009 0.926 1.0695 0.726 (0.798,1.2349) (0.853,1.195) (0.7346,1.5572) 1911830 EXON 15 0.9957 0.969 1.014 0.895 1.0879 0.661 (0.8004,1.2387) (0.857,1.201) (0.7461,1.5863) 6473337 INTRON 0.979 0.849 0.991 0.965 1.0285 0.898 (0.7876,1.217) (0.831,1.181) (0.6702,1.5784) 7837449 EXON 19 0.7385 0.048 0.769 0.052 3.0987 0.367 (0.5467,0.9978) (0.574,1.03) J (0.3215,29.8679) F STXBP3 1124426 UP 0.8364 0.112 0.858 0.256 0.8053 0.380 (0.6709,1.0428) (0.715,1.03) (0.496,1.3075) 1124427 EXON 1 0.8439 0.131 0.893 0.299 0.9761 0.916 (0.6769,1.0521) (0.747,1.066) (0.6241,1.5268) 6604120 INTRON 0.8343 0.108 0.884 0.262 0.9568 0.846 (0.6689,1.0407) (0.74,1.055) (0.6131,1.4931) 1277044 INTRON 0.8542 0.162 0.89 0.376 0.9067 0.663 (0.6849,1.0652) (0.746,1.062) (0.5836,1.4086) 1277042 INTRON 0.8555 0.166 0.89 0.384 0.9007 0.642 (0.6857,1.0672) (0.746,1.062) (0.5798,1.3994) 1690734 INTRON 0.8583 0.177 0.885 0.388 0.8586 0.503 (0.6876,1.0713) (0.741,1.057) (0.5495,1.3415) 1298092 INTRON 0.8653 0.199 0.893 0.432 0.8825 0.580 (0.694,1.079) (0.748,1.066) (0.5665,1.3749) 12132867 EXON 14 0.8796 0.259 0.895 0.488 0.84 0.471 (0.704,1.0991) (0.746,1.074) (0.5227,1.3497) - 48 -
Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p STXBP3 1277239 INTRON 0.8543 0.162 0.89 0.376 0.9066 0.663 (0.6852,1.0652) (0.746,1.062) (0.5836,1.4084) 1149154 EXON 19 0.8563 0.168 0.892 0.387 0.9081 0.668 (0.6867,1.0678) (0.747,1.064) (0.5846,1.4108) 1149153 DOWN 0.8677 0.205 0.889 0.406 0.838 0.408 (0.6966,1.0809) (0.747,1.057) (0.5509,1.2747) STXBP4 9902718 UP 1.3158 0.216 1.313 0.214 2.0595 0.620 (0.8513,2.0337) (0.866,1.989) J (0.1863,22.7683) F 17817382 UP 0.9964 0.976 1.051 0.443 1.1865 0.235 (0.7913,1.2548) (0.901,1.226) (0.8946,1.5736) 9303362 UP 0.935 0.568 0.991 0.654 1.0704 0.630 (0.7427,1.1772) (0.85,1.155) (0.8113,1.4124) NEW140 UP 0.8905 0.356 0.892 0.599 0.7655 0.547 (0.696,1.1393) (0.712,1.117) (0.3203,1.8295) 17818028 UP 1.2808 0.261 1.245 0.2627J 1.0281 1.000 (0.831,1.9739) (0.829,1.868) (0.1444,7.3208) F 10468513 UP 1.3137 0.218 1.272 0.220 1.0266 1.000 (0.85,2.0305) (0.846,1.914) J (0.1442,7.31) F 1156287 EXON 3 1.3092 0.219 1.269 0.221 1.0282 1.000 (0.851,2.0142) (0.847,1.902) J (0.1444,7.3211) F 11658717 INTRON 1.3049 0.225 1.265 0.226 1.025 1.000 (0.8482,2.0075) (0.844,1.897) J (0.1439,7.2986) F 1894936 INTRON 0.9368 0.596 0.929 0.716 0.7791 0.457 (0.7357,1.1928) (0.753,1.144) (0.4028,1.5069) 17818142 EXON 10 0.9485 0.646 1.018 0.177 1.5514 0.109 (0.757,1.1884) (0.842,1.23) (0.9034,2.6642) UCP1 3811790 UP 0.9061 0.384 0.893 0.289 0.7752 0.128 (0.7259,1.1312) (0.76,1.049) (0.5584,1.0762) NEW149 UP 0.9924 0.961 0.994 0.962 1.0254 1.000 (0.7297,1.3495) (0.745,1.327) J (0.2553,4.1176) F 2270565 EXON 5 1.2756 0.079 1.281 0.130 2.0696 0.177 (0.972,1.674) (0.997,1.646) (0.7035,6.0887) UCP2 659366 UP 0.7939 0.057 0.902 0.147 0.9677 0.803 (0.6259,1.0071) (0.777,1.048) (0.7479,1.252) 17174145 UP 1.3808 0.113 1.381 0.110 2.5906 0.281 (0.9251,2.061) (0.956,1.995) J (0.5008,13.4008) F NEW156 EXON 1 0.9562 0.765 1.001 0.358 2.4147 0.219 (0.7134,1.2816) (0.762,1.316) (0.6217,9.3792) F 660339 EXON 4 0.8918 0.368 0.932 0.650 0.9343 0.616 (0.6949,1.1445) (0.795,1.092) (0.7161,1.2189) UCP3 1800849 UP 0.8912 0.301 0.935 0.531 1.0255 0.904 (0.7166,1.1085) (0.787,1.111) (0.6819,1.5422) NEW159 EXON 3 0.9034 0.360 0.93 0.658 0.9495 0.800 (0.7267,1.123) (0.784,1.104) (0.6358,1.4179) 2075576 INTRON 0.8882 0.288 0.925 0.553 0.9829 0.935 (0.7137,1.1054) (0.778,1.101) (0.651,1.4841) - 49 -
Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p UCP3 1726747 INTRON 1.25 0.084 1.127 0.222 1.0974 0.473 (0.9702,1.6105) (0.964,1.317) (0.8516,1.4141) 3781907 INTRON 1.0175 0.880 1.046 0.685 1.144 0.390 (0.8115,1.276) (0.892,1.227) (0.8416,1.5549) 2734828 INTRON 0.9469 0.634 0.941 0.620 1.000 (0.7563,1.1856) (0.752,1.178) J F 2075577 EXON 5 1.2471 0.086 1.113 0.228 1.0614 0.649 (0.9689,1.6052) (0.952,1.302) (0.8213,1.3717) 647126 EXON 7 1.2536 0.080 1.117 0.214 1.066 0.625 (0.9735,1.6142) (0.955,1.306) (0.8252,1.377) 7930460 DOWN 0.8583 0.169 0.918 0.307 1.0561 0.791 (0.6903,1.0673) (0.773,1.091) (0.7061,1.5795) VAMP2 NEW167 INTRON 1.2726 0.331 1.18 0.339 0.500 (0.7817,2.0717) (0.74,1.88) J F 11548690 EXON 5 0.9481 0.736 0.939 0.849 0.7334 0.597 (0.6954,1.2927) (0.707,1.246) (0.2316,2.3229) 1150 EXON 5 1.1577 0.248 1.14 0.216 1.2325 0.102 (0.9031,1.4841) (0.98,1.328) (0.9591,1.5839) VAMP3 707457 UP 1.2236 0.306 1.2 0.308 1.025 1.000 (0.8309,1.8018) (0.831,1.734) J (0.1439,7.2986) F NEW173 EXON 1 0.9488 0.640 0.96 0.896 0.966 0.884 (0.7615,1.1822) (0.802,1.15) (0.6076,1.5358) 2301489 INTRON 0.9826 0.882 0.938 0.411 0.8348 0.195 (0.7789,1.2395) (0.805,1.093) (0.6351,1.0973) NEW175 EXON 5 1.2258 0.302 1.202 0.304 1.0266 1.00 (0.8323,1.8051) (0.832,1.736) J (0.1442,7.3103) F J: Jonckheere-Terpstra Test, F: Fisher's Exact test SNP 과당뇨병과의관련성에대한분석결과는첨부되는 CD excel 파일의 SNP 전체결과및 SNP 요약결과 sheet 에자세히나타내었음 - 50 -
다. 17 개 SNP set 분석 - 6개유전자의 17 개 KSNP를 6,653명환자, 내당장애및정상인집단에서분석하였다. 전체적인분석현황은다음과같음 GeneName rs# Pass Fail CallRate XX XY YY MAF HWE p-value INSR 6510976 6610 43 99.4 3849 2409 352 0.235 0.321 NEW202 6597 55 99.2 5113 1277 207 0.128 0.000 2860178 6631 22 99.7 6515 115 1 0.009 0.497 2303672 6637 16 99.8 25 750 5862 0.940 0.847 IRS1 1801123 6528 125 98.1 458 2570 3500 0.733 0.640 1801278 6631 22 99.7 6515 115 1 0.009 0.497 IRS2 1865434 6565 88 98.7 326 2384 3855 0.769 0.083 NEW208 6599 54 99.2 3883 2385 331 0.231 0.151 CBL 2510152 6606 47 99.3 145 1675 4786 0.851 0.913 RAPGEF1 11243480 6615 38 99.4 3972 2298 345 0.226 0.594 4740174 6614 39 99.4 4904 1572 138 0.140 0.362 17148121 6546 107 98.4 2474 3076 996 0.387 0.432 2274600 6615 38 99.4 5864 733 18 0.058 0.329 11243446 6625 28 99.6 5034 1479 112 0.129 0.779 943851 6568 85 98.7 1232 3261 2075 0.564 0.435 PIK3CA 2699895 6639 14 99.8 4398 2030 211 0.185 0.209 21142T/C 6598 55 99.2 0 2328 4270 0.824 0.000-51 -
- 대량분석결과각각의 SNP 빈도는다음과같음 GeneName rs# SNP discovery-ksnp DNALINK INSR 6510976 G 0.783 A 0.217 G 0.765 A 0.235 New202 G 0.813 C 0.188 G 0.872 C 0.128 2860178 T 0.696 C 0.304 C 0.991 T 0.009 2303672 A 0.938 G 0.063 A 0.94 G 0.06 IRS1 1801123 A 0.761 G 0.239 A 0.733 G 0.267 novel(1801278) G 0.991 A 0.009 IRS2 1865434 A 0.870 G 0.130 A 0.769 G 0.231 New208 C 0.875 D 0.125 C 0.769 D 0.231 CBL 2510152 T 0.875 G 0.125 T 0.851 G 0.149 RAPGEF1 11243480 G 0.848 A 0.152 G 0.774 A 0.226 4740174 G 0.854 A 0.146 G 0.86 A 0.14 17148121 G 0.659 A 0.341 G 0.613 A 0.387 2274600 G 0.891 A 0.109 G 0.942 A 0.058 11243446 G 0.795 A 0.205 G 0.871 A 0.129 943851 T 0.688 C 0.313 T 0.564 C 0.436 PIK3CA 2699895 C 0.896 A 0.104 G 0.815 A 0.185 novel(21142t/c) T 0.870 C 0.130 T 0.824 C 0.176 Chr 19 : INSR www.hapmap.org 19p13.2 r2 = 0.001 Caucasian Chinese Japanese Af-American KR_DNALink rs2303672 A G 1 0 0.91 0.09 0.96 0.05 1 0 0.94 0.06 rs6510976 C T 0.48 0.52 0.80 0.20 0.82 0.18 0.30 0.70 0.78 0.22-52 -
Chr 2 : IRS1 2q36.3 r 2 = 0.043 Caucasian Chinese Japanese Af-American KR-DNAlink rs1801278 G A 0.94 0.06 0.98 0.02 0.92 0.08 0.90 0.10 0.99 0.01 rs1801123 A G 0.95 0.05 0.74 0.26 0.67 0.33 0.58 0.42 0.73 0.27 Chr 9 : RAPGEF1-53 -
표. RAPGEF1 유전자의 International HapMap 의다인종에서의발현빈도 rs943851 rs11243446 rs2274600 rs17148121 rs11243480 C T C T C T C T C T Caucasian 0.22 0.78 0.98 0.03 0.99 0.01 0.85 0.15 0.66 0.34 Chinese 0.39 0.61 0.96 0.04 0.90 0.10 0.59 0.41 0.78 0.22 Japaneses 0.41 0.59 0.92 0.09 0.90 0.10 0.73 0.27 0.69 0.31 Af -American 0.27 0.73 0.91 0.09 0.93 0.08 0.48 0.53 0.89 0.11 KR_DNALink 0.436 0.564 0.871 0.129 0.942 0.058 0.613 0.387 0.774 0.226-54 -
5. 검토사항 가. Minor allele frequency 차이 - 국립보건원 SNP discovery 결과의 SNP 빈도와대량분석의 genotyping 결과의빈도가유의 적인차이를보이는경우가있음. - CYP11B2 rs11779229에대해서 Long PCR product를이용한분석과이용하지않은분석으 로나누어동일한 PCR PRIMER를사용하여 1232개 sample의분석결과를비교해보았음. Long PCR를실시하지않은 genotyping결과는 SNP discovery 결과와유사한빈도를보였 으나 Long PCR를실시한결과와비교시분석결과의일치율은 70% 였음다. Method Genotyping Long PCR +genotyping CC 867 1208 CT 332 24 TT 33 0 C:T 0.838:0.162 0.99:0.01 - SNP discovery 에서 polymorphism 이발견되었다고보고된 CYP11B2 rs13276226, rs13268153, new30 등도높은 gene homology 에의한 false positive 결과로생각된다. - CYP11B2의유전자는동일염색체 8번에위치한 CYP11B1와높은 homology를나타내기때문에 CYP11B2 특이적인염기서열을이용한 Long PCR을실시한후그 product를이용하여 target SNP에대한 genotyping 분석을실시해야정확한분석결과를얻을수있음을확인하였음. - 55 -
- 제공된 SNP discovery의빈도와대량분석및일본인에서보고된빈도와의비교시 SNP discovery결과의빈도와차이남을확인할수있음. HapMap Japanese의알려진빈도와비교한결과는다음과같음 SNP discovery DNALINK Japanese(HapMap) CYP11B2 NEW30 T 0.708 C 0.292 T 1.000 C 0.000 13276226 C 0.833 T 0.167 C 1.000 T 0.000 G 0.913 A 0.088 13268153 G 0.833 A 0.167 G 1.000 A 0.000 11779229 C 0.833 T 0.167 C 0.990 T 0.010 FABP1 NEW36 T 0.696 A 0.261 T 1.000 A 0.000 STXBP3 NEW127 T 0.429 C 0.2 T 1.000 C 0.000 NEW134 G 0.938 A 0.063 G 0.998 A 0.002 FABP2 10034579 T 0.723 G 0.255 G 0.576 T 0.424 1397613 C 0.875 A 0.125 A 0.577 C 0.423 A 0.614 C 0.386 SLC27A1 8110830 A 0.75 G 0.25 A 0.583 G 0.417 PPARD 2016520 T 0.917 C 0.083 T 0.759 C 0.241 T 0.795 C 0.205 2076168 T 0.917 G 0.083 T 0.758 G 0.242 T 0.795 G 0.205 2076167 T 0.917 C 0.083 T 0.758 C 0.242 T 0.795 C 0.205 3734254 T 0.915 C 0.064 T 0.761 C 0.239 1053049 T 0.917 C 0.083 T 0.778 C 0.222 760783 G 0.87 T 0.087 G 0.784 T 0.216 G 0.86 T 0.14 SNTG1 1508620 A 0.917 G 0.083 A 0.681 G 0.319 INSR rs2860178 T 0.696 C 0.304 C 0.991 T 0.009 C 0.919 T 0.081 나. Novel SNP 여부 SNP discovery 에서 Novel SNP 으로보고된 site 중 FABP2 의 New45 는 rs1973598, New48 은 rs1799888 가같은 site 였으며 ASPSCR1 의 New89 는 rs13087, PPARD 의 New102 는 rs2076186, STXBP3 New135 는 rs1149154, IRS1 의 G971R 은 rs1801278 과같은 site 로확인되었다. 다. phenotype 자료 6500 명에대한추가 Phenotype 자료가없어더이상의통계분석이어려움. 자료가추가되는 대로 follow-up 하겠음. - 56 -
6. 연구결과에대한기여도, 기대효과및활용방안 가. 기대효과 - 한국인당뇨병관련유전자발굴을통한환자의진단, 치료방법및신약개발의기초제공. - 난치성질환의진단, 치료에필요한정보를제공함으로써국가의료서비스향상 - 기존에밝혀진 SNP 및새로운 SNP 들에대한 validation 을통한한국인특이 SNP 의체계 적인발굴을통한국내 SNP 연구의활성화 - 관련 bioinformatics 기법발전 - Genome information 에근거한신개념의신약개발활성화 나. 활용방안 - 한국인당뇨병관련유전자분석을통한조기진단, 예후예측용 DNA chip 제작에필요한 contents 제공. - 한국인특이 SNP 을중심으로한당뇨병관련 SNP database 구축을통한연구 infra 제공 - 특정유전자또는유전체부위에대한 haplotype mapping, 또는 linkage disequilibrium block mapping 에응용 - 질병의발병, 예후약물반응등을예측할수있는 marker set 의개발을통한예측의학분야 및 personalized medicine 분야에활용. - 57 -
부록 1. Assay Information Gene Primer Type Primer sequence ACAS2 Forward Primer GTGAAATGATGGTGAGGTGG (NEW1) Reverse Primer AGCTTCTCTCTAGCCCCT Extension Primer(F) GAGCGAGTGACGCATACTATTGTGGTGAGTGCAGGTGAGAGATG ACAS2 Forward Primer TCAGTGAGGAGAAGAGTGGG (rs2295096) Reverse Primer TTTTCCATTTTCAAAGAACCG Extension Primer(R) TCCATCTCCTCACCCACCCCTCAGC ACAS2 Forward Primer CATTCCTTCGGTACAACTTTG (rs4911163) Reverse Primer TTTTTCTCATGGACATTTCGA Extension Primer(R) CGTGCCGCTCGTGATAGAATTAGTTGCTCCTTTCATCCACTCAAT ACAS2 Forward Primer TTCTTTCTGTTCCATTCCATC (NEW4) Reverse Primer AAGAACTGTGAACAGCTCAAGAT Extension Primer(R) AGATAGAGTCGATGCCAGCTGAGGTGGGACAGGCAGGTAGGAGTA ACAS2 Forward Primer TAGCTGGGGGATGGACAA (rs3746450) Reverse Primer AAGATACCATCCTAGYCAGGATAC Extension Primer(F) AGATAGAGTCGATGCCAGCTGGTGACTACCTCCCAAGGCTGCTTG ACAS2 Forward Primer TGCCCCATCGTGGACACC (rs3818273) Reverse Primer AGTCAGAGATGAGAAATTTGAGTCA Extension Primer(F) GACCTGGGTGTCGATACCTACTGGCAAACAGAGACAGTGAGTGAA ACAS2 Forward Primer TAGTTAGATAGTGGAGAACTGGACTG (rs2273683) Reverse Primer ATTGCAGGAGCTACACCAAA Extension Primer(F) ACGCACGTCCACGGTGATTTTGTGAAGAATTTGGGGTTCTGGGGC CD36 Forward Primer AGAATTTTTTTCTATTTACCCATGC (rs1049654) Reverse Primer ATGTTTTATTTACGATACCTTATTCACA Extension Primer(F) AGCGATCTGCGAGACCGTATGATTCTTTCTGTGACTCATCAGTTC CD36 Forward Primer CCTGTACTTACTACAAAGACATAACCC (rs3173798) Reverse Primer TTCTGTGCCTGTTTTAACCC Extension Primer(R) ACGCACGTCCACGGTGATTTCCTTCTTCGAGGACAACTTGCTATG CD36 Forward Primer AGGAAGTGATGATGAACAGCA (rs3765187) Reverse Primer AAATACATGGTCAAGGTAAGAGTGT Extension Primer(F) GGCTATGATTCGCAATGCTTCAACATTCAAGTTAAGCAAAGAGGT CD36 Forward Primer TATTATAAACTTACCAGTATCAAATAACTTCC (rs3211864) Reverse Primer AAAACTGATTTTTTTTTCCTCAGC Extension Primer(F) AGCGATCTGCGAGACCGTATGAAGTTAGTGTTTTGTTATTGTGGC CD36 Forward Primer TAAAATGTGCATGTATGTTACTAGGG (rs3211908) Reverse Primer TAAAGAGTTAATACCTAGCAGAACAGG Extension Primer(F) AGAGCGAGTGACGCATACTAAAGACATAGGCATCAACCTRTAGAA CD36 Forward Primer ATCACATTCTTGAAAGTTACTGAA (rs1527483) Reverse Primer TCACATATACTCTTTGACTTTTGC Extension Primer(F) TCTTGAAAGTTACTGAAACTTAGGTC CD36 Forward Primer TTGTGCCAAAGTTGTCCAA (rs3211956) Reverse Primer TCTTATGAAACYATTTCCTAGTCAATTAT Extension Primer(F) AGCGATCTGCGAGACCGTATTTGACTGGTTCATTTCTCAATTATA CYP11B2 Forward Primer ATGATAAAAAGATCGGAGATGT (NEW15) Reverse Primer TATTTTCACTGTCTTAAAAAGAGG Extension Primer(F) CAGGGACTGGGGCAGGAGGGATGAGC CYP11B2 Forward Primer TCAGGGACTGGGGCAGGA (rs11781082) Reverse Primer TGCAGGATCCTAGGAGTATTTTC Extension Primer(F) GATGAGCAGGCAGAGCACAGGTTTT CYP11B2 Forward Primer TGGTTGATAACAACGTATCGA (rs10087214) Reverse Primer AAAAACTGCAGAAAATGTCCAC Extension Primer(F) CGTGCCGCTCGTGATAGAATATTCCTCACATGGAACCAGTGCGCT - 59 -
Gene Primer Type Primer sequence CYP11B2 Forward Primer TGAACTAAATCTGTGGTATAAAAATA (rs1799998) Reverse Primer GTCCATGCTGGTGGAAGG Extension Primer(F) GCGGTAGGTTCCCGACATATAAAGTCTATTAAAAGAATCCAAGGC CYP11B2 Long PCR Forward Primer CACCTGaCTTCTCCTTCaTC (NEW19) Long PCR Reverse Primer CCTGTCTCTGTGAAGCATCT Forward Primer GGCACTGGGCACTAGAGC Reverse Primer AGCCTCAGCCACCTGTTG Extension Primer(F) CTCRGGCCCCTAGGACGGTGCTGCC CYP11B2 Long PCR Forward Primer CACCTGaCTTCTCCTTCaTC (rs13276226) Long PCR Reverse Primer CCTGTCTCTGTGAAGCATCT Sequencing Forward Primer ACTTTGGATTGGGACTGCA Reverse Primer CCCAGACCGCAAGAGAAA CYP11B2 Long PCR Forward Primer CACCTGaCTTCTCCTTCaTC (rs13268153) Long PCR Reverse Primer CCTGTCTCTGTGAAGCATCT Sequencing Forward Primer ACTTTGGATTGGGACTGCA Reverse Primer CCCAGACCGCAAGAGAAA CYP11B2 Long PCR Forward Primer CACCTGaCTTCTCCTTCaTC (rs11779229) Long PCR Reverse Primer CCTGTCTCTGTGAAGCATCT Forward Primer TGCAGAGAAGACGCAGAG Reverse Primer CCCAGACCGCAAGAGAAA Extension Primer ç GCAGCCGCCGAGGTTGGTGCAGAGC CYP11B2 Long PCR Forward Primer AGATGCTTCACAGAGACAGG (rs4539) Long PCR Reverse Primer CTCATCCAACTTTCTTCCAG Forward Primer TCCCGATGGTGGATGCAG Reverse Primer ACGTCCAGGGTCAGGCTC Extension Primer(F) GCCAGGGACTTYTCCCAGGCCCTGA CYP11B2 Long PCR Forward Primer AGATGCTTCACAGAGACAGG (rs6399) Long PCR Reverse Primer CTCATCCAACTTTCTTCCAG Forward Primer GCATCTTCCACTACACCATAG Reverse Primer TCAGGCTGGCAGAACTG Extension Primer(F) GGAGTGTGGGGAGGCAGCCAGGAGG CYP11B2 Long PCR Forward Primer AGATGCTTCACAGAGACAGG (rs4536) Long PCR Reverse Primer CTCATCCAACTTTCTTCCAG Forward Primer AAAATCTAYCAGGAACTGGCC Reverse Primer ATAGAGTTGGCCTTGATGGC Extension Primer(F) CTCAACACTACACAGGCATCGTGGC CYP11B2 Long PCR Forward Primer AGTGGTCATCAAGGTTTCAG (rs4736320) Long PCR Reverse Primer AGATCCTACCCTGTCTTGCT Forward Primer CTCAGCAGGTGCAAGGAA Reverse Primer ATCCAYAGAAAGGGTCCTCA Extension Primer(F) GCGGGGTCTTSGGTAGGAAGGGTGC CYP11B2 Long PCR Forward Primer AGTGGTCATCAAGGTTTCAG (rs4545) Long PCR Reverse Primer AGATCCTACCCTGTCTTGCT Forward Primer CCTGAGCGGTATAATCCCC Reverse Primer ACTGGCGCATGCCAAAGC Extension Primer(R) AAGGGCACGTGGTGGAAGTTCCTGC CYP11B2 Long PCR Forward Primer ATTGACACCTGGGAACAGT (rs6431) Long PCR Reverse Primer GTGCTGACTAGAGGAGTTGG Forward Primer AGGGAGGATCTGTGCTGAG Reverse Primer CTCCACCCCACTCCCCTA Extension Primer(F) AAGACAGGGTAGGATCTGGGTGAGG CYP11B2 Long PCR Forward Primer ATTGACACCTGGGAACAGT (rs3097) Long PCR Reverse Primer GTGCTGACTAGAGGAGTTGG - 60 -
Gene Primer Type Primer sequence CYP11B2 Forward Primer ACCAGCTTCCCTCTGCCT (rs3097) Reverse Primer TTCCATTTGTGCAGGAGC Extension Primer(R) GAGGGGTGACTCAGGAAGCTGTGCA CYP11B2 Long PCR Forward Primer ATTGACACCTGGGAACAGT (NEW30) Long PCR Reverse Primer GTGCTGACTAGAGGAGTTGG Sequencing Forward Primer ACCAGCTTCCCTCTGCCT Reverse Primer GTGCTGACTAGAGGAGTTGG CYP11B2 Long PCR Forward Primer ATTGACACCTGGGAACAGT (rs3802230) Long PCR Reverse Primer GTGCTGACTAGAGGAGTTGG Forward Primer TGACCCCAGGCAGCTCGA Reverse Primer GATAAGTGACCACAGGGGC Extension Primer(F) GAGCTCTCTGAAAGTTGTCACCCTG CYP11B2 Long PCR Forward Primer ATTGACACCTGGGAACAGT (NEW32) Long PCR Reverse Primer GTGCTGACTAGAGGAGTTGG Sequencing Forward Primer ACCAGCTTCCCTCTGCCT Reverse Primer GTGCTGACTAGAGGAGTTGG CYP11B2 Long PCR Forward Primer ATTGACACCTGGGAACAGT (rs3802228) Long PCR Reverse Primer GTGCTGACTAGAGGAGTTGG Sequencing Forward Primer ACCAGCTTCCCTCTGCCT Reverse Primer GTGCTGACTAGAGGAGTTGG FABP1 Forward Primer AATCTGCATGGCAGGACA (rs2970902) Reverse Primer TTAAGGTGTACCCTGCCACA Extension Primer(F) CATTAGTGAGTGTTCTCTGCCCACA FABP1 Forward Primer AAGGTAGACTGGAGTTGCCTTT (rs2970901) Reverse Primer TCACGTGACGGGTCATGA Extension Primer(F) AGAGCGAGTGACGCATACTACAAAAATTAGAACTCATGATAGGGA FABP1 Forward Primer ATCATGACCCGTCACGTG (NEW36) Reverse Primer CCACTGCTCCAGGCATGA Extension Primer(F) AGATAGAGTCGATGCCAGCTACTAGGCCATCCAGGACCTGTGGCC FABP1 Forward Primer CAGACAAAGAAGGGAGAGA (rs2919872) Reverse Primer TTTGTTGTGAAGCACAGAGAG Extension Primer(F) CGACTGTAGGTGCGTAACTCTCTAGGGGTGAGGGGGTGCTTGTAA FABP1 Forward Primer TTCCTCACAGGTCACCTCTC (rs2241883) Reverse Primer ACACGCTCAGAGCACCAC Extension Primer(F) GGAAGGTGACAATAAACTGGTGACA FABP1 Forward Primer TWACTGAGGATCCATCATGTCC (rs1545224) Reverse Primer ATGCAGGGCAGGTGGAGG Extension Primer(F) AGGGTCTCTACGCTGACGATGGCAGTTTACACACATTATCACTAA FABP1 Forward Primer TATTGCTCACCATATTCTCGTAAA (rs2919867) Reverse Primer AGCATGGGTTTTGTGTTCA Extension Primer(F) AGATAGAGTCGATGCCAGCTCGTAAATGTTGATAATCCACATGAT FABP2 Forward Primer ATTAAAACTCTTTACTTCACACCAAGTT (rs13306357) Reverse Primer AGGGTATAGAGGGTAGCTAGAAAGG Extension Primer(F) AGGGTCTCTACGCTGACGATGCCTGAAGTAGGGAAAGGAGAGATA FABP2 Forward Primer AGCTACCCTCTATACCCTGAC (rs10034579) Reverse Primer ATCCAGTTTTAGCTGTGCCA Extension Primer(F) GTGATTCTGTACGTGTCGCCCTACTGGGTTTGGGGATGAGAGGCA FABP2 Forward Primer TAAACACAATGCAAAAAGACATATTAG (rs2282688) Reverse Primer ATATATCAAATAACATCTGGGAAATGA Extension Primer(R) AGAGCGAGTGACGCATACTATTTATGATGAAATACAGTTGAAAAG FABP2 Forward Primer TATAAATTATATTRCCTCCAACATAGTCTG (rs6857641) Reverse Primer TCAACTGAATTAAAACATGAGAAGC Extension Primer(F) GGATGGCGTTCCGTCCTATTGAAATAAGTATGCCCACAAACTGTA - 61 -
Gene Primer Type Primer sequence FABP2 Forward Primer AAGATCTCAGGAATGTACAGAACG (rs1397614) Reverse Primer GGGCGAGAATTTATTATATTTCTTATC Extension Primer(F) AGGGTCTCTACGCTGACGATCTGAACTTTAAGCTTCCACATCACA FABP2 Forward Primer ACATTAGGAAGAATCCACTATCTCAC (rs1397613) Reverse Primer TTTCTTACTGTAAACAACAAAAAAAGTC Extension Primer(F) CGTGCCGCTCGTGATAGAATAAAAAGATCCAGAAATGTTTCCTTC FABP2 Forward Primer AAAAGGAAGCTTGCAGCTC (rs1799883) Reverse Primer TAATTAAAGGTGACACCAAGTTCA (new48) Extension Primer(F) AGCGATCTGCGAGACCGTATTAAATTCACAGTCAAAGAATCAAGC FABP2 Forward Primer TTTTCGAAACATTGAAGTTGTTT (rs4834770) Reverse Primer GCTCATAAAAAAAAAAATTCTTACCC Extension Primer(F) GGATGGCGTTCCGTCCTATTAACTTGGTGTCACCTTTAATTACAA FABP2 Forward Primer CCAATATTAAAATGATTTCTGCTCTAT (rs11935130) Reverse Primer ATAATGTAGACAAACTTCATTGGCT Extension Primer(R) GGCTATGATTCGCAATGCTTTGGCTTCTTCAGTTAGTGAAGGAGA FABP2 Forward Primer AAGCCAATGAAGTTTGTCTACATT (rs12510269) Reverse Primer TAATTCTTACTGTTCAATTCAGATTCTT Extension Primer(F) AGGGTCTCTACGCTGACGATCAAATTGGCAGGRTATTTAAATATG FABP2 Forward Primer ACAGTCCAAAATACAAATTGGA (rs2964) Reverse Primer TAATTAGCTTTTACTTCTTTTGCTTTG Extension Primer(F) CGTGCCGCTCGTGATAGAATAGAAGATCTATATTGTACCAGAACT FABP2 Forward Primer GCAAAAGAAGTAAAAGCTAATTAGGA (rs9999724) Reverse Primer TGTCAATTCATATTCACATACTTTTTC Extension Primer(F) CGTGCCGCTCGTGATAGAATTTTAAAAAACAAAAATAAATATTGC FABP2 Forward Primer CATGTTGCTTTATATGTAGCCTTG (rs11724758) Reverse Primer TAAGACCCTCTCTCTACGAAAAC Extension Primer(F) ACGCACGTCCACGGTGATTTAAGTATGTGAATATGAATTGACAGA FABP2 Forward Primer ACATACCACTTTGAATAGGATGATAG (rs10212775) Reverse Primer TCTCAAAGACAAGTTAGTGGACA Extension Primer(R) CGTGCCGCTCGTGATAGAATAGATGATCCTGATTTTCAGCCAAGC FABP2 Forward Primer TGATGCAGCCTGTCCAC (rs10213267) Reverse Primer GGAAAATTGGCAATGTATGTGTT Extension Primer(R) GACCTGGGTGTCGATACCTAGTTAAAAATAACATTGATAACTGAT FABP2 Forward Primer CTTTTATTCATATTTTCACATGACGTA (NEW57) Reverse Primer MCCCCTCTCCATCCCCTA Extension Primer(F) GTGATTCTGTACGTGTCGCCATGACGTAAAATTTTATGTGCTTCA FABP2 Forward Primer TTTTTCACATAAACACATACATTG (NEW58) Reverse Primer CCTAATATGTACCCTGCTAAAATA Extension Primer(F) ATGACGTAAAATTTTATGTGCTTCAY FABP2 Forward Primer GTTTCCTTTTATTCATATTTTCACATG (rs9996644) Reverse Primer TGAAAAAGAATTTGTCAAAAGTCTATAG Extension Primer(F) GCGGTAGGTTCCCGACATATCATATTAGGGGATGGAGAGGGGKTA FABP2 Forward Primer TATTAATCTCAATACAAAAACTGAGAGAGA (NEW60) Reverse Primer TAGGCTGAAAGTCAAGACCTACA Extension Primer(R) GGATGGCGTTCCGTCCTATTTAATTCTTCTGTTCATCATGGACTT FABP4 Forward Primer TGTTTCTCTTTCATCCCCTTT (rs2290200) Reverse Primer AAGAAAATAATTGGCTAACATACGG Extension Primer(F) AGGGTCTCTACGCTGACGATTAAATTAAGCAATAACTTGGAAAAA FABP4 Forward Primer ATGGTTACGTTGGTTAAATAAACTTT (rs16909196) Reverse Primer CTTCACTCAGTTATGACTAAGAATACATC Extension Primer(F) GTGATTCTGTACGTGTCGCCGATTTAGAAGGTGATGTAATGATGT FABP4 Forward Primer TTTCATAATAATTCAAGGTAAAAAGGG (rs1054135) Reverse Primer AGCTATGRTAAAGAAGGATTTAATATTCA Extension Primer(F) AGATAGAGTCGATGCCAGCTTATCTATGGATTTCCCTAGGTAGGA - 62 -
Gene Primer Type Primer sequence SLC27A1 Forward Primer TTCTAGGAAGGATGGGGAC (rs8109373) Reverse Primer TTAACACGTGTGATTCAAGGAC Extension Primer(F) GACCTGGGTGTCGATACCTACTCCCTGGGATCCTGGGGTGTGACG SLC27A1 Forward Primer TTTTTAAGACCGAAAGAGGCTT (rs8110830) Reverse Primer AAAAAWATCCCAGTCCACACC Extension Primer(F) GGCTATGATTCGCAATGCTTGGGGCTTGGATTTAGGACAAGCTGG SLC27A1 Forward Primer CAACGCCCGAAGCACCTTA (NEW66) Reverse Primer TGACCAGTTGGGGGTCTC Extension Primer(F) GAGGGGGCTACAGGGGACACTTTAAG SLC27A1 Forward Primer TGCCCCTCAGCTCCTGTG (NEW67) Reverse Primer TGATGCCTTTGTCCAGCC Extension Primer(F) GTGATTCTGTACGTGTCGCCCTCCATGTTACCCTGGGGACAGAGA SLC27A1 Forward Primer AATCAGGCCCCACCTCCT (rs10418156) Reverse Primer ACATAAGGGAGTTAAGGGAATAGAG Extension Primer(R) CGGGGCCTGGCTGGGAGAGGACGGG SLC27A1 Forward Primer AGGCTCTATTCCCTTAACTCC (rs7251644) Reverse Primer AACAGCCATTCCTTAAGCC Extension Primer(F) GGCTATGATTCGCAATGCTTTTATGTGCGCCCTCCTGTCCACCTG SLC27A1 Forward Primer ACAATGGAGCTGCTGCG (rs3746318) Reverse Primer CCTAGGGAGAAGGCCATG Extension Primer(R) TTCAGGCTGCGCTAGGCAGGCAGGG SLC27A1 Forward Primer TACCTCTCAGGTGCGCAG (rs11879407) Reverse Primer AAGTACATGTAGCCCAGCTCAT Extension Primer(F) ACGCACgTCCACggTgATTTTGACTGGCTGTGCGGATGGGGATCC SLC27A1 Forward Primer CTTGAGCCAGACAGCGCT (NEW72) Reverse Primer TCTGAGGTTCCAGTTTCTCAGT Extension Primer(F) TAGTACACACCCACCTGGCCGAGCT SLC27A1 Forward Primer CCGGCCTTTCCTTTTTCC (rs4808657) Reverse Primer AAAAGCAGCTGGACCCTAC Extension Primer(F) AGAGCGAGTGACGCATACTATCCTCTCCTGCCGAGAGTGGAACAC SLC27A1 Forward Primer GATGGCTCTGACACCGT (rs15401) Reverse Primer TGTTCGAGATGAGCCACC Extension Primer(F) AGAGCGAGTGACGCATACTAATGTCCCAGACAATCCCACCAGGAC SLC27A1 Forward Primer ATCCACGCCAGCCTTTC (rs9137) Reverse Primer CAGGAAAGTTCACATGAGACG Extension Primer(R) GGCTATGATTCGCAATGCTTGTGCTGCTGGAGGGAGGACAGACGG SLC27A4 Forward Primer AGGTTCAAGCAATTCTCCTG (rs7030121) Reverse Primer AAGTAACCATGTTGGGCATC Extension Primer(R) TCCACAGTTGCCACTTGTCCTTAAA SLC27A4 Forward Primer CTCACCTCTGCCTGAAACC (NEW77) Reverse Primer AAAACCCGGAGCACGCTC Extension Primer(F) CCCTCTTAGCGAGCTTCTAGGTGTG SLC27A4 Forward Primer AACAGCAGCCTCTGCTCC (NEW78) Reverse Primer AGCCTCAAGTTAGGGAGATTC Extension Primer(R) ACGCACgTCCACggTgATTTTTCCCTGGGAGAAGACTTTGACCTT SLC27A4 Forward Primer ATCTTTCAGCAGAGGGTCC (rs2240953) Reverse Primer ATCTGTGAGGTCCATGCC Extension Primer(F) CTTCTGCTCTGGCTCCTGGGAGCCC SLC27A4 Forward Primer GTGTCCATTTGTGTGACCCT (NEW80) Reverse Primer TGCCGGATGTGTAGATGTAG Extension Primer(F) AGATAGAGTCGATGCCAGCTTGAGCTGCCCCCGACAGCCACTCGC SLC27A4 Forward Primer TTTTGCCTTCTTGGAGTTTTAG (NEW81) Reverse Primer TTTTTGCTTTCCTATCAGCTTG Extension Primer(F) GACCTGGGTGTCGATACCTAATCGACCACATAATCTGTCATGAGA - 63 -
Gene Primer Type Primer sequence SLC27A4 Forward Primer CTCCTAGCCTGTGTGCAAA (rs7048106) Reverse Primer AATGGGCCAGGGGGTTGG Extension Primer(R) AAGCGGCCACAGTGGGGCTGTGGGG ASPSCR1 Forward Primer TTCATCATTGCCCCAAAC (NEW83) Reverse Primer TGCAACCTCAGAACCTTACTG Extension Primer(F) GCGGTAGGTTCCCGACATATAGGAACAGACTCTTCAAAAACGCCA ASPSCR1 Forward Primer GGTGAGACTCAAGTTCGCT (NEW84) Reverse Primer CAGAATCTGGCAGCCCTA Extension Primer(F) CCCACGGTTCTGAAACCGGTCTCCC ASPSCR1 Forward Primer GTTCACCATCCTTTCATCTCAC (NEW85) Reverse Primer CAAGTGAGAGCCAGTATGTTC Extension Primer(R) AGCGATCTGCGAGACCGTATGGGGCACAGATATACTCAGACCCTG ASPSCR1 Forward Primer TGTACACGAGGGATGAGGTA (NEW86) Reverse Primer GACAGCAGATTTTCCAGTTGG Extension Primer(F) CGACTGTAGGTGCGTAACTCGCGGCCTGCTCTTGCTCACCCAGTC ASPSCR1 Forward Primer GAGCACACTCAGGAGAAGCA (rs8074498) Reverse Primer GAGAGGGACTTCGGCAAC Extension Primer(F) CCTTTCTCGGGTGGGGGACAGAGAC ASPSCR1 Forward Primer AGGAGCAGCATCTGTGG (NEW88) Reverse Primer GTCTCCACACAGACACCA Extension Primer(F) GCAGCATCTGTGGCCTCCCGCCCAG ASPSCR1 Forward Primer GTGTCTACCTGGAGCCTG (rs13087) Reverse Primer AAGACACAGAGTGGTGAGTGC (NEW89) Extension Primer(R) CCGGCACTTACCTGGCCACCAGCAC CACNA1G Forward Primer TCTCCGCAGTGGCGCAAG (rs7501581) Reverse Primer CCAACATCTGCGAGAGGTAG Extension Primer(R) GACCTGGGTGTCGATACCTACCAAAATCTCCGGAGAGGGAACTGA CACNA1G Forward Primer CGAAGAATCAGAGTATTCGGC (NEW91) Reverse Primer AGAGCTGAGCTCCKTCCTC Extension Primer(F) GCTGCCCAAATCAGGGGTCGTGGAC CACNA1G Forward Primer TCATCCTGCTGCCCCTA (rs12946808) Reverse Primer GCTGAGAAGCTGACGTTC Extension Primer(F) CGACTGTAGGTGCGTAACTCAAGCAGGATTCCTCATTGACCTCTT CACNA1G Forward Primer CAATGGGACGCTCAGGG (rs12449998) Reverse Primer CTAAGTGGCAGTCGGCAT Extension Primer(R) CGTGCCGCTCGTGATAGAATGTCGAGGGTGGTGGCAGCATGAGCC CACNA1G Forward Primer CAGACTCCCTGACTCATTTTAC (rs198551) Reverse Primer GAACAGGAGAGTAGAGGTGAGA Extension Primer(F) ACTCATTTTACACATACTCASAGGGC CACNA1G Forward Primer CCATGGGCCTGAGCATCC (rs12936750) Reverse Primer GATCCAGTCCTCCAAACATG Extension Primer(R) GAGCGAGTGACGCATACTAACACCAGGGAGAGGAGGAGCAGGAA CACNA1G Forward Primer AGAGCAGAGCACCCCAC (rs3744521) Reverse Primer ACACACATTCCCAGAGACAG Extension Primer(R) AGAGCGAGTGACGCATACTAAGCTGTGACCACAGGAAGCCCAGGA CACNA1G Forward Primer TATAGGAGCAGCGGCAGC (NEW97) Reverse Primer GCTTGCACAGGGCTCCAA Extension Primer(F) AGATAGAGTCGATGCCAGCTCCCAGGAGTGACACGGCCCAAAACA CACNA1G Forward Primer TGAAGAAGTGCTACAGCGTG (rs739925) Reverse Primer TGACGGCGATAGAGTGTCTC Extension Primer(R) AGGGTCTCTACGCTGACGATTCCTCTGCTCATCCAGCCAGGACGT CACNA1G Forward Primer GCCTAGTATCACCATAGACCC (rs9893223) Reverse Primer ACTCAGCACATCTTTCTTTGG Extension Primer(F) AgCgATCTgCgAgACCgTATGTATCTGCCTCCGGAGGAGGGCTCC - 64 -
Gene Primer Type Primer sequence PPARD Forward Primer AAGGTGCGTGGTGAACA (rs9658117) Reverse Primer ATTCACATAACCCTCAAGTATCAG Extension Primer(R) AGGGTCTCTACGCTGACGATACATAACCCTCAAGTATCAGAGGAG PPARD Forward Primer TKACCTCTTCCTGTCTTCTCCT (rs2016520) Reverse Primer AGCTCTGGCATCGTCTGG Extension Primer(R) CGACTGTAGGTGCGTAACTCCTGAAYGCAGATGGACCTCTACAGG PPARD Forward Primer CCAGGCGTGGGGTAGTGT (rs2076168) Reverse Primer ATCTGAGACAGGCTTGAGTTTC (new102) Extension Primer(R) GACCTGGGTGTCGATACCTAATCAGTTCCCTTCTGGGCCAGAATG PPARD Forward Primer ATGCCGGAGGCTGAGAAG (rs2076167) Reverse Primer TTCTTTTTGGTCATGTTGAAGTT Extension Primer(F) GACCTGGGTGTCGATACCTAAGCTGGTGGCAGGGCTGACTGCAAA PPARD Forward Primer ATCTTCACACTGGTTTGCCA (rs9658173) Reverse Primer GAGGGAACCCTGCCTACTT Extension Primer(R) GCGGTAGGTTCCCGACATATTCCAAGCTGGGAGAGTGCCGTCCAG PPARD Forward Primer TTTMTCTAGCTGGCTCCACG (rs3734254) Reverse Primer CTGCAGGTAAAGTGATTTCAGTG Extension Primer(F) CGTGCCGCTCGTGATAGAATCCCCACTCCCCCTGAAGCTGCC PPARD Forward Primer CTGGTCCTCCCTCCCAAG (rs1053049) Reverse Primer GAATCCTGCCAGCAGAGAG Extension Primer(F) ACGCACGTCCACGGTGATTTTGGAAGTGCCCAGCCCCTGCCCCTA PPARD Forward Primer ATTTAAGAGCTGACTGGAAGC (rs760783) Reverse Primer AAGCCTCCTTTGCCCTC Extension Primer(F) AGGGTCTCTACGCTGACGATTTACTTGCATGTTTTTCCTGGGGCT PPARG Forward Primer CCAACCTAACAGCGTAAGTC (rs12486170) Reverse Primer CAGCTTATCCATCAATAGCAACT Extension Primer(F) AGCGATCTGCGAGACCGTATTYGTTTAAACATCAATTGATGTTCA PPARG Forward Primer GTTAGAAATCTCCAAGTCATCCAC (rs13306747) Reverse Primer GCCCTGAAAGATGCGGA Extension Primer(R) CCACCTCTTTGCTCTGCTCCTGCAG SNAP23 Forward Primer AGCTTTTCTACTAGAGTTATTAGTGTCATC (rs9302112) Reverse Primer ATGTTGTCTTATAAGCCTTGCC Extension Primer(F) CGTGCCGCTCGTGATAGAATTTGGTATCTCCCTACACTCCTGACT SNTG1 Forward Primer GGAAGATCCCCTACCTGGA (rs3812426) Reverse Primer AAGTCCTAGACGTTGTGCTTTC Extension Primer(R) GGATGGCGTTCCGTCCTATTCTTGCTTCCATCAAGGATTAGAGCG SNTG1 Forward Primer TCCCCTTAATTGGTAATCAAGAT (rs2247368) Reverse Primer TTCTTTGGAAAGCTCGTGG Extension Primer(R) GCGGTAGGTTCCCGACATATTCTTTAATTACATAAGATGAAAAGG SNTG1 Forward Primer TCTAGATAATTTACAGGGATTTGAGC (rs4873415) Reverse Primer AAATGTATCTACTTTACCCATGAGGA Extension Primer(F) GGCTATGATTCGCAATGCTTGTGGAAAATTGTGTATGATTTGAGT SNTG1 Forward Primer TGTATTAGTCTACACTTCTAAGASACAATTG (rs4873417) Reverse Primer TGCTTCCATAACAAGAGGTTTT Extension Primer(F) CGACTGTAGGTGCGTAACTCTCTTTGATATTAATCTTTTTGAAGC SNTG1 Forward Primer CGGCCCTGTACGAGAACC (rs4367560) Reverse Primer TGATGACAGCTAGGAGTCATAGAC Extension Primer(F) GCGGTAGGTTCCCGACATATGCCTGGGGTCCATGAACACATATTT SNTG1 Forward Primer GCATTAGTGGCTGAGTCTGC (rs1481475) Reverse Primer TAACTCATACCTGCTTTCAAGGG Extension Primer(R) GGATGGCGTTCCGTCCTATTTTCCCCTTCTGTGAGAGAAGTCCTC SNTG1 Forward Primer ATCTTTTAAGATGCTAACCATATAAGACA (rs7817955) Reverse Primer AAAGTTCCGCTGTGAATGAA Extension Primer(R) GCGGTAGGTTCCCGACATATAAGCACAATTGATAATGCATTGACT - 65 -
Gene Primer Type Primer sequence SNTG1 Forward Primer ATTAGGCAGATATGACATTTATCCA (rs12542191) Reverse Primer AATTCTTCAGAACACATTTCATCC Extension Primer(F) CGTGCCGCTCGTGATAGAATTATCAAATTTTCAAATGTTCTCTTA SNTG1 Forward Primer TTTAACACCCTTGAAAAGTCTGG (rs1508620) Reverse Primer ATAAACAAAATACACTGACATATGCTTG Extension Primer(R) AGATAGAGTCGATGCCAGCTAATTTGATCCATTATTTTATAGGTT SNTG1 Forward Primer ATATTATGCAAATCAAATACAGTGGTAA (rs1911830) Reverse Primer ATCTTGCACATAATCTCATAAACTGA Extension Primer(F) AGAGCGAGTGACGCATACTATAAAGGTGACCACCTGGGACTGGAC SNTG1 Forward Primer CGTCATGGCCAGAAGAATA (rs6473337) Reverse Primer AACTAAGAAAYGCAATTAGTTTTGTG Extension Primer(F) GGATGGCGTTCCGTCCTATTACTTACTTTAAGATTTTATTGGCAA SNTG1 Forward Primer AGGATGCACTTTAATTTGCTGAT (rs7837449) Reverse Primer TCCTTTGGAAGTTCTTCGGAA Extension Primer(F) GCGGTAGGTTCCCGACATATTGCCCTTGTGTTCTCATCCCATGAT STXBP3 Forward Primer GGCGGAAATTCCCTTGCT (rs1124426) Reverse Primer TACTTTGGCCGCAGAAGC Extension Primer(F) GGATGGCGTTCCGTCCTATTACGCGCGTGACGTCACCGGAGGGCG STXBP3 Forward Primer AAAGTAGGTTGGGAGTGGA (rs1124427) Reverse Primer ACGCTCTTTAGCCCCCTC Extension Primer(R) CTGCCACCGGCGGCGCCATCTTCCC STXBP3 Forward Primer TTGAGAGAGGGAAGGCCG (rs6604120) Reverse Primer TCCCAAAGCAACAAGTGC Extension Primer(F) GGCTATGATTCGCAATGCTTCGGGCCTCGTTTTGCAGCCCCAACC STXBP3 Forward Primer ACCTTAAAAAGCAGGTTCATCA (rs1277044) Reverse Primer TTTATGGCAATATTTTTCTACTAAAGTG Extension Primer(F) AGCGATCTGCGAGACCGTATTTTCACCATTGTTATGGTTTACTAA STXBP3 Forward Primer CAGGAAACAGCTATGACCcataagtcttggtggttcgt (NEW127) Reverse Primer TGTAAAACGACGGCCAGTacattttgggctgattttta STXBP3 Forward Primer TATATTTACTTCACTGACTGTAAGTCTTTTAA (rs1277042) Reverse Primer ACATTTTGGGCTGATTTTTAAA Extension Primer(F) GGATGGCGTTCCGTCCTATTAAGTTATTGCTTCATTGTTCAAAAT STXBP3 Forward Primer TGTTTGCTTCCTTAACTGTGT (rs1690734) Reverse Primer AWTGATAGTGAGCCTGGTATTTACTT Extension Primer(F) GGCTATGATTCGCAATGCTTTACAGTAGAGCTACATTTAAACACA STXBP3 Forward Primer TAATAGTGAAGGTGAATTTTAAAGTTTG (rs1298092) Reverse Primer TATTGAAACTAGGCAATGGGTAC Extension Primer(F) GTGATTCTGTACGTGTCGCCTTTTTATTTTTGGAAAGTTCTAACA STXBP3 Forward Primer AATGAATTTTGGTTATAGATCACTGAC (rs12132867) Reverse Primer TTTGTTGAGTAGAACTGGAAGGA Extension Primer(F) AATATTTTATTTTCATTAAGGACCT STXBP3 Forward Primer AGCAAATATTGGTGTAATAAGTAAATGA (rs1277239) Reverse Primer TTTGTTTGGCATAAAGGTCAA Extension Primer(F) GTGATTCTGTACGTGTCGCCAAGGTTTGTGAGTTTCAAAGAAAAG STXBP3 Forward Primer atattgttcccagtgtccag (NEW134) Reverse Primer ctgctatgtgaaggaagagg Extension Primer(F) CGACTGTAGGTGCGTAACTCAAAATCAATGAAATTTTCATTCTAC STXBP3 Forward Primer AATAAACCCAAGGATAAAGTCTCC (rs1149154) Reverse Primer CAACATTTTCTTTCTATTTTAGTTCAAC (new135) Extension Primer(R) CGTGCCGCTCGTGATAGAATAATCTCTAAACCCTCCAAAAAGAAA STXBP3 Forward Primer TTTTACTTGCCYTCATAGTGTTCT (rs1149153) Reverse Primer TATGTACCCAAACAACGCAG Extension Primer(F) GCGGTAGGTTCCCGACATATATGGCAGACCATAGTTTTATTGATT - 66 -
Gene Primer Type Primer sequence STXBP4 Forward Primer ATTCAGTTAATTTACGGAAGAATCTATG (rs9902718) Reverse Primer AGAGTCCCAGAGAAATTGAAGA Extension Primer(F) GCGGTAGGTTCCCGACATATGGATGATCCTCTTGATTTTAGTCTT STXBP4 Forward Primer AATCAGTTGCTGATGTGCTG (rs17817382) Reverse Primer AAGAAGCTGAAATTTAGGGCA Extension Primer(F) AGATAGAGTCGATGCCAGCTATATTACCATTATGTGTGTGCTCAG STXBP4 Forward Primer AAAGTGAGACAGTTTGTTACATTT (rs9303362) Reverse Primer TTATAGTCTACAGGATGGACCTTGA Extension Primer(F) GACCTGGGTGTCGATACCTAACATGTATCCATTTGTGAAATCATC STXBP4 Forward Primer AATCAAGGCACCTACTGTACCTC (NEW140) Reverse Primer TGTTTTTCTTCTCCTAACTAGAACTCAGG Extension Primer(R) GACCTGGGTGTCGATACCTATAGGYCTGGGTTCTCATTTGAAATT STXBP4 Forward Primer TATAACCTGAGTTCTAGTTAGGAGAAGAA (rs17818028) Reverse Primer ATCATTTAGTACAACTCTCTTCCT Extension Primer(F) ACGCACGTCCACGGTGATTTTTTGTGAGGGAAAACCACACAGTCT STXBP4 Forward Primer TTATGAAAAGGTGGTAAATCTCTGT (rs10468513) Reverse Primer TTCTTAAATGGCTTTCCTATGAAG Extension Primer(F) GTGATTCTGTACGTGTCGCCAATACAGTTTGTTTTAATTAAATGA STXBP4 Forward Primer AAGGAATCTATGATTGGTGTATCAT (rs1156287) Reverse Primer ATTCTAAATGACTCATTCTATCTCATGG Extension Primer(F) AGAGCGAGTGACGCATACTATGAAGAAGCAAAAAGCATAATTACC STXBP4 Forward Primer TTTAATAACATCTGAAACATTATCTTGG (rs11658717) Reverse Primer AGATTTTCTGGCTGAATGTTGT Extension Primer(R) AGCGATCTGCGAGACCGTATCTCCCAAGCAGATTCTAACCTAGTA STXBP4 Forward Primer TGGGGCTTAATTTTATTTTTAATG (rs1894936) Reverse Primer TTCACACTAAATGTTGCACAGATAT Extension Primer(F) ACGCACGTCCACGGTGATTTTATTCAGTTGGCCTTTAGAGAATTA STXBP4 Forward Primer GCCAGGGCAGAATTAGATG (rs17818142) Reverse Primer CATGAAAAAAAATGTATTTCCCA Extension Primer(R) CGACTGTAGGTGCGTAACTCCCCAAAGAACTCACAGGATCCAAAA UCP1 Forward Primer AATTTCCCAGTCAGTCAGGC (rs3811790) Reverse Primer AGGCAAATAAGTGGCATGC Extension Primer(F) GGATGGCGTTCCGTCCTATTAGAATGCATTCTATATACGCGACTT UCP1 Forward Primer ACCTTGTCCATTTCCCTCTT (rs3811789) Reverse Primer TTTCCTAGTTCGATCTTTGTGC Extension Primer(F) AGGGTCTCTACGCTGACGATCCTATTATTTCCCACTGTTTTTTTT UCP1 Forward Primer CTGCCCTGTCCTTTCCAC (NEW149) Reverse Primer AAATAGTACATTGCTAAAGCACCG Extension Primer(R) GACCTGGGTGTCGATACCTAGTACAAAATGAGATACCACCTGTTC UCP1 Forward Primer TTGACCATGACTACTACCAAATTCT (rs2071415) Reverse Primer TAGGATAGGACAGAACCCCC Extension Primer(R) GTGATTCTGTACGTGTCGCCGAGAGGCCAATATTCTCAGCTCTTA UCP1 Forward Primer AATGCCCGACGTCCAGTG (NEW151) Reverse Primer AGGCAGCCCGCTGTAGAG Extension Primer(R) AGATAGAGTCGATGCCAGCTATCCGCCCTTCTGTTTTTACCACAG UCP1 Forward Primer AAGTGTAACACACAGAGGAGT (rs7688743) Reverse Primer ACTTCCATGACTGGGTGAC Extension Primer(R) CGACTGTAGGTGCGTAACTCACTTTTTTCTATGAACAATGAGCTAAT UCP1 Forward Primer ATGTTGACCTGTTTCATCGATC (rs2270565) Reverse Primer AGAATTAATAAATCTGGTTTTTACTACATCC Extension Primer(R) AGAGCGAGTGACGCATACTATTTACTACATCCACCGGGGAGGACA UCP2 Forward Primer ACGTGTTTGTCCCGGCCA (rs659366) Reverse Primer AAAGGCGTCAGGAGATGG Extension Primer(F) GGGCCCAATTGTTGGCTGTTCACGC - 67 -
Gene Primer Type Primer sequence UCP2 Forward Primer TGTAACCAATCGACAGCGA (rs17174145) Reverse Primer CCTACGCTGGGCAAACTAC Extension Primer(F) CCAGTCCCGCCCTGCAGGAGCCAGC UCP2 Forward Primer ACTGCGAAGCCCAGCTGC (NEW156) Reverse Primer TGCSGCTGTGTCTGTCGG Extension Primer(R) TGGCGGAGGGCGCGTCGGACGAGCC UCP2 Forward Primer TACGCACCATGGTCAGAAT (rs660339) Reverse Primer ATCGAGATGACTGGAGGTG Extension Primer(F) CATCACACCGCGGTACTGGGCGCTG UCP3 Forward Primer AGCCTCTTGTCAAGTGATCAG (rs1800849) Reverse Primer TCCTTGTCATTCACTGTTGTCT Extension Primer(F) AGCGATCTGCGAGACCGTATTCTAGGATAAGGTTTCAGGTCAGCC UCP3 Forward Primer CCTGCAGCGCCAGATGAG (NEW159) Reverse Primer TCACTGTCCGCGCCTTTG Extension Primer(F) GACCTGGGTGTCGATACCTATCGCCTCCATCCGCATCGGCCTCTA UCP3 Forward Primer AAAGTGTGTGGGTCAAGAGAC (rs2075576) Reverse Primer TTGATRTCCACTCAGAGCCTC Extension Primer(R) CGACTGTAGGTGCGTAACTCAGCGTCCGGTACCAGCTTCCCCCCG UCP3 Forward Primer TATGAGGAGGCTCTGAGTGG (rs1726747) Reverse Primer ATCCGGGTAGTGAGGCTG Extension Primer(R) GACCTGGGTGTCGATACCTAGGGCCAGTGTCCCCTACTAAGCAGC UCP3 Forward Primer ATGAGGACAGCAGAGGAGATAA (rs3781907) Reverse Primer TTAGGTGTCACCTTCTCTGCA Extension Primer(R) GTGATTCTGTACGTGTCGCCGAGCTCCACCTCTGGGGCAACCCCT UCP3 Forward Primer CAGCMAGGGCATCCATTT (rs2734828) Reverse Primer CAAAGTTCCTGTTAGGAAGGC Extension Primer(F) CCCATTTCCCATTCCTCCCTCCCCA UCP3 Forward Primer TGAGGTGGTGACCTACGAC (rs2075577) Reverse Primer AGAGCTGCCTGCCTGGAG Extension Primer(F) AGCGATCTGCGAGACCGTATTCCTCAAGGAGAAGCTGCTGGACTA UCP3 Forward Primer AGATAATGAATGATAAGCAGGAAGG (rs647126) Reverse Primer CATGTGTGATGGTGCAGC Extension Primer(F) CGTGCCGCTCGTGATAGAATAGATCAGCCACGGAGCCTGGAGGGA UCP3 Forward Primer TACCAGAGAAGTGGGAATAACAAT (rs7930460) Reverse Primer TTAAGCCTCCCAGGTAGCA Extension Primer(F) GGCTATGATTCGCAATGCTTCTGCTTCACAGGCTTGTTGTGAGGG VAMP2 Forward Primer AGTGATTTGCGCCATCAT (NEW167) Reverse Primer TCCACTGGGGAAACCTCT Extension Primer(R) AGGGTCTCTACGCTGACGATCCAGGGAAAGGGCCCCGGCCATTCT VAMP2 Forward Primer AACCCTCAGCCGTTCCTC (rs1061032) Reverse Primer ACAGCTGGCTATTTACAGGG Extension Primer(F) AGATAGAGTCGATGCCAGCTCTCTCAGCCATATCTTTCAGCCCCC VAMP2 Forward Primer CAATTCCTGCCTGAAATCTCTG (NEW169) Reverse Primer AGGAGGGTTTTCAGTCCAAC Extension Primer(F) ACGCACGTCCACGGTGATTTTTCCTGCCTGAAATCTCTGGCCTCA VAMP2 Forward Primer TACTAGTCACTGCCAATGTGTGTAT (rs11548690) Reverse Primer ATTTAAAAAGACAATAGACCCTTCCT Extension Primer(R) GGATGGCGTTCCGTCCTATTTCTCTCTGGGCTCAATAGTCCTGGA VAMP2 Forward Primer ATATCTAAGCTGTGGTGTGAAAGG (rs1150) Reverse Primer GTTACTCATTACACATTTATTGTACATTTTC Extension Primer(F) ATGCTGGAGGTGGGGGTGCTGTGTT VAMP3 Forward Primer ATCTGTCTCTAATACTGTTAGAAATGGTC (rs707457) Reverse Primer AATACAGCTCCCTGTCCG Extension Primer(F) GACCTGGGTGTCGATACCTACCTCACTTATCCACGCAGTCCCTCG - 68 -
Gene Primer Type Primer sequence VAMP3 Forward Primer GCCCTCAAGTTCCCAAGT (NEW173) Reverse Primer AGAGAAGCGAAGTTGGGAC Extension Primer(R) AGGCCAGGGAGATGGGTGGGACGGC VAMP3 Forward Primer AGTCATCAGCCCCTCCCA (rs2301489) Reverse Primer ATTCACAGAACTACGAGAAAACTGA Extension Primer(F) CTCTACCCCAGCAGGGCCCCAAACA VAMP3 Forward Primer AAGCAACAATTCTGTAGCCTCA (NEW175) Reverse Primer ATAATTTAGAAAACTGATTTAGGAAGCA Extension Primer(F) GGCTATGATTCGCAATGCTTTCTGGCCCAAATATGAAGATAAACT INSR Forward Primer TGAATGTTCTTCTTGCTTGG (rs6510976) Reverse Primer TCACTTCTATAATCAGATAAGAACTAAA Extension Primer(F) ACGCACgTCCACggTgATTTGGGAGAGGAAAAGGAGTGGAAAACT INSR Forward Primer AGAGGAGAAGGAACGTGGG (NEW202) Reverse Primer AAAACAGGGTGCAAAAGTAGC Extension Primer(F) TGTGACGATGCCGCTTGTTAAAACT INSR Forward Primer CTGAAGACCAATGGGGAC (rs2860178) Reverse Primer CACCAGTCCATGGAAAAAC Extension Primer(F) TCACTGGTCCCCAACCTTTTTGGCA INSR Forward Primer TCAGTCTTACAAGTCCATATGCG (rs2303672) Reverse Primer AGTCAACTGAATAAGCGCAGTC Extension Primer(F) CGTGCCGCTCGTGATAGAATTCACCCTTGGAAATCCCTTCCTCCA IRS1 Forward Primer ATCAGCACCTCCGCCTT (rs1801123) Reverse Primer ATGTGGAAGGCTGGGCT Extension Primer(F) AGCGATCTGCGAGACCGTATGTCGCCTTCTCTATGCTGCAACAGC IRS1 Forward Primer TGAGGAGTACATGAAGATGGAC (rs1801278) Reverse Primer TCATCTGCATGGTCATGTAGTC Extension Primer(F) GGGCAGACTGGGCCCTGCACCTCCC IRS2 Forward Primer TCTCTGCTAACTTCCCTCCT (rs1865434) Reverse Primer TTCCAAACACAGTCATTGCTC Extension Primer(F) GCGGTAGGTTCCCGACATATTCACCAGGATGTCACATGTCAGAGA IRS2 Forward Primer ATATAATGTTAGCTGGAAGGTCAAT (NEW208) Reverse Primer ACCCATTGATGCCTATTCC Extension Primer(R) CAAAACTAAATAAAAACTTCAGGAT CBL Forward Primer TCCCCAAGACCATATCAAAG (rs2510152) Reverse Primer CTATAAAGAATATACTAAAGCTTGTGTCCA Extension Primer(F) GACCCAGGTGAGTTTTGTTTCACAT RAPGEF1 Forward Primer AAAGACTTGAGAATGAGATTCCTC (rs11243480) Reverse Primer CGAGTCACTGGCTGAAAGG Extension Primer(F) GGCTATGATTCGCAATGCTTGAGGCTGCCTGGGTTCTGCCGCTGG RAPGEF1 Forward Primer ATATTTCACACAGGCTTGAAGTATT (rs4740174) Reverse Primer CTTTAAAAGTTCTTTGTCATTATGAAAC Extension Primer(F) AGGGTCTCTACGCTGACGATGTTTCCAAGGATCTTCTGTGAGCGC RAPGEF1 Forward Primer AAAGAAACCCATGGTCTTCAG (rs17148121) Reverse Primer GAACTTGGACCGTGTCCTG Extension Primer(F) GTGATTCTGTACGTGTCGCCTGGTGTTTGTCACATCGAGGAGTGC RAPGEF1 Forward Primer TTGCTGGAGGACTACTCGG (rs2274600) Reverse Primer TATACCTCCATGAGGAGCTTGT Extension Primer(F) GACCTGGGTGTCGATACCTACGCAGCCCTCTATGTTCTACCAGAC RAPGEF1 Forward Primer CTTGGCCTCCAGGCATC (rs11243446) Reverse Primer CTGGACATAATTTCGTTGTGGT Extension Primer(F) AGTCGGCTCAGTCGGA RAPGEF1 Forward Primer AACAGACCCTGTGCCTGG (rs943851) Reverse Primer TCATGTGCTGCCTGGGCC Extension Primer(F) GCCCAGGCAATTCCCCAGGCCCTCA - 69 -
Gene Primer Type Primer sequence PIK3CA Forward Primer AGGTAAAGTCAAATGCTGATGCT (rs2699895) Reverse Primer TGGATGTTCTCCTAACCATCTGA Extension Primer(R) ACAGGTCAACAGATTACTGTATAGT PIK3CA Forward Primer AGAAACTGACCCTGATTTGTTTT (21142T/C) Reverse Primer TTTGATTAGTCAATGCTTTCTTCA Extension Primer(R) AATCTCACAAGCAAGTTATCCAAAT Extension Primer의소문자 20mer는 UHT 분석을위한 tag sequence임. - 70 -
부록 2. CYP11B2 유전자 Blast 결과 CYP11B2 Original Sequence 000000001 ctgcatcctttggaggatggggaaggagtgcagcacatgctggtctgtgg 000000050 <<<<<<<<< <<<<<<<<< 143957887 ctgcatcctttggaagatggggaaggagtgcagcacgtgctggtctgtgg 143957838 CYP11B1 Sequence 000000051 tgctgccagggcaggggatagtgcagagaaaaccccagctcactgcagag 000000100 <<<<<<<<< <<<<<<<<< 143957837 cgctgccagggcaggggatggtgcagagcaaatcccagctcgctgcagag 143957788 000000101 agggcaggactcagaagcactaaagttgaaaggttccagggagccagcag 000000150 <<<<<<<<< <<<<<<<<< 143957787 agggcaggactcagaggcactgaagttaagaggttccgggcagtcagcaa 143957738 000000151 gagggctttagctgtgaagccgctaatccaggagcagggagggtggacag 000000200 <<<<<<<<< <<<<<<<<< 143957737 gagggctttagctgtgaagccgctaatccaggagaggggagggtggacag 143957688 Primer 000000201 gagacactttggattgggactgcagggtggggccacgagggacatgaccc 000000250 <<<<<<<<< <<<<<<<<< 143957687 gagacactttggattgggactgcagggtggggctagcggggacatggtcc 143957638 rs13276226 000000251 Ctccagcagggcctcctgcttggccccacaggtacaacttgggaggacc 000000300 <<<<<<<<< <<<<<<<<< 143957637 catccagcacggcctcgtgcttggccccacaggtacgacttgggaggagc 143957588 Primer 000000301 acgcatggtgtgtgtgatgctgccggaggatgtggagaagctgcaacagg 000000350 <<<<<<<<< <<<<<<<<< 143957587 aggcatggtgtgtgtgatgctgccggaggacgtggagaagctgcaacagg 143957538 000000351 tggacagcctgcatccctgcaggatgatcctggagccctgggtggcctac 000000400 <<<<<<<<< <<<<<<<<< - 71 -
143957537 tggacagcctgcatccccacaggatgagcctggagccctgggtggcctac 143957488 000000401 agacaacatcgtgggcacaaatgtggcgtgttcttgttgtaagcggcgag 000000450 <<<<<<<<< <<<<<<<<< 143957487 agacaacatcgtgggcacaaatgtggcgtgttcttgctgtaagcggc... 143957441 000000451 ttgggagctgagagctgggagcagggtgggcagcctgggtgtagggggga 000000500 <<<<<<<<< <<<<<<<<< 143957440...gagctgagagctgggagcagggtgggcagcctgggtgtagggggga 143957395 000000501 g 000000501 <<<<<<<<< <<<<<<<<< 143957394 g 143957394 #21 rs13268153 : chr8: 143995228~143995628 chr8:143957158-143957649 를포함한 800bp 이상에서 homology 보임 000000003 gggacatgaccccgtccagcagggcctcctgcttggccccacaggtacaa 000000052 <<<<<<<<< <<<<<<<<< 143957649 gggacatggtcccatccagcacggcctcgtgcttggccccacaggtacga 143957600 000000053 cttgggaggaccacgcatggtgtgtgtgatgctgccggaggatgtggaga 000000102 <<<<<<<<< <<<<<<<<< 143957599 cttgggaggagcaggcatggtgtgtgtgatgctgccggaggacgtggaga 143957550 000000103 agctgcaacaggtggacagcctgcatccctgcaggatgatcctggagccc 000000152 <<<<<<<<< <<<<<<<<< 143957549 agctgcaacaggtggacagcctgcatccccacaggatgagcctggagccc 143957500 Primer 000000153 tgggtggcctacagacaacatcgtgggcacaaatgtggcgtgttcttgtt 000000202 <<<<<<<<< <<<<<<<<< 143957499 tgggtggcctacagacaacatcgtgggcacaaatgtggcgtgttcttgct 143957450 rs13268153-72 -
000000203 gtaagcggcgagttgggagctgagagctgggagcagggtgggcagcctgg 000000252 <<<<<<<<< <<<<<<<<< 143957449 gtaagcggc...gagctgagagctgggagcagggtgggcagcctgg 143957407 Primer 000000253 gtgtaggggggaggcgagagaggtaggacccaaaagcacatctgccctgg 000000302 <<<<<<<<< <<<<<<<<< 143957406 gtgtaggggggaggcgagagaggcaggacccaaaagcacatctgccctgg 143957357 000000303 gcccctgtggtgggcagtgagggtgagcacccggcccagaggacggccat 000000352 <<<<<<<<< <<<<<<<<< 143957356 gcccctgtggtgggcagtgagggtgagcacccgacccagaggacggccat 143957307 000000353 cctgtggggtcgcgtctgcactgtgggttggggaagcagggcggtggtgg 000000402 <<<<<<<<< <<<<<<<<< 143957306 tccgtggggtcgtgtctgccctgtgggttggggaagcagggcggtggtgg 143957257 000000403 agaaatgggcacgggcacctctgcagagaagacgcagagcaatgagccct 000000452 <<<<<<<<< <<<<<<<<< 143957256 agaaatgggcacgggcacctctgcagagaagatgcagagcaatgagccct 143957207 000000453 tctgtgtagtgagaacccgctctgcaccaacctcggcggctgctttctc 000000501 <<<<<<<<< <<<<<<<<< 143957206 tctgtgtagtgagaacccgctctgcaccaaccttggccgatgctttctc 143957158 #22 rs11779229 chr8:143956944-143957439 000000001 agctgagagctgggagcagggtgggcagcctgggtgtaggggggaggcga 000000050 <<<<<<<<< <<<<<<<<< 143957439 agctgagagctgggagcagggtgggcagcctgggtgtaggggggaggcga 143957390 000000051 gagaggtaggacccaaaagcacatctgccctgggcccctgtggtgggcag 000000100 <<<<<<<<< <<<<<<<<< 143957389 gagaggcaggacccaaaagcacatctgccctgggcccctgtggtgggcag 143957340-73 -
000000101 tgagggtgagcacccggcccagaggacggccatcctgtggggtcgcgtct 000000150 <<<<<<<<< <<<<<<<<< 143957339 tgagggtgagcacccgacccagaggacggccattccgtggggtcgtgtct 143957290 000000151 gcactgtgggttggggaagcagggcggtggtggagaaatgggcacgggca 000000200 <<<<<<<<< <<<<<<<<< 143957289 gccctgtgggttggggaagcagggcggtggtggagaaatgggcacgggca 143957240 Primer 000000201 cctctgcagagaagacgcagagcaatgagcccttctgtgtagtgagaacc 000000250 <<<<<<<<< <<<<<<<<< 143957239 cctctgcagagaagatgcagagcaatgagcccttctgtgtagtgagaacc 143957190 rs11779229 Primer 000000251 Cgctctgcaccaacctcggcggctgctttctcttgcggtctggggactgt 000000300 <<<<<<<<< <<<<<<<<< 143957189 cgctctgcaccaaccttggccgatgctttctcttgcggtctggggactgt 143957140 000000301 ccttcccataggtcagaaaactgaggccctgagaaggggacttccactgg 000000350 <<<<<<<<< <<<<<<<<< 143957139 ccttcccataggtcagaaaactgaggccctgagaagggtactcccactgg 143957090 000000351 cccaggtcacaggctgagtgctgagcctggtgttcgccggggccacagcc 000000400 <<<<<<<<< <<<<<<<<< 143957089 cccaggtcacaggctgagtactgagcctggtgttcgccggggccgcagcc 143957040 000000401 tccctcagggcgctcagggtccctgcagtcctggcaaaccttcctgatgg 000000450 <<<<<<<<< <<<<<<<<< 143957039 tccctcagggcgctcagggtccctgcagtcctggcaaaccttcctgatgg 143956990 000000451 ggacagtccggggcaggaggcaggtggggacgcaggtggctggtgg 000000496 <<<<<<<<< <<<<<<<<< 143956989 ggacagtccggggcaggaggcaggtggggatgcaggtggctggtgg 143956944-74 -
#30 New30 chr8:143952265-143952744 000000002 tctgcctgaccccaggccacctgtcttctctcccacgtgcacagcttcct 000000051 <<<<<<<<< <<<<<<<<< 143952744 tctgcctgaccccaggccacccctcttctctcccacatgcacagcttcct 143952695 000000052 gagtcacccctctgtccagccagctcctgcacaaatggaactccccaggg 000000101 <<<<<<<<< <<<<<<<<< 143952694 gagtcacccctctgtctaaccagccccagcacaaatggaactcccgaggg 143952645 000000102 cctccaggactggggcttgccaggct 000000127 <<<<<<<<< <<<<<<<<< 143952644 cctctaggaccagggtttgccaggct 143952619 000000136 agcaaggccagggcacagctggagacgatcttgctg 000000171 <<<<<<<<< <<<<<<<<< 143952614 agcaatgccagggcacagctggggaagatcttgctg 143952579 primer 000000183 ccttgtccccagctccacctggccccttctccagcaagcagtgccctctg 000000232 <<<<<<<<< <<<<<<<<< 143952577 ccttgtccccagccccacctggccctttctccagcaagcactgtcctctg 143952528 000000233 g 000000233 <<<<<<<<< <<<<<<<<< 143952527 g 143952527 new 30 000000248 ctcccagcgctggctccaggctcctcatgaggccatgcaagggtgctgtg 000000297 <<<<<<<<< <<<<<<<<< 143952509 ctcccagtgctggctccaggctcctcgtgtggccatacaagggtgctgtg 143952460 primer - 75 -
000000298 attttgtcccttgccttcctgcctagtctcacatgtccctgtccctctcg 000000347 <<<<<<<<< <<<<<<<<< 143952459 gttttgtcccttgccttcctgcctagtctcacatgtccctgttcctcttc 143952410 000000348 ccctggccagggcctctgtgcagacagtgtcagagtcattaagcgggatc 000000397 <<<<<<<<< <<<<<<<<< 143952409 ccctggccagggcccctgcgcagac..tgtcagagtcattaagcgggatc 143952362 000000398 ccagcatctcagagtccagtcaagttccctcctgcagcctgacccc.agg 000000446 <<<<<<<<< <<<<<<<<< 143952361 ccagcatctcagagtccagtcaagttccctcctgcagcctgacccctagg 143952312 000000447 cagctcgagcatgccctgagctctctgaaagttgtcaccctggaata 000000493 <<<<<<<<< <<<<<<<<< 143952311 cagctcgagcatgccctgagctctctgaaagttgtcaccctggaata 143952265 #32 New32 chr8:143951812-143952316 000000001 ccaggcagctcgagcatgccctgagctctctgaaagttgtcaccctggaa 000000050 <<<<<<<<< <<<<<<<<< 143952316 ctaggcagctcgagcatgccctgagctctctgaaagttgtcaccctggaa 143952267 000000051 tacgatcctgcagggtagactaaaaaggcccctgtggtcacttatcctga 000000100 <<<<<<<<< <<<<<<<<< 143952266 tagggtcctgcagggtagaataaaaaggcccctgtggtcacttgtcctga 143952217 000000101 ca...cattttcaagtgatacaactgagtctcgaggggcgtgtgttccc 000000146 <<<<<<<<< <<<<<<<<< 143952216 catccccattttcaagtgatacaactgagtctcgagggacgtgtgttccc 143952167 000000147 cagctgatcatgtcagcctcatgccccaggcctcgtctttcatggaccag 000000196 <<<<<<<<< <<<<<<<<< - 76 -
143952166 cagctgatcgtgtcagcctcatgccccaggcctcatctttcatggaccag 143952117 primer 000000197 gtcttgttcaagcagcgagtgttgggtcctctgcttcctgagctgtcccc 000000246 <<<<<<<<< <<<<<<<<< 143952116 gccttgttccaggagtgggtgttgggtcctctgcttcctgtgctgtcccc 143952067 new 32 primer 000000247 tggagaaggtcccgaggatgctgtcaggagatggaagagtcatgtggggc 000000296 <<<<<<<<< <<<<<<<<< 143952066 tggggaaggtcccgaggatgctgtcaggagatggaagagtcatgtggggt 143952017 000000297 gggaacctggggtgtggttccagaaatgtttttggcaacaggagagacag 000000346 <<<<<<<<< <<<<<<<<< 143952016 gggaacctggggtgtggttccagaaatgtttttggcaacaggagagacag 143951967 000000347 gattgggccaacaaggactcagatgagttttattgactcattcctctggt 000000396 <<<<<<<<< <<<<<<<<< 143951966 gattgggccaacaaggactcagacgagttttattgactcatttctctggt 143951917 000000397 tgatacggagccatgtcatgtgccacgacctggggtgggcacagggaggc 000000446 <<<<<<<<< <<<<<<<<< 143951916 tgatacggagccatgtcatgtgccacgacctggggtgggcacagggaggc 143951867 000000447 tgcagttccctacgtgaacctgccttgggcctcatctgctcctagcccag 000000496 <<<<<<<<< <<<<<<<<< 143951866 tgcagtttcctgcgtgaacctgccttgggcctcatctgctcctagcccag 143951817 000000497 cagag 000000501 <<<<<<<<< <<<<<<<<< 143951816 cagag 143951812 #33 rs3802228 chr8:143951566-143951869 - 77 -
000000001 ggctgcagttccctacgtgaacctgccttgggcctcatctgctcctagcc 000000050 <<<<<<<<< <<<<<<<<< 143951869 ggctgcagtttcctgcgtgaacctgccttgggcctcatctgctcctagcc 143951820 000000051 cagcagagagagttgacccctcctgaactggccactccccagtgctcctg 000000100 <<<<<<<<< <<<<<<<<< 143951819 cagcagagagagttgacccctcctgaactggccactccccagtgctcccg 143951770 000000101 tgcagggataggaag 000000115 <<<<<<<<< <<<<<<<<< 143951769 tgcagggagaggaag 143951755 000000136 cccagccacatcatctttatctcctgggattttcattagggcaaagatct 000000185 <<<<<<<<< <<<<<<<<< 143951735 cccagccagactgtctttatctccatggattttcatcagggcaaagatca 143951686 primer 000000186 cagcagccagctcctggtagctgatgaggatcagagcgtttgttccccca 000000235 <<<<<<<<< <<<<<<<<< 143951685 cagcagccagctcctggtggctgatgaggatcagagcatttgttccccca 143951636 rs3802228 primer 000000236 tgaaaggggaaatacttaggtaatcattccaggtgtgttcagtagttcca 000000285 <<<<<<<<< <<<<<<<<< 143951635 tgaaaggggaaatccctatggcatcattccagtggtggtcagtagatcca 143951586 000000286 ggac 000000289 <<<<<<<<< <<<<<<<<< 143951585 ggac 143951582 000000294 ggactcag 000000301 <<<<<<<<< <<<<<<<<< 143951573 ggactcag 143951566-78 -
부록 3. Genotyping Technology 1) SNPstreamR UHT SNP genotyping technology A true multiplex SNP genotyping technology (12 plex reaction) 384 plate의 well 하나에 16개의고유한 oligonucleotide( 또는 tag) 이 array되어있는 SNPstreamR Tag Array plate를이용하여 multiplexed reactions이이루어짐. 16개의 tag중 4 개는 control이붙는위치이기때문에 12 plex reaction이이루어짐. plate 위의각 tag은 12개 extension primer의 tag들과상보적으로결합하게되어, 각각의 SNP은 well position에의해구분되어분석됨. < SNPstreamR Tag Array plate > SNP- Primer POS 1,2 SNP 1 SNP 2 POS 2 SNP 3 SNP 4 SNP 5 SNP 6 SNP 7 SNP 8 SNP 9 SNP 10 POS 1 SNP 11 SNP 12 NEG Plate surface < SNP hybridization to the complementary Tag > Single-base primer extension technology in a tagged oligonucleotide fluorescent assay system Single-base primer extension technology 를이용하여각 genotype 당 0.17 nm 의 DNA 를사용 하여 99% 이상의정확도를나타냄. - 79 -
Tag SNP-IT primer SNP site 3 5 PCR target 5 Labeled terminating NTP 3 5 SNP-IT Primer Substrate Tag complement for Hybridization capture < Tag Array Process의개요 > Specification 1 Throughput : Up to 800,000 genotyping per day 2 Assay conversion rate : 80-90% with quality input sequences 3 Genotyping success rate : >95% callable - 실험방법 1 PCR setup ᄀ PCR mix composition Component Concentration Volume 10X PCR Buffer Ⅱ 1 X 0.5 μl MgCl 2 (25mM Stock) 5 mm 1 μl dntp mix (2.5mM each) 75 um each 0.15 μl Primer pool (10uM each) 50 nm each 0.025 μl Genomic DNA 2 ng/2μl Dried Tag Gold (5U/ μl ) 0.1 U/ μl 0.1 μl Water Add to final vol of 3μl 1.225 μl ᄂ Thermal Cycling Step Temp Time 1 95 1:00 2 95 0:30 3 55 0:30 4 72 1:00 5 Go to Step 2 for 34 Times 6 4 forever - 80 -
2 Clean-up (384 plate 1개기준 ) Manual Automatic Number of plates 2 4 2 4 SNP-IT CleanUp Reagent 125μl 250μl 200μl 320μl SNP-IT CleanUp Diluent 3mL 6mL 4.8mL 7.68mL Total 3.13mL 6.25mL 5mL 8mL ᄀ 25X SNP-IT Clean Up Reagent 62.5μl + SNP-IT Clean Up Diluent 1.5mL로희석해서 384 PCR Plate 각 Well에 3μl씩분주한다. ᄂ Microfilm A film으로 sealing하고간단하게 spin-down 한다. ᄃ Clean-up program - 37 30min/ 96 10min/ hold at 4 / store at -20 3 primer Extension ᄀ각각의 panel에맞는 extension mix(1 out of the 6 types) 를준비한다. - Extension mix 90μl를 extension mix diluent 1.69mL로희석 - DNA polymerase 17.6μl첨가하고섞어준다. Manual Automatic Number of plates 2 4 2 4 SNP-IT primer pool 27μl 54μl 35μl 58μl H 2O 2673μl 5346μl 3436μl 6872μl Extension Mix 180μl 360μl 230μl 460μl Extension mix diluent 3385μl 6770μl 4324μl 8648μl DNA polymerase 18.8μl 37.6μl 24μl 48μl Total 6.3mL 12.6mL 8.0mL 16mL ᄂ 384 plate의각각의 well에 7ul의 extension mix를분주하고 spin-down한다. ᄃ Micro A film으로 sealing 한다. ᄅ Thermal Cycling Step Temp Time 1 96 3:00 2 94 0:20 3 40 0:11 4 Go to Step 2 for 45 Times 5 4 forever - 81 -
4 Hybridization ᄀ Preparation of SNP-IT Plate - 20X SNPware UHT Wash Buffer를 DI H 2 O로 final 농도가 1X가되도록희석 - plate washer로 SNP-IT plate를 wash buffer I 으로세번 washing한다. (manual로 washing 할경우 10μl /well 로 washing ) - plate를 vacuum을이용하여 dry한다. ᄂ Preparation of Hybridization Solution Manual Automatic Number of plates 2 4 2 4 2Xhybridization solution. 9.45mL 18.9mL 18.9mL 33.1mL Hybridization additive 550μl 1.1mL 1.1mL 1.9mL Total 10mL 20mL 20mL 35mL Wash 30mL 60mL - hybridization solution 4.725mL + Hybridization additive 275μl (Total 5mL) SNPstreamR의 Multimek 이용 - PCR plate 각 well에 a 8μl를첨가한후 pipetting으로잘섞어준다. (8times) SNPstreamR의 Multimek 이용 - 위 mixture 중 8μl를 SNP-IT plate에옮긴다. SNPstreamR의 Multimek 이용ᄃ Hybridization SNP-IT plate를 humidified incubator(or a covered tray humidified with wetted paper tower in an oven) at 42 for 2 hours(+/- 15 mins) ᄅ Post-hybridization wash - one vol의 64X SNPware UHT Stringent Wash Solution과 63vol의 DI H 2O를섞어서 washing solution을만든다. - Hybridization이완료되면 SNP-IT plate를 well당 10μl washing solution으로세번 washing한다. - SNP-IT plate를 vacuum을이용하여완전하게 dry시킨다. - SNPstreamR UHT Genotyping System에서 imaging하기전까지 SNP-IT plate를어두운곳에보관한다. 분석단계분석장비ᄀ SNPstreamR UHT Array Imager - SNP microarrays의형광 signal을측정하고 image를 capture함. - 82 -
- 3 종류의 laser를사용함. 20 mw, 488 nm "blue" argon gas tube laser : bodipy-fluorescein dye를여기시킴 20 mw, 532 nm "green" solid state diode laser : TAMRA dye를여기시킴 5 mw 650 nm "red" solid state diode laser : bar code 라벨을읽을때사용함 Blue channel well image Green channel well image org fail < Image of spots : Pass = green circle, Fail = red circle ("org fail") > Spot Layout Imaging Channel 1 (Allele X) Imaging Channel 2 (Allele Y) POS 1,2 SNP 1 SNP 2 POS 2 G G C C SNP 3 SNP 4 SNP 5 SNP 6 G G C C C C SNP 7 POS 1 SNP 8 SNP 11 SNP 9 SNP 12 SNP 10 NEG G G G G G C C C GC CC GG CC CC GC GC GC GG GG GG CC CC GG < Spot Layout and image data calling > : GetGenos 을통해 uploade 된 raw data 로부터 genotype 이 call 되며, QCreview 를 통해 assay 의 pass/fail 여부를결정하고최종적으로데이터를분석함. - 83 -
< QC Review > - 84 -
2) SNP-IT assay using SNPstreamR 25K system - PCR 이후의모든과정이완전자동화된 system으로하루 25,000여 genotyping이가능한 high-throughput SNP scoring system임 ( 아래그림참조 ). - PCR 이후 primer extension과정을실온에서실행하므로추가의 PCR 과정을거치지않음. 이에따른 cross-contamination을방지. - ELISA reader를이용하여발색반응을탐지하는 assay system으로충분한 test가끝난 technology를이용하므로유지관리가용이하고매우안정적인 system임. < SNPstreamR 25K > - SNP-IT assay는미국 Beckman Coulter사의 SNP scoring technology로 primer extension assay를이용한기술임 ( 아래그림참조 ). - PCR 반응에사용하는 primer중한개에 phosphorotioate를붙여서 PCR 반응을함. - PCR 산물을 exonuclease III로 digestion : 이때한쪽 strand는 phosphorotioation에의하여보호되어 single strand product가남음. - Single stranded product를 SNP-IT plate에 overlay시켜서 hybridization이일어나도록함. - 보고자하는 SNP에맞는 extension mix를첨가하여 single nucleotide extension이일어나게함. 이때 biotin 또는 FITC가 tagging된 acycloterminator를이용. - Extension된산물에 anti-fitc-ap (alkaline phosphatase) 또는 streptavidin-hrp (horse radish peroxidase) 를 binding 시킨후여기에 substrate로서 PNPP (p-nitrophenyl Phosphate) 또는 TMB (tetramethylbenzidine) 를반응시켜발색반응시킴. - 발색반응으로나오는각각의 absorbance 값은 fluorstar plate reader가읽어자동으로결과를내게되고 analysis program(qc Review) 상에서결과를확인 - 85 -
SNP-IT PCR ssss A Exonuclease ssss +exo A Hybridization ssss A SNP-IT Primer Plate + ddttp tagged with reporter and DNA polymerase Extension ssss A T* Plate Detection Detection of primer extension using antifluorescein-ap or streptavidin-hrp conjugates and standard colorimetric methods < Biochemistry of SNP-IT Assay > - 86 -
- 실험방법 1 PCR amplification - 384 well microtiter PCR plate에서진행. - DNA sample을제외한시료를섞은 Master Mix를제조하여 5ul 씩 liquid handler (Biomek FX workstation) 를이용하여분주 - PCR mix composition (DNA 10ng) 10X Buffer MgCl 2 (25mM) dntp(10mm) Primers(100pM) Taq(5Unit/ul) dh 2 O Total 0.5ul [1X] 0.6ul [3mM] 0.125ul [0.25mM] 0.025/0.025ul [0.5pM] 0.025ul [0.125unit] 3.7ul 5 ul - PCR Cycle Condition (Tm: autoprimer에서지정해준값을사용 ) 95 10mins 35 Cycles: 95 30s, Tm 1 min, 72 1 min 72 7mins 2 SNP-IT assay - SOP에맞추어필요한양의시약을각각의용기에분주 - Multidrop dispenser 및 microtiter plate washer 등의장비를사용 buffer로 priming. - PCR 산물과보고자하는 SNP을조사할수있는 SNP-IT primer가 coating 된 plate를 system의 microtiter plate hotel에넣은후 control program을실행. - Assay가끝난후결과를 database server로 upload. - 87 -
<SNPstream 25K SOP> Date: 2005.05.02 1.Exonuclease Treatment TargEx (ul of 50U/ul) 472.5ul Lot # TargEx Dilution Buffer 25.448ml Lot # 2. Hybridization Salts Hybridization Solution 48.9ml Lot # 3. Extension A/G A/C T/A T/C T/G G/C 10X Extension Mix 2.66ml Lot # Extension Buffer 24.22ml Lot # 10X Extension Mix False ml Lot # Extension Buffer False ml Lot # 10X Extension Mix False ml Lot # Extension Buffer False ml Lot # 10X Extension Mix 2.66ml Lot # Extension Buffer 24.22ml Lot # 10X Extension Mix False ml Lot # Extension Buffer False ml Lot # 10X Extension Mix False ml Lot # Extension Buffer False ml Lot # 4. Detection Complex I Detection Complex(1:1500) 31ul Lot # Detection Dilution Buffer 46.08ml Lot # 5. Detection Substrate I Substate I 115.20ml Lot # 6. Detection Complex II Detection Complex II(1:4000) 11.52ul Lot # Detection Dilution Buffer 46.08ml Lot # 7. Detection Substrate II Substrate II 115.20ml Lot # - 88 -
3 결과분석 : QC review program - SNPstreamR 전용분석 program인 QC review program으로각각의 genotyping 실험결과를 confirm. - 아래그림의각점은개개의샘플을표현. 한 plate에서분석된샘플의전체적인경향을볼수있고 system에의한 genotype call을사용자가확인하고필요에따라서는정정할수있음. < QC review window > - 89 -
3) Single base primer extension assay using SNaPshot kit Single-base primer extension technology는 dideoxynucleotides(terminator) 의존재하에 cycle sequencing을하는것이핵심기술이며 terminator가 DNA strand로끼어들어가면더이상의 extension은진행되지않음. 이때사용되는 dideoxynucleotides를각각다른 dye로 labeling함으로써 cycle sequencing을통해 extension된단하나의특정 nucleotide를 detection할수있음. 1 PCR amplification SNP을포함하는부위를먼저 PCR로증폭함. 2 PCR product purification : SAP& Exo I treatment (10μl) Material volume( μl ) SAP(1unit/ μl ) 5 Exo I (10 unit/ μl ) 0.2 PCR product 4 Deionized water Adjust to 10 Total 10 reaction temp. reaction time 37 1hr 72 15min 3 SNaPshot Reaction : one base extension 을위한 PCR 과정 SNP 바로앞까지합성된 primer를 denature된 PCR product와 anneal 시킨후위에서 제시된 4가지형광색으로표지된 dideoxynucleotide와 taq polymerase를첨가하여다음의 조건에서 cycling. material volume( μl ) SNaPshot Ready Reaction Premix 5 Genotyping primer(0.15pmol/ μl ) 1 Purified PCR product 1 Deionized water 3 Total 10-90 -
reaction temp. reaction time 96 10sec 50 5sec 60 30sec 25cycles ( 이반응을통해 labeling되지않은 primer가 template에붙어 SNP 부위바로앞까지 anneal되고 SNP에해당하는부위에는 dideoxynucleotide가들어가합성되므로 SNP 부위까지만합성된 product가반복되어만들어짐.) 4 SAP treatment : 반응하고남은 oligonucleotide 제거과정 material volume( μl ) SAP(1unit/ μl ) 1 Deionized water 1 reaction temp. reaction time 37 1hr 72 15min 5 Running ABI 3700등의 automatic sequencer로분석함. 분석결과의형광색으로 SNP 위치에어떤 nucleotide sequence가있는지를조사함. 6 Data analysis Single base extension이끝난 product를 automatic sequencer인 ABI 3700을통해분석하 면 labeling 된부분만을 detection 하여다음과같은 peak 으로나타나게 됨. Primer extension assay 후 automatic sequencer 에서의분석에는한번에약 10 분밖에소요되지 않으므로 ABI 3700 과같은 high-throughput automatic sequencer 를사용할경우하루최 소한 2,000 개이상의 SNP 을 typing 할수있음. - 91 -
- 92 -
4) direct Sequencing 을이용한 genotyping & validation 1 Direct PCR sequencing 을위한 PCR primer 제작 - 선정된각각의유전자의전체 coding(exon) 부위및 intron에위치하는 splice donor/ acceptor site를포함하도록 primer를 design. - Exon1의 upstream 약 1.5kb 정도의 promoter 부위를 sequencing할수있도록 primer set 을 design. 바로옆의 fragment와최소한 100bp 정도는 overlap 되도록 primer를 design. - 1회분석부위가최소 500bp가되고약 700bp 정도의 amplicon을생성할수있도록 design. - Pseudogene, repeat, family member 등과 high homology region을공유하는부위를피하도록하고 pick up된 primer의위치에알려진 SNP 유무를확인. - 대부분의유전자부위는 1개의 384 plate에서동시에증폭할수있도록 annealing temperature를동일한온도에서설정. - Pick up된 primer sequence의 5 말단에 universal primer를포함하는 tail sequence를첨부하여 sequencing 반응시동일한 master mix를제조하여경제적이고효율적인 sequencing이가능하도록함 ( 아래그림참조 ) - 93 -
Liquid handler를이용한 PCR master mix의분주 - 384 well microtiter PCR plate에서진행. - DNA sample과 primer를제외한시료를섞은 master mix를제조하여 8μl씩 Biomek FX workstation을이용하여분주 - PCR mix composition (DNA 10ng) 10X Buffer MgCl 2(25mM) dntp(10mm) Primers(5pM) Taq(5unit/ μl ) dh 2O Total 1μl [1X] 1.2μl [3mM] 0.25μl [0.25mM] 1/1μl [5pM] 0.05μl [0.25unit] 5.5μl 10μl - PCR Cycle Condition (Tm : Primer3에서지정해준값을사용 ) 95 10mins 35 Cycles: 95 30s, Tm 1 min, 72 1 min 72 7mins - 증폭된 PCR product를 agarose gel에서확인 - 94 -
5) PCR Clean Up PCR 이후에과잉으로남아있는 primer와 dntp 등의불순물을 magnetic bead를이용한방식으로제거함. magnetic bead kit는 Agencourt사의 AMPure 를이용하며, automation 장비는 Beckman Coulter사의 Biomek FX를사용. <AMPure 를이용한 PCR clean up 의개요 > - PCR 이끝난 384 plate 2 개, SPRIPlate384 (384 magnetic plate) 2 개, 96set 20 μl tip 1 통, 70% 에탄올및 AMPure (magnetic bead 용액 ) 가들어있는 reservoir 각 1 개를아래그 림과같이 Biomek FX (96 head) 의 deck 에배치. <Deck Layout> - AMPure Startup Dialog 창을띄워 reaction format, number of plates, quadrants, PCR reaction volume, incubation time 등의 parameter 에대한값들을입력. - 95 -
<AMPure Startup Dialog> - 18 μl의 AMPure magnetic bead 용액을 384 plate 의각 well 에담겨있는 10 μl의 PCR product 에넣고 15 회 pipette mix. 이때 PCR product 는 magnetic bead 와결합하고나 머지불순물들은용액에남아있게됨. ( 용액을옮기고 pipette mix 를하는작업들은모 두 96 head 의 Biomek FX 장비를이용하며, tip 을사용하는각단계가끝날때마다 wash station에서 tip wash. - 위의 plate를 SPRIPlate384 (384 magnetic plate) 위에 3~5분동안올려놓고 magnetic bead가용액과분리되도록함. 이과정에서 magnetic bead와결합한 PCR product는 SPRIPlate384의자석링과결합. - Plate에남아있는용액을모두빨아내어제거. - 70% 에탄올 30μl를각 well에넣고실온에서 30초간 incubation 시킨후빨아내어제거. ( 단, PCR plate는그대로 SPRIPlate384 위에놓아둔채로진행 ) - 위의에탄올 wash 과정을한번더반복. - 남아있는소량의에탄올을제거하기위해실온에서 10-20분간 air-dry. - 30μl의물을각 well에넣고 shaking 하여 elution. - Elution한 PCR clean-up product를 agarose gel에서확인. 6) Sequencing reaction - Liquid handler (Biomek FX, Beckman Coulter) 를이용하여 PCR clean-up product를 384 plate의각 well에 2μl씩분주. - PCR clean-up product를제외한시료를섞은 master mix를제조하여 8μl씩 liquid handler (Biomek FX, Beckman Coulter) 를이용하여분주. - Sequencing reaction master mix composition - 96 -
5X Sequencing Buffer BigDye Terminator Sequencing Primers(1.6pM) dh 2 O Total 2μl [1X] 0.5μl 1.6μl [1pM] 3.9μl 8μl - Cycle Sequencing Condition 25 Cycles: 96 10s, 50 5s, 60 4 min - Sequencing primer 는 PCR 에사용했던 primer 의 5 에위치한 tail sequence 에 complement 한 primer 를이용. 7) Dye-Terminator Removal sequencing reaction 이후에사용되고남아있는 dye-terminator를 magnetic bead를이용한방식으로제거. magnetic bead kit는 Agencourt사의 CleanSEQ 를이용하며, automation 장비는 Beckman Coulter사의 Biomek FX를사용. <CleanSEQ 를이용한 dye-terminator removal 의개요 > - Sequencing reaction 이끝난 384 plate 3 개, SPRIPlate384 (384 magnetic plate) 3 개, final elution 용액을옮겨놓을빈 384 plate 3 개, 96set 20μl tip 1 통, deionized water ( 또는 elution buffer), 85% 에탄올, CleanSEQ (magnetic bead 용액 ) 가들어있는 reservoir 각 1 개를아래그림과같이 Biomek FX (96 head) 의 deck 에배치. - 97 -
<Deck Layout> - CleanSEQ Startup Dialog 창을띄워 reaction format, number of plates, quadrants, sequencing reaction volume, elution time, ethanol concentration 등의 parameter 에대한 값들을입력. <CleanSEQ Startup Dialog> - 5μl의 CleanSEQ magnetic bead 용액을 sequencing reaction (10μl) 이끝난 384 plate의각 well에넣음. ( 용액을옮기고 pipette mix를하는작업들은모두 96 head의 Biomek FX 장비를이용하며, tip을사용하는각단계가끝날때마다 wash station에서 tip wash.) - 85% 에탄올 42μl를각 well에넣고 14회 pipette mix 한다. 이때 sequencing reaction product는 magnetic bead와결합하고과잉의 dye-terminator는용액에남아있게됨. - 위의 plate를 SPRIPlate384 (384 magnetic plate) 위에 2분이상올려놓고 magnetic bead 가용액과분리되도록함. 이과정에서 magnetic bead와결합한 sequencing reaction product는 SPRIPlate384의자석링과결합. - Plate에남아있는용액을모두빨아내어제거. - 98 -
- 85% 에탄올 30μl를각 well에넣고 1~3회 pipette mix. - 실온에서 30초간 incubation 시킨후빨아내어제거. ( 단, sequencing reaction plate는그대로 SPRIPlate384 위에놓아둔채로진행.) - 위의에탄올 wash 과정을한번더반복. - 남아있는소량의에탄올을제거하기위해실온에서 10분간 air-dry. - 15μl의 deionized water ( 또는 elution buffer) 를각 well에넣고 5분간실온에서 incubation 하여 elution 한다. 8) 염기서열분석 - High-throughput automated sequencing machine(abi 3730xl, ABI3700) 을이용하여염기서 열분석 (2304 reactions/day). 9) Sequence Data Analysis 분석된 sequence 결과를 Phred and Phrap 및 Polyphred software package 를이용하여 multiple align 하여 SNP 위치를찾은후각 sample 에대한 genotype 을 call. SNP 분석 - Automatic DNA sequence analyzer에서 sequencing 결과생성된각샘플의 ab1 파일을 Polyphred 프로그램에 input파일로입력하여, sequencing된각샘플들의데이터를 align 한후 SNP 유무를확인. - 아래그림의예에서분석된 152번째염기에서 SNP이발견되었으며 ( 상단눈금아래의붉은색 tag으로표시된부분 ) 이들의 electropherogram 데이터를확인할수있음. - 99 -
<Polyphred : SNP 발굴> - 발견된 SNP 위치에서 각 샘플의 genotype 결과(wild, hetero, homo mutant)를 얻음. <Wild> <Hetero> <Homo mutant> - 100 -