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1 질병관리본부유전체센터 학술연구용역사업 한국인질병위험도측정및 질병예측을위한바이오마커발굴 (I) 당뇨병위험도분석을위한형질마커발굴 결과보고서 (2006 년 4 월 28 일 ) 디엔에이링크 질병관리본부유전체센터

2 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 검토사항 55 가. Minor allele frequency 차이 55 나. Novel SNP 여부 56 다. phenotype 자료 연구결과에대한기여도, 기대효과및활용방안 57 가. 기대효과 57

3 나. 활용방안 연구용역사업비 58 부록 1. Assay Information 59 부록 2. Gene blast Information 71 부록 3. Genotyping Technology 79

4 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 5q 와당뇨병발생과의관련성을새롭게보고하였음. 제 2 형당뇨병은인슐린저항성과베타세포의기능부전을특징으로하며당뇨병발생과관 련한원인유전자는인슐린분비경로및작용경로에관여하는호르몬, 대사관여물질과관 여한유전자들및인슐린에의해발현이조절되는유전자들로알려져있음

5 다. 연구필요성 최근미국등의서구사회뿐아니라우리나라에서도사회경제적발전과식생활의서구화 및생활수준의향상으로당뇨병의유병률이급격히증가하고있고당뇨병으로인한합병 증및사망률증가로보건사회적, 경제적부담이급증하고있음. 당뇨병은유전적인성향이강한질병으로가족, 특히형제, 자매중에당뇨병을앓고있는분이있는경우에는현재본인이정상이라할지라도당뇨병에걸릴확률은일반인들에비해무려 40% 정도증가함. 그러나최근까지밝혀진당뇨병의원인유전자는전체의 5% 미만에불과함. 질환은유전적소인과환경적소인에의하여발생하며유전적소인의대부분은개인간유전적차이인 SNP에의해나타나고있어, 그러한유전적소인이밝혀진다면이를바탕으로질병발생위험도측정과발생예측이가능하게되며, 이로써발병전중재를통하여적극적인예방, 조기진단, 종합적예후향상을기대할수있음. 한국인에서중요한대표적생활습관질환인당뇨병에대한병인요소연구에의해서최근 밝혀진유전체지표를이용하여당뇨병발생의위험도를측정할수있을것으로판단됨

6 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 -

7 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

8 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

9 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 -

10 자세한분석원리및분석과정은부록 3 에나타내었음. <High-throughput Liquid Handling> - 7 -

11 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

12 <Data Flow Chart> - 9 -

13 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 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 중복시료수 % 이상 SNP결과 fail시료수 6 7 미분석시료수 2 2 최종분석시료수 ) Genotyping 분석현황 - 175개 set에대한분석은 6개의 SNP을제외한 169개의 SNP에대한분석이완료되었음. 분석이완료된 SNP 중 new36, new134, rs 의 frequency가 monomorphic, 0.002, 0.01 로나타나 setting시 monomorphic한결과를보인 SNP에대한대량분석은실시하지않음. 분석제외된 SNP은 sequencing으로빈도를재확인하였고 CYP11B2의 rs , rs , new30와 STXBP3의 new127 등 4개 SNP은 monomorphic한결과를보였음. assay가만들어지지않은 SNP은 FABP2의 rs , STXBP3의 rs 개임

14 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 Snapshot UHT NEW4 UHT UHT UHT UHT CD UHT UHT UHT UHT UHT Snapshot UHT CYP11B2 NEW15 25K Snapshot UHT UHT NEW19 25K sequencing sequencing K K K K K K K snapshot NEW30 sequencing K NEW32 sequencing sequencing FABP Snapshot UHT NEW36 Mono UHT K UHT UHT FABP UHT UHT UHT UHT assay fail UHT UHT UHT UHT UHT UHT UHT

15 rs number platform Pass Fail CallRate XX XY YY X Y Hetero HWE zygosity p-value FABP UHT UHT UHT UHT NEW57 UHT NEW58 25K UHT NEW60 UHT FABP UHT UHT UHT SLC27A UHT UHT NEW66 Snapshot NEW67 25K snapshot UHT K UHT NEW72 Snapshot UHT UHT UHT SLC27A K NEW77 25K NEW78 UHT Snapshot NEW80 UHT NEW81 UHT K ASPSCR1 NEW83 UHT NEW84 25K NEW85 UHT NEW86 UHT K NEW88 Snapshot K CACNA1G UHT NEW91 Snapshot UHT UHT K UHT UHT NEW97 UHT UHT UHT PPARD UHT UHT UHT UHT UHT UHT UHT

16 Gene rs number platform Pass Fail CallRate XX XY YY X Y Hetero HWE zygosity p-value PPARD UHT PPARG Snapshot Snapshot SNAP UHT SNTG UHT UHT UHT UHT UHT UHT UHT UHT UHT UHT UHT UHT STXBP UHT snapshot UHT UHT NEW127 sequencing UHT UHT UHT assay fail snapshot UHT NEW134 UHT UHT UHT STXBP UHT UHT UHT NEW140 UHT UHT UHT UHT UHT UHT UHT UCP UHT UHT NEW149 UHT UHT NEW151 UHT UHT UHT UCP K K NEW156 Snapshot K UCP UHT NEW159 UHT UHT

17 Gene rs number platform Pass Fail CallRate XX XY YY X Y Hetero HWE zygosity p-value UCP UHT UHT K UHT UHT UHT VAMP2 NEW167 UHT UHT NEW169 UHT UHT snapshot VAMP UHT NEW173 snapshot K NEW175 UHT 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

18 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< p< p< Total

19 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 Control INTRON AA AG GG DM Control EXON2 Phe91Phe CC CT TT DM Control INTRON NEW4 GG AG AA DM Control INTRON GG GT TT DM Control INTRON GG AG AA DM Control INTRON CC CT TT DM Control CD36 UP AA AC CC DM Control INTRON TT CT CC DM Control EXON3 Pro89Ser CC CT TT DM Control INTRON AA AG GG DM Control INTRON CC CT TT DM Control INTRON GG AG AA DM Control DOWN TT GT GG DM Control CYP11B2 UP NEW15 AA AT TT DM Control

20 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p CYP11B2 UP CC CT TT DM Control UP CC CT TT DM Control UP TT CT CC DM Control EXON 1 Pro36Pro NEW19 GG AG AA DM <.0001 Control <.0001 EXON 3 Lys172Arg 4539 AA AG GG DM <.0001 Control <.0001 INTRON 6399 CC CT TT DM <.0001 Control <.0001 EXON 5 Ala290Ala 4536 GG AG AA DM <.0001 Control <.0001 INTRON TT AT AA DM Control EXON 8 Ser434Gly 4545 AA AG GG DM Control INTRON 6431 TT CT CC DM <.0001 Control <.0001 EXON GG AG AA DM Control EXON GG GT TT DM Control EXON 9 NEW32 CC CT TT DM Control EXON AA AG GG DM Control FABP1 UP CC CG GG DM Control UP CC AC AA DM Control

21 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p FABP1 UP GG AG AA DM Control EXON 3 Thr39Ala AA AG GG DM Control INTRON CC CT TT DM Control DOWN TT CT CC DM Control FABP2 UP TT CT CC DM Control UP GG GT TT DM Control UP GG AG AA DM Control UP GG AG AA DM Control UP GG AG AA DM Control INTRON AA AC CC DM Control EXON 2 Ala54Thr GG AG AA DM Control EXON 2 Asn71Asn CC CT TT DM Control INTRON GG GT TT DM Control INTRON TT CT CC DM Control EXON AA AG GG DM Control EXON GG AG AA DM Control

22 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p FABP2 EXON CC CT TT DM Control EXON CC CG GG DM Control EXON TT CT CC DM Control EXON 4 NEW57 CC CT TT DM Control EXON 4 NEW58 AA AG GG DM Control EXON CC AC AA DM Control DOWN NEW60 AA AG GG DM Control FABP4 INTRON AA AC CC DM Control DOWN AA AT TT DM Control DOWN AA AG GG DM Control SLC27A1 UP GG GT TT DM Control UP AA AG GG DM Control INTRON NEW66 GG AG AA DM Control INTRON NEW67 GG AG AA DM Control INTRON TT CT CC DM Control INTRON CC CG GG DM Control

23 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p SLC27A1 INTRON GG AG AA DM Control INTRON TT GT GG DM Control EXON 12 NEW72 GG GT TT DM Control EXON GG AG AA DM Control EXON GG AG AA DM Control EXON TT CT CC DM Control SLC27A4 UP GG AG AA DM Control UP NEW77 GG CG CC DM Control INTRON NEW78 CC CT TT DM Control EXON 4 Gly208Ser GG AG AA DM Control INTRON NEW80 GG AG AA DM Control INTRON NEW81 TT CT CC DM Control DOWN AA AG GG DM <.0001 Control <.0001 ASPSCR1 UP NEW83 CC AC AA DM Control UP NEW84 GG CG CC DM Control INTRON NEW85 GG GT TT DM Control

24 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p ASPSCR1 INTRON NEW86 CC AC AA DM Control EXON 7 Leu251Gln TT AT AA DM Control INTRON NEW88 GG GT TT 0 DM Control EXON 14 Asp486Asp TT CT CC DM Control CACNA1G UP GG CG CC DM Control UP NEW91 AA AG GG DM Control INTRON GG GT TT DM Control EXON 8 Arg530Arg AA AC CC DM Control INTRON GG AG AA DM Control INTRON GG AG AA DM Control INTRON CC CG GG DM Control INTRON NEW97 AA AG GG DM Control EXON 36 Pro2223Pro TT CT CC DM Control EXON 36 Pro2301Pro GG AG AA DM Control PPARD INTRON GG GT TT DM Control EXON TT CT CC DM Control

25 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p PPARD INTRON TT GT GG DM Control EXON 7 Asn162Asn TT CT CC DM Control INTRON GG AG AA DM Control EXON TT CT CC DM Control EXON TT CT CC DM Control DOWN GG GT TT DM Control PPARG UP AA AG GG DM Control EXON 6 Pro296Pro CC CG GG DM Control SNAP23 INTRON TT CT CC DM Control SNTG1 UP AA AG GG DM Control UP CC CT TT DM Control INTRON GG AG AA DM Control INTRON TT CT CC DM Control INTRON TT CT CC DM Control INTRON GG AG AA DM Control INTRON TT CT CC DM Control

26 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p SNTG1 INTRON TT AT AA DM Control INTRON AA AG GG DM Control EXON 15 Thr329Thr AA AG GG DM Control INTRON CC AC AA DM Control EXON TT GT GG DM Control STXBP3 UP CC AC AA DM Control EXON CC CG GG DM Control INTRON CC CT TT DM Control INTRON CC CG GG DM Control INTRON GG CG CC DM Control INTRON GG GT TT DM Control INTRON TT CT CC DM Control EXON 14 Leu371Leu GG CG CC DM Control INTRON GG AG AA DM Control INTRON NEW134 GG AG AA DM Control EXON GG AG AA DM Control

27 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p STXBP3 DOWN GG CG CC DM Control STXBP4 UP TT CT CC DM Control UP GG AG AA DM Control UP GG AG AA DM Control UP NEW140 AA AG GG DM Control UP CC CG GG DM Control UP CC AC AA DM Control EXON 3 Arg14Gly AA AG GG DM Control INTRON AA AG GG DM Control INTRON AA AG GG DM Control EXON AA AG GG DM Control UP GG GT TT DM Control UCP1 UP AA AT TT DM Control UP NEW149 GG AG AA DM Control INTRON TT CT CC DM Control EXON 2 Ala63Thr NEW151 GG AG AA DM Control

28 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p UCP1 INTRON CC CT TT DM Control EXON 5 Met228Leu AA AT TT DM Control UCP2 UP GG AG AA DM Control UP CC CT TT DM Control EXON 1 NEW156 CC CT TT DM Control EXON 4 Ala54val CC CT TT DM Control UCP3 UP CC CT TT DM Control EXON 3 NEW159 TT CT CC DM Control INTRON GG AG AA DM Control INTRON AA AT TT DM Control INTRON TT CT CC DM Control INTRON CC CT TT DM <.0001 Control <.0001 EXON 5 Tyr209Tyr TT CT CC DM Control EXON CC CT TT DM Control DOWN TT CT CC DM Control VAMP2 INTRON NEW167 GG CG CC DM Control

29 Gene Position Amino acid Hetero HWE rs number Genotype MAF change zygosity p VAMP2 EXON CC AC AA DM Control EXON 5 NEW169 CC CT TT DM Control EXON CC CT TT DM Control EXON CC CT TT DM Control VAMP3 UP CC AC AA DM Control EXON 1 NEW173 CC AC AA DM Control INTRON CC CG GG DM Control EXON 5 NEW175 AA AG GG DM Control

30 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< MAF< MAF< MAF< MAF< MAF< Total ) Haplotype 빈도 - 20 개유전자에서분석된 SNP 의수와 Haplotype block 및 5% 이상발현되는 Haplotype 의 수는다음과같음. SNP수 Block수 Haplotype 수 ACAS ASPSCR CACNA1G CD CYP11B FABP FABP FABP PPARD SLC27A SLC27A SNTG STXBP STXBP UCP UCP UCP VAMP VAMP

- FABP4 의 LD 및 Haplotype frequency Haplotype Frequency A-A 0.65260 A-G 0.28051 T-G 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.

31 - FABP4 의 LD 및 Haplotype frequency Haplotype Frequency A-A A-G T-G SNP1 SNP2 D' r^2 rs rs rs rs rs rs SCL27A4 의 Haplotype frequency Haplotype Frequency G-G-C-G-G-T A-G-T-A-G-C A-C-C-G-G-T A-C-C-G-G-T A-G-C-G-G-T G-G-T-A-G-C A-G-T-G-G-C G-G-T-G-G-T G-C-C-G-A-T G-G-C-G-A-T G-C-C-G-G-T

32 - SCL27A4의 LD SNP1 SNP2 D' r^2 rs NEW rs NEW rs rs rs NEW rs NEW rs rs NEW77 NEW NEW77 rs NEW77 NEW NEW77 NEW NEW77 rs NEW78 rs NEW78 NEW NEW78 NEW NEW78 rs rs NEW rs NEW rs rs NEW80 NEW NEW80 rs NEW81 rs

- 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.

33 - UCP1 의 Haplotype block 과 haplotype frequency Haplotype Frequency G-C G-T A-T A-C UCP1 의 LD SNP1 SNP2 D' r^2 rs rs rs NEW rs rs rs NEW rs rs rs rs rs NEW rs rs rs NEW rs rs rs rs NEW149 rs NEW149 NEW NEW149 rs NEW149 rs rs NEW rs rs rs rs NEW151 rs NEW151 rs rs rs

- VAMP2의 LD SNP1 SNP2 D' r^2 NEW167 rs1061032 1 0.023 NEW167 NEW169 0.637 0.

096 rs1061032 rs11548690 1 0.073 rs1061032 rs1150 0.998 0.894 NEW169 rs11548690 1 0.

34 - VAMP2의 LD SNP1 SNP2 D' r^2 NEW167 rs NEW167 NEW NEW167 rs NEW167 rs rs NEW rs rs rs rs NEW169 rs NEW169 rs rs rs VAMP2 의 Haplotype block 과 haplotype frequency

35 1 ACAS2 유전자 Haplotype Frequency GACGGGC TGTGTAT GATGTAT GGTGTAT GACAGGC TATGTAT GGTATAT GATATAT CD36 유전자

36 3 CYP11B2 유전자

4 FABP1 유전자 5 FABP2 유전자 Haplotype Frequency

37 4 FABP1 유전자 5 FABP2 유전자 Haplotype Frequency TGGGGAGCGCGATGCTGA TTAAGCATGTAGCCTCAC TGGGGAGCTCGATGCTGA CTAAACGTGTAGCCTCAC TTAAGCGTGTAGTCTCAC TGGGGAGCGCGATGCTAA TGGGGAGCTTAGCCTTAC TTAAGCATGTAGCCTTAC

38 6 SCL27A1 유전자

39 7 ASPSCR1 유전자 8 CACNA1C 유전자 Haplotype Frequency GAG TCA TCG TAG GCA GCG TAA

9 PPARD 유전자 Haplotype Frequency TTTGTTG 0.

01565 CGCGCCG 0.00625 CGCGTTG 0.00392 TGCGCCT 0.

00042 TGCGCCG 0.00041 TTCGCCG 0.00038 TTTGCTG 0.

40 9 PPARD 유전자 Haplotype Frequency TTTGTTG CGCGCCT TTTATTG CGCGCTG CGCGCCG CGCGTTG TGCGCCT CTTGTTG CGCACCT TTTGCCT TGCGCCG TTCGCCG TTTGCTG TGCACCT CGCACTG SNTG1 유전자

11 STXBP3 유전자 Haplotype Freq CCCCGGTGGGGG 0.74113 AGTGCTCCAGAC 0.21587 AGTGCTCGAGAC 0.01448 CCCCGGTGGGGC 0.01232 CGTGCTCCAGAC 0.00375 AGTCGGTGGGGG 0.

00038 ACTGCTCGAGAC 0.00038 AGTGGGTGGGGG 0.00038 AGTGCGTGGGGG 0.00038 CGTGCTCCAAAC 0.00035 12 STXBP4 유전자 Haplotype Frequency GACCAAAA 0.

41 11 STXBP3 유전자 Haplotype Freq CCCCGGTGGGGG AGTGCTCCAGAC AGTGCTCGAGAC CCCCGGTGGGGC CGTGCTCCAGAC AGTCGGTGGGGG AGTGCTCCAAAC CCCCGGCGGGGG CCCGCTCCAGAC CGTGCTCGAGAC ACTGCTCCAGAC CCCCGGTGGAAC CCCGGTCCAGAC ACTGCTCGAGAC AGTGGGTGGGGG AGTGCGTGGGGG CGTGCTCCAAAC STXBP4 유전자 Haplotype Frequency GACCAAAA GACCAAAG AACCAAGA AGCCAAAA AACCAAAA AAGAGGAA GACCGGAA AAGAAAAA AGCCGGAA AGGAAAAA

block 와 haplotype frequency 는 CD 의 excel 파일 의

42 13 UCP2 유전자 Haplotype Frequency GCC ACC ACT GTC GCT ATC GTT ATT 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

43 나. 당뇨병발생과의관련성 - 175개 SNP set의당뇨-대조군의 phenotype 자료를이용하여각 SNP과당뇨병발생과의관련성에대한분석을진행하였음. Monomorphic하거나 MAF 0.01미만인 rs , , new30, new36, new127, new134, re 와 assay fail된 rs , rs 를제외한 166개 SNP에대한 association 분석을진행하였음. - FABP1, SLC27A1, UCP1, VAMP2 유전자와당뇨병발생위험증가와유의한관련성을보였고 CACNA1G 유전자는당뇨병발생위험감소와유의한관련성을나타냄. 각모델별로유전자별당뇨발생에대한 OR 의 p value 를나타낸그림임. rs ( ) rs ( ) Dominant model rs ( ) rs ( ) p=0.05 -Log 10 (p-value)

44 Co-dominant model rs ( ) rs ( ) New51,rs New169 p=0.05 -Log 10 (p-value) Recessive model rs ( ) p=0.05 -Log 10 (p-value)

45 SNP 및 Haplotype 분석결과요약 Gene SNP Position DM Control DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p FABP INTRON (1.0308,1.6804) (0.983,1.324) (0.846,1.3924) DOWN (1.0519,1.7165) (0.966,1.304) (0.7863,1.2999) h1(act) (0.7504,1.2403) (0.764,1.032) (0.5886,0.9631) h2(atc) (0.8718,1.3521) (0.877,1.239) (0.6314,1.4296) h3(gtc) (0.9219,1.453) (0.938,1.389) (0.6867,2.2383) FABP DOWN (0.9319,1.454) (0.998,1.392) (1.0003,2.0137) DOWN (0.5823,1.117) (0.59,1.097) J (0.0935,2.808) F h1(a-a) (0.4965,0.9996) (0.721,1.005) (0.6919,1.0799) h2(a-g) (1.0564,1.6405) (1.075,1.528) (1.0001,2.337) h3(t-g) (0.5764,1.1183) (0.585,1.098) J (0.0939,2.8183) F SCL27A INTRON (1.0294,1.6232) (1.037,1.534) CA (0.8162,2.6269) INTRON (0.934,1.4936) (0.952,1.304) (0.8388,1.477) h1(gt) (0.6667,1.1755) (0.762,1.045) (0.6672,1.0678) h2(gg) (0.8132,1.2676) (0.806,1.169) (0.4461,1.2337) h3(ag) (1.0034,1.5855) (1.013,1.502) CA (0.7827,2.5414) EXON (0.6178,1.0099) (0.64,0.996) CA (0.2868,1.4141) CACNA1G UP (0.7077,1.1681) (0.694,1.083) (0.1859,0.9843) PPARD INTRON (1.0039,2.9681) UCP UP ) (1.0394,1.8651) (1.044,1.802 CA (0.5267,6.2056) INTRON (0.9986,1.7012) (1.022,1.653) CA (0.7642,4.8639) NEW151 EXON (1.0041,1.8387) (1.026,1.805) CA (0.6226,9.3929) F

46 Gene SNP Position DM Control DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p UCP INTRON (0.8407,1.3138) (0.873,1.259) (0.6801,1.7832) h1(g-c) (0.5405,1.4273) (0.785,1.133) (0.7538,1.1788) h2(g-t) (0.7138,1.174) (0.743,1.145) (0.4298,1.7223) h3(a-t) (0.9665,1.7777) (0.984,1.743) J (0.5187,8.3652) F VAMP EXON (0.9284,1.5013) (0.956,1.297) (0.8683,1.4643) NEW169 EXON (0.9988,1.8451) (0.94,1.66) (0.1133,1.7091) F h1(c-c) (0.6844,1.1551) (0.774,1.05) (0.6704,1.0844) h2(a-c) (0.8766,1.3749) (0.888,1.213) (0.7226,1.3118) h3(a-t) (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

47 Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p ACAS2 NEW1 UP (0.7706,1.1923) (0.835,1.183) (0.7401,1.7293) INTRON (0.8014,1.2402) (0.867,1.203) (0.7802,1.5885) EXON (0.7434,1.2084) (0.862,1.174) (0.8341,1.407) NEW4 INTRON (0.4726,1.7817) (0.473,1.782) INTRON (0.7281,1.1916) (0.857,1.17) (0.8372,1.4161) INTRON (0.7291,1.1898) (0.851,1.162) (0.8212,1.3891) INTRON (0.7397,1.2072) (0.863,1.18) (0.841,1.4329) CD UP (0.8107,1.2526) (0.842,1.185) (0.6527,1.4402) INTRON (0.6617,1.0277) (0.721,1.008) (0.5587,1.1496) EXON (0.8251,1.5468) (0.829,1.507) J (0.2068,5.113) F INTRON (0.6841,1.3681) (0.754,1.438) J (0.7452, ) F INTRON (0.7797,1.2846) (0.798,1.259) (0.4265,2.4959) INTRON (0.7807,1.2256) (0.818,1.213) (0.6136,2.1018) DOWN (0.787,1.2311) (0.832,1.227) (0.682,2.2225) CYP11B2 NEW15 UP (0.8591,1.3285) (0.867,1.219) (0.6327,1.3786) UP (0.7592,1.3184) (0.762,1.256) (0.2724,1.9032) UP (0.8619,1.334) (0.865,1.217) (0.6229,1.3535) UP (0.8529,1.3215) (0.855,1.205) (0.6026,1.3228) NEW19 EXON (0.5227,1.35) (0.649,1.351) (0.4911,3.5842) 4539 EXON (0.7896,1.2285) (0.851,1.131) (0.7297,1.2501) 6399 INTRON (0.8282,1.2828) (0.882,1.171) (0.7723,1.326) 4536 EXON (0.7709,1.1989) (0.865,1.145) (0.7997,1.3509) INTRON (0.8803,1.4329) (0.847,1.153) (0.6507,1.0976) J: Jonckheere-Terpstra Test, F: Fisher's Exact test

48 Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p CYP11B EXON (0.7421, (0.834,1.137) (0.7578,1.2684) 6431 INTRON (0.7598,1.2219) (0.76,1.222) 3097 EXON (0.661,1.294) (0.68,1.282) J (0.2055,5.0821) F EXON (0.8099,1.2572) (0.826,1.165) (0.5967,1.2979) NEW32 EXON (0.8649,1.3386) (0.868,1.22) (0.624,1.3558) EXON (0.8497,1.3219) (0.85,1.193) (0.6043,1.2733) FABP UP (0.7816,1.311) (0.771,1.223) (0.2661,1.4097) UP (0.6997,1.101) (0.743,1.087) (0.5182,1.5161) UP (0.7154,1.127) (0.753,1.103) (0.5124,1.4994) EXON (0.9674,1.5131) (0.962,1.413) (0.6363,1.9727) FABP UP (0.6067,1.2105) (0.613,1.176) J (0.0936,2.8081) F UP (0.6858,1.0887) (0.748,1.02) (0.5982,1.0538) UP (0.6889,1.0913) (0.6007,1.0577) UP (0.6865,1.0886) (0.746,1.019) (0.5933,1.0511) UP (0.6088,1.2148) (0.615,1.18) J (0.0938,2.8169) F INTRON (0.6889,1.0913) (0.748,1.019) (0.5952,1.0473) EXON (0.733,1.1341) (0.784,1.09) (0.6186,1.2635) EXON (0.6899,1.0936) (0.751,1.023) (0.6008,1.0572) INTRON (0.8501,1.3325) (0.849,1.237) (0.5118,1.4599) INTRON (0.9548,1.4892) (0.973,1.339) (0.8582,1.6218) 2964 EXON (0.9586,1.4956) (0.975,1.343) (0.8598,1.6248) EXON (0.9586,1.4956) (0.975,1.343) (0.8598,1.6248) EXON (0.918,1.4501) (0.946,1.293) (0.8432,1.5035) J: Jonckheere-Terpstra Test, F: Fisher's Exact test

49 Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p FABP EXON (0.9577,1.4959) (0.979,1.349) (0.8717,1.6536) EXON (0.9615,1.5047) (0.978,1.349) (0.8632,1.632) NEW57 EXON (0.954,1.4895) (0.964,1.327) (0.83,1.5656) NEW58 EXON (0.9391,1.4586) (0.985,1.36) (0.9354,1.845) EXON (0.9561,1.4923) (0.976,1.345) (0.8648,1.6424) NEW60 DOWN (0.563,1.2301) (0.575,1.23) J (0.0644, ) F FABP INTRON (0.5932,1.4051) (0.586,1.368) J F SLC27A UP (0.7713,1.3455) (0.793,1.327) (0.3998,3.5789) UP (0.9315,1.4824) (0.94,1.292) (0.8023,1.4415) NEW66 INTRON (0.7737,1.3416) (0.803,1.333) (0.4761,3.9997) NEW67 INTRON (0.6544,1.2128) (0.682,1.239) J (0.3211, ) F INTRON (0.9651,1.5422) (0.967,1.322) (0.8455,1.4792) INTRON (0.9678,1.5471) (0.982,1.344) (0.8837,1.5494) NEW72 EXON (0.5701,1.7122) (0.617,1.703) J (0.186, ) F EXON (0.9529,1.5072) (0.956,1.426) (0.6366,2.2163) 9137 EXON (0.6263,1.0252) (0.656,1.021) (0.3417,1.645) SLC27A UP (0.8953,1.3832) (0.943,1.319) (0.8647,1.8656) NEW77 UP (0.7577,1.4516) (0.742,1.367) J (0.0793,2.1215) F NEW78 INTRON (0.8868,1.3903) (0.892,1.293) (0.6082,1.6499) EXON (0.8951,1.4044) (0.913,1.322) (0.6907,1.8485) NEW80 INTRON (0.6936,1.4324) (0.677,1.343) J (0.0285,2.2957) F NEW81 INTRON (0.8974,1.4074) (0.911,1.319) (0.6687,1.8014) DOWN (0.9576,1.4841) (0.957,1.484)

50 Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p ASPSCR1 NEW83 UP (0.8736,1.4243) (0.907,1.234) (0.7998,1.3437) NEW84 UP (0.7131,1.7194) (0.699,1.66) J F NEW85 INTRON (0.5334,1.279) (0.533,1.279) NEW86 INTRON (0.7447,1.4679) (0.749,1.456) J (0.0641, ) F EXON (0.746,1.1665) (0.766,1.117) (0.4665,1.3597) NEW88 INTRON F (0.6624,1.3579) (0.642,1.286) J EXON (0.7906,1.2215) (0.833,1.165) (0.6663,1.4249) CACNA1G NEW91 UP (0.6468,1.7543) (0.645,1.641) J (0.0465,5.6834) F INTRON (0.6633,1.2851) (0.67,1.259) J (0.1128,4.0666) F EXON (0.6877,1.3243) (0.692,1.295) J (0.1145,4.1287) F INTRON (0.673,1.3739) (0.665,1.348) J F INTRON (0.7305,1.298) (0.742,1.267) (0.2607,2.8277) INTRON (0.6645,1.118) (0.711,1.139) (0.519,3.0638) NEW97 INTRON (0.8265,1.348) (0.81,1.271) (0.2329,1.5221) EXON (0.7524,1.2219) (0.756,1.167) (0.3221,1.5192) EXON (0.6186,1.195) (0.642,1.18) J (0.2196,3.0724) F PPARD INTRON (0.6703,1.2741) (0.71,1.313) J (0.4592, ) F EXON (0.8287,1.2909) (0.831,1.201) (0.5165,1.3772) INTRON (0.8035,1.2473) (0.809,1.165) (0.4984,1.314) EXON (0.7928,1.2322) (0.802,1.155) (0.4984,1.3142) EXON (0.7966,1.2369) (0.788,1.138) (0.4213,1.1526) EXON (0.7937,1.2391) (0.783,1.133) (0.4035,1.1159) DOWN (0.8038,1.2554) (0.803,1.179) (0.4282,1.3566)

51 Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p PPARG UP (0.8575,1.3589) (0.865,1.294) (0.5184,1.8553) EXON (0.9815,1.9687) (0.998,1.952) J (0.3215, ) F SNAP INTRON (0.9024,1.3981) (0.915,1.313) (0.6809,1.7412) SNTG UP (0.6908,1.0901) (0.746,1.095) (0.5765,1.6985) UP (0.6452,1.0315) (0.697,1.048) (0.5127,1.8352) INTRON (0.6965,1.1093) (0.739,1.113) (0.5272,1.9843) INTRON (0.7547,1.1893) (0.799,1.184) (0.6155,2.051) INTRON (0.7705,1.2691) (0.809,1.253) (0.5709,2.4373) INTRON (0.766,1.2444) (0.768,1.177) (0.3448,1.4384) INTRON (0.7572,1.2288) (0.76,1.164) (0.3399,1.4176) INTRON (0.7329,1.15) (0.77,1.126) (0.5356,1.58) INTRON (0.798,1.2349) (0.853,1.195) (0.7346,1.5572) EXON (0.8004,1.2387) (0.857,1.201) (0.7461,1.5863) INTRON (0.7876,1.217) (0.831,1.181) (0.6702,1.5784) EXON (0.5467,0.9978) (0.574,1.03) J (0.3215, ) F STXBP UP (0.6709,1.0428) (0.715,1.03) (0.496,1.3075) EXON (0.6769,1.0521) (0.747,1.066) (0.6241,1.5268) INTRON (0.6689,1.0407) (0.74,1.055) (0.6131,1.4931) INTRON (0.6849,1.0652) (0.746,1.062) (0.5836,1.4086) INTRON (0.6857,1.0672) (0.746,1.062) (0.5798,1.3994) INTRON (0.6876,1.0713) (0.741,1.057) (0.5495,1.3415) INTRON (0.694,1.079) (0.748,1.066) (0.5665,1.3749) EXON (0.704,1.0991) (0.746,1.074) (0.5227,1.3497)

52 Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p STXBP INTRON (0.6852,1.0652) (0.746,1.062) (0.5836,1.4084) EXON (0.6867,1.0678) (0.747,1.064) (0.5846,1.4108) DOWN (0.6966,1.0809) (0.747,1.057) (0.5509,1.2747) STXBP UP (0.8513,2.0337) (0.866,1.989) J (0.1863, ) F UP (0.7913,1.2548) (0.901,1.226) (0.8946,1.5736) UP (0.7427,1.1772) (0.85,1.155) (0.8113,1.4124) NEW140 UP (0.696,1.1393) (0.712,1.117) (0.3203,1.8295) UP J (0.831,1.9739) (0.829,1.868) (0.1444,7.3208) F UP (0.85,2.0305) (0.846,1.914) J (0.1442,7.31) F EXON (0.851,2.0142) (0.847,1.902) J (0.1444,7.3211) F INTRON (0.8482,2.0075) (0.844,1.897) J (0.1439,7.2986) F INTRON (0.7357,1.1928) (0.753,1.144) (0.4028,1.5069) EXON (0.757,1.1884) (0.842,1.23) (0.9034,2.6642) UCP UP (0.7259,1.1312) (0.76,1.049) (0.5584,1.0762) NEW149 UP (0.7297,1.3495) (0.745,1.327) J (0.2553,4.1176) F EXON (0.972,1.674) (0.997,1.646) (0.7035,6.0887) UCP UP (0.6259,1.0071) (0.777,1.048) (0.7479,1.252) UP (0.9251,2.061) (0.956,1.995) J (0.5008, ) F NEW156 EXON (0.7134,1.2816) (0.762,1.316) (0.6217,9.3792) F EXON (0.6949,1.1445) (0.795,1.092) (0.7161,1.2189) UCP UP (0.7166,1.1085) (0.787,1.111) (0.6819,1.5422) NEW159 EXON (0.7267,1.123) (0.784,1.104) (0.6358,1.4179) INTRON (0.7137,1.1054) (0.778,1.101) (0.651,1.4841)

53 Gene SNP Position DOMINANT CODOMINANT RECESSIVE OR(95% CI) p OR(95% CI) p OR(95% CI) p UCP INTRON (0.9702,1.6105) (0.964,1.317) (0.8516,1.4141) INTRON (0.8115,1.276) (0.892,1.227) (0.8416,1.5549) INTRON (0.7563,1.1856) (0.752,1.178) J F EXON (0.9689,1.6052) (0.952,1.302) (0.8213,1.3717) EXON (0.9735,1.6142) (0.955,1.306) (0.8252,1.377) DOWN (0.6903,1.0673) (0.773,1.091) (0.7061,1.5795) VAMP2 NEW167 INTRON (0.7817,2.0717) (0.74,1.88) J F EXON (0.6954,1.2927) (0.707,1.246) (0.2316,2.3229) 1150 EXON (0.9031,1.4841) (0.98,1.328) (0.9591,1.5839) VAMP UP (0.8309,1.8018) (0.831,1.734) J (0.1439,7.2986) F NEW173 EXON (0.7615,1.1822) (0.802,1.15) (0.6076,1.5358) INTRON (0.7789,1.2395) (0.805,1.093) (0.6351,1.0973) NEW175 EXON (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

54 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 NEW IRS IRS NEW CBL RAPGEF PIK3CA T/C

55 - 대량분석결과각각의 SNP 빈도는다음과같음 GeneName rs# SNP discovery-ksnp DNALINK INSR G A G A New202 G C G C T C C T A G A 0.94 G 0.06 IRS A G A G novel( ) G A IRS A G A G New208 C D C D CBL T G T G RAPGEF G A G A G A G 0.86 A G A G A G A G A G A G A T C T C PIK3CA C A G A novel(21142t/c) T C T C Chr 19 : INSR 19p13.2 r2 = Caucasian Chinese Japanese Af-American KR_DNALink rs A G rs C T

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.

56 Chr 2 : IRS1 2q36.3 r 2 = Caucasian Chinese Japanese Af-American KR-DNAlink rs G A rs A G Chr 9 : RAPGEF1-53 -

57 표. RAPGEF1 유전자의 International HapMap 의다인종에서의발현빈도 rs rs rs rs rs C T C T C T C T C T Caucasian Chinese Japaneses Af -American KR_DNALink

58 5. 검토사항 가. Minor allele frequency 차이 - 국립보건원 SNP discovery 결과의 SNP 빈도와대량분석의 genotyping 결과의빈도가유의 적인차이를보이는경우가있음. - CYP11B2 rs 에대해서 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 CT TT 33 0 C:T 0.838: : SNP discovery 에서 polymorphism 이발견되었다고보고된 CYP11B2 rs , rs , new30 등도높은 gene homology 에의한 false positive 결과로생각된다. - CYP11B2의유전자는동일염색체 8번에위치한 CYP11B1와높은 homology를나타내기때문에 CYP11B2 특이적인염기서열을이용한 Long PCR을실시한후그 product를이용하여 target SNP에대한 genotyping 분석을실시해야정확한분석결과를얻을수있음을확인하였음

59 - 제공된 SNP discovery의빈도와대량분석및일본인에서보고된빈도와의비교시 SNP discovery결과의빈도와차이남을확인할수있음. HapMap Japanese의알려진빈도와비교한결과는다음과같음 SNP discovery DNALINK Japanese(HapMap) CYP11B2 NEW30 T C T C C T C T G A G A G A C T C T FABP1 NEW36 T A T A STXBP3 NEW127 T C 0.2 T C NEW134 G A G A FABP T G G T C A A C A C SLC27A A 0.75 G 0.25 A G PPARD T C T C T C T G T G T G T C T C T C T C T C T C T C G 0.87 T G T G 0.86 T 0.14 SNTG A G A G INSR rs T C C T C T 나. Novel SNP 여부 SNP discovery 에서 Novel SNP 으로보고된 site 중 FABP2 의 New45 는 rs , New48 은 rs 가같은 site 였으며 ASPSCR1 의 New89 는 rs13087, PPARD 의 New102 는 rs , STXBP3 New135 는 rs , IRS1 의 G971R 은 rs 과같은 site 로확인되었다. 다. phenotype 자료 6500 명에대한추가 Phenotype 자료가없어더이상의통계분석이어려움. 자료가추가되는 대로 follow-up 하겠음

60 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

61 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 (rs ) Reverse Primer TTTTCCATTTTCAAAGAACCG Extension Primer(R) TCCATCTCCTCACCCACCCCTCAGC ACAS2 Forward Primer CATTCCTTCGGTACAACTTTG (rs ) Reverse Primer TTTTTCTCATGGACATTTCGA Extension Primer(R) CGTGCCGCTCGTGATAGAATTAGTTGCTCCTTTCATCCACTCAAT ACAS2 Forward Primer TTCTTTCTGTTCCATTCCATC (NEW4) Reverse Primer AAGAACTGTGAACAGCTCAAGAT Extension Primer(R) AGATAGAGTCGATGCCAGCTGAGGTGGGACAGGCAGGTAGGAGTA ACAS2 Forward Primer TAGCTGGGGGATGGACAA (rs ) Reverse Primer AAGATACCATCCTAGYCAGGATAC Extension Primer(F) AGATAGAGTCGATGCCAGCTGGTGACTACCTCCCAAGGCTGCTTG ACAS2 Forward Primer TGCCCCATCGTGGACACC (rs ) Reverse Primer AGTCAGAGATGAGAAATTTGAGTCA Extension Primer(F) GACCTGGGTGTCGATACCTACTGGCAAACAGAGACAGTGAGTGAA ACAS2 Forward Primer TAGTTAGATAGTGGAGAACTGGACTG (rs ) Reverse Primer ATTGCAGGAGCTACACCAAA Extension Primer(F) ACGCACGTCCACGGTGATTTTGTGAAGAATTTGGGGTTCTGGGGC CD36 Forward Primer AGAATTTTTTTCTATTTACCCATGC (rs ) Reverse Primer ATGTTTTATTTACGATACCTTATTCACA Extension Primer(F) AGCGATCTGCGAGACCGTATGATTCTTTCTGTGACTCATCAGTTC CD36 Forward Primer CCTGTACTTACTACAAAGACATAACCC (rs ) Reverse Primer TTCTGTGCCTGTTTTAACCC Extension Primer(R) ACGCACGTCCACGGTGATTTCCTTCTTCGAGGACAACTTGCTATG CD36 Forward Primer AGGAAGTGATGATGAACAGCA (rs ) Reverse Primer AAATACATGGTCAAGGTAAGAGTGT Extension Primer(F) GGCTATGATTCGCAATGCTTCAACATTCAAGTTAAGCAAAGAGGT CD36 Forward Primer TATTATAAACTTACCAGTATCAAATAACTTCC (rs ) Reverse Primer AAAACTGATTTTTTTTTCCTCAGC Extension Primer(F) AGCGATCTGCGAGACCGTATGAAGTTAGTGTTTTGTTATTGTGGC CD36 Forward Primer TAAAATGTGCATGTATGTTACTAGGG (rs ) Reverse Primer TAAAGAGTTAATACCTAGCAGAACAGG Extension Primer(F) AGAGCGAGTGACGCATACTAAAGACATAGGCATCAACCTRTAGAA CD36 Forward Primer ATCACATTCTTGAAAGTTACTGAA (rs ) Reverse Primer TCACATATACTCTTTGACTTTTGC Extension Primer(F) TCTTGAAAGTTACTGAAACTTAGGTC CD36 Forward Primer TTGTGCCAAAGTTGTCCAA (rs ) Reverse Primer TCTTATGAAACYATTTCCTAGTCAATTAT Extension Primer(F) AGCGATCTGCGAGACCGTATTTGACTGGTTCATTTCTCAATTATA CYP11B2 Forward Primer ATGATAAAAAGATCGGAGATGT (NEW15) Reverse Primer TATTTTCACTGTCTTAAAAAGAGG Extension Primer(F) CAGGGACTGGGGCAGGAGGGATGAGC CYP11B2 Forward Primer TCAGGGACTGGGGCAGGA (rs ) Reverse Primer TGCAGGATCCTAGGAGTATTTTC Extension Primer(F) GATGAGCAGGCAGAGCACAGGTTTT CYP11B2 Forward Primer TGGTTGATAACAACGTATCGA (rs ) Reverse Primer AAAAACTGCAGAAAATGTCCAC Extension Primer(F) CGTGCCGCTCGTGATAGAATATTCCTCACATGGAACCAGTGCGCT

62 Gene Primer Type Primer sequence CYP11B2 Forward Primer TGAACTAAATCTGTGGTATAAAAATA (rs ) 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 (rs ) Long PCR Reverse Primer CCTGTCTCTGTGAAGCATCT Sequencing Forward Primer ACTTTGGATTGGGACTGCA Reverse Primer CCCAGACCGCAAGAGAAA CYP11B2 Long PCR Forward Primer CACCTGaCTTCTCCTTCaTC (rs ) Long PCR Reverse Primer CCTGTCTCTGTGAAGCATCT Sequencing Forward Primer ACTTTGGATTGGGACTGCA Reverse Primer CCCAGACCGCAAGAGAAA CYP11B2 Long PCR Forward Primer CACCTGaCTTCTCCTTCaTC (rs ) 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 (rs ) 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

63 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 (rs ) 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 (rs ) Long PCR Reverse Primer GTGCTGACTAGAGGAGTTGG Sequencing Forward Primer ACCAGCTTCCCTCTGCCT Reverse Primer GTGCTGACTAGAGGAGTTGG FABP1 Forward Primer AATCTGCATGGCAGGACA (rs ) Reverse Primer TTAAGGTGTACCCTGCCACA Extension Primer(F) CATTAGTGAGTGTTCTCTGCCCACA FABP1 Forward Primer AAGGTAGACTGGAGTTGCCTTT (rs ) Reverse Primer TCACGTGACGGGTCATGA Extension Primer(F) AGAGCGAGTGACGCATACTACAAAAATTAGAACTCATGATAGGGA FABP1 Forward Primer ATCATGACCCGTCACGTG (NEW36) Reverse Primer CCACTGCTCCAGGCATGA Extension Primer(F) AGATAGAGTCGATGCCAGCTACTAGGCCATCCAGGACCTGTGGCC FABP1 Forward Primer CAGACAAAGAAGGGAGAGA (rs ) Reverse Primer TTTGTTGTGAAGCACAGAGAG Extension Primer(F) CGACTGTAGGTGCGTAACTCTCTAGGGGTGAGGGGGTGCTTGTAA FABP1 Forward Primer TTCCTCACAGGTCACCTCTC (rs ) Reverse Primer ACACGCTCAGAGCACCAC Extension Primer(F) GGAAGGTGACAATAAACTGGTGACA FABP1 Forward Primer TWACTGAGGATCCATCATGTCC (rs ) Reverse Primer ATGCAGGGCAGGTGGAGG Extension Primer(F) AGGGTCTCTACGCTGACGATGGCAGTTTACACACATTATCACTAA FABP1 Forward Primer TATTGCTCACCATATTCTCGTAAA (rs ) Reverse Primer AGCATGGGTTTTGTGTTCA Extension Primer(F) AGATAGAGTCGATGCCAGCTCGTAAATGTTGATAATCCACATGAT FABP2 Forward Primer ATTAAAACTCTTTACTTCACACCAAGTT (rs ) Reverse Primer AGGGTATAGAGGGTAGCTAGAAAGG Extension Primer(F) AGGGTCTCTACGCTGACGATGCCTGAAGTAGGGAAAGGAGAGATA FABP2 Forward Primer AGCTACCCTCTATACCCTGAC (rs ) Reverse Primer ATCCAGTTTTAGCTGTGCCA Extension Primer(F) GTGATTCTGTACGTGTCGCCCTACTGGGTTTGGGGATGAGAGGCA FABP2 Forward Primer TAAACACAATGCAAAAAGACATATTAG (rs ) Reverse Primer ATATATCAAATAACATCTGGGAAATGA Extension Primer(R) AGAGCGAGTGACGCATACTATTTATGATGAAATACAGTTGAAAAG FABP2 Forward Primer TATAAATTATATTRCCTCCAACATAGTCTG (rs ) Reverse Primer TCAACTGAATTAAAACATGAGAAGC Extension Primer(F) GGATGGCGTTCCGTCCTATTGAAATAAGTATGCCCACAAACTGTA

64 Gene Primer Type Primer sequence FABP2 Forward Primer AAGATCTCAGGAATGTACAGAACG (rs ) Reverse Primer GGGCGAGAATTTATTATATTTCTTATC Extension Primer(F) AGGGTCTCTACGCTGACGATCTGAACTTTAAGCTTCCACATCACA FABP2 Forward Primer ACATTAGGAAGAATCCACTATCTCAC (rs ) Reverse Primer TTTCTTACTGTAAACAACAAAAAAAGTC Extension Primer(F) CGTGCCGCTCGTGATAGAATAAAAAGATCCAGAAATGTTTCCTTC FABP2 Forward Primer AAAAGGAAGCTTGCAGCTC (rs ) Reverse Primer TAATTAAAGGTGACACCAAGTTCA (new48) Extension Primer(F) AGCGATCTGCGAGACCGTATTAAATTCACAGTCAAAGAATCAAGC FABP2 Forward Primer TTTTCGAAACATTGAAGTTGTTT (rs ) Reverse Primer GCTCATAAAAAAAAAAATTCTTACCC Extension Primer(F) GGATGGCGTTCCGTCCTATTAACTTGGTGTCACCTTTAATTACAA FABP2 Forward Primer CCAATATTAAAATGATTTCTGCTCTAT (rs ) Reverse Primer ATAATGTAGACAAACTTCATTGGCT Extension Primer(R) GGCTATGATTCGCAATGCTTTGGCTTCTTCAGTTAGTGAAGGAGA FABP2 Forward Primer AAGCCAATGAAGTTTGTCTACATT (rs ) Reverse Primer TAATTCTTACTGTTCAATTCAGATTCTT Extension Primer(F) AGGGTCTCTACGCTGACGATCAAATTGGCAGGRTATTTAAATATG FABP2 Forward Primer ACAGTCCAAAATACAAATTGGA (rs2964) Reverse Primer TAATTAGCTTTTACTTCTTTTGCTTTG Extension Primer(F) CGTGCCGCTCGTGATAGAATAGAAGATCTATATTGTACCAGAACT FABP2 Forward Primer GCAAAAGAAGTAAAAGCTAATTAGGA (rs ) Reverse Primer TGTCAATTCATATTCACATACTTTTTC Extension Primer(F) CGTGCCGCTCGTGATAGAATTTTAAAAAACAAAAATAAATATTGC FABP2 Forward Primer CATGTTGCTTTATATGTAGCCTTG (rs ) Reverse Primer TAAGACCCTCTCTCTACGAAAAC Extension Primer(F) ACGCACGTCCACGGTGATTTAAGTATGTGAATATGAATTGACAGA FABP2 Forward Primer ACATACCACTTTGAATAGGATGATAG (rs ) Reverse Primer TCTCAAAGACAAGTTAGTGGACA Extension Primer(R) CGTGCCGCTCGTGATAGAATAGATGATCCTGATTTTCAGCCAAGC FABP2 Forward Primer TGATGCAGCCTGTCCAC (rs ) 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 (rs ) Reverse Primer TGAAAAAGAATTTGTCAAAAGTCTATAG Extension Primer(F) GCGGTAGGTTCCCGACATATCATATTAGGGGATGGAGAGGGGKTA FABP2 Forward Primer TATTAATCTCAATACAAAAACTGAGAGAGA (NEW60) Reverse Primer TAGGCTGAAAGTCAAGACCTACA Extension Primer(R) GGATGGCGTTCCGTCCTATTTAATTCTTCTGTTCATCATGGACTT FABP4 Forward Primer TGTTTCTCTTTCATCCCCTTT (rs ) Reverse Primer AAGAAAATAATTGGCTAACATACGG Extension Primer(F) AGGGTCTCTACGCTGACGATTAAATTAAGCAATAACTTGGAAAAA FABP4 Forward Primer ATGGTTACGTTGGTTAAATAAACTTT (rs ) Reverse Primer CTTCACTCAGTTATGACTAAGAATACATC Extension Primer(F) GTGATTCTGTACGTGTCGCCGATTTAGAAGGTGATGTAATGATGT FABP4 Forward Primer TTTCATAATAATTCAAGGTAAAAAGGG (rs ) Reverse Primer AGCTATGRTAAAGAAGGATTTAATATTCA Extension Primer(F) AGATAGAGTCGATGCCAGCTTATCTATGGATTTCCCTAGGTAGGA

65 Gene Primer Type Primer sequence SLC27A1 Forward Primer TTCTAGGAAGGATGGGGAC (rs ) Reverse Primer TTAACACGTGTGATTCAAGGAC Extension Primer(F) GACCTGGGTGTCGATACCTACTCCCTGGGATCCTGGGGTGTGACG SLC27A1 Forward Primer TTTTTAAGACCGAAAGAGGCTT (rs ) 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 (rs ) Reverse Primer ACATAAGGGAGTTAAGGGAATAGAG Extension Primer(R) CGGGGCCTGGCTGGGAGAGGACGGG SLC27A1 Forward Primer AGGCTCTATTCCCTTAACTCC (rs ) Reverse Primer AACAGCCATTCCTTAAGCC Extension Primer(F) GGCTATGATTCGCAATGCTTTTATGTGCGCCCTCCTGTCCACCTG SLC27A1 Forward Primer ACAATGGAGCTGCTGCG (rs ) Reverse Primer CCTAGGGAGAAGGCCATG Extension Primer(R) TTCAGGCTGCGCTAGGCAGGCAGGG SLC27A1 Forward Primer TACCTCTCAGGTGCGCAG (rs ) Reverse Primer AAGTACATGTAGCCCAGCTCAT Extension Primer(F) ACGCACgTCCACggTgATTTTGACTGGCTGTGCGGATGGGGATCC SLC27A1 Forward Primer CTTGAGCCAGACAGCGCT (NEW72) Reverse Primer TCTGAGGTTCCAGTTTCTCAGT Extension Primer(F) TAGTACACACCCACCTGGCCGAGCT SLC27A1 Forward Primer CCGGCCTTTCCTTTTTCC (rs ) 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 (rs ) 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 (rs ) 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

66 Gene Primer Type Primer sequence SLC27A4 Forward Primer CTCCTAGCCTGTGTGCAAA (rs ) 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 (rs ) 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 (rs ) Reverse Primer CCAACATCTGCGAGAGGTAG Extension Primer(R) GACCTGGGTGTCGATACCTACCAAAATCTCCGGAGAGGGAACTGA CACNA1G Forward Primer CGAAGAATCAGAGTATTCGGC (NEW91) Reverse Primer AGAGCTGAGCTCCKTCCTC Extension Primer(F) GCTGCCCAAATCAGGGGTCGTGGAC CACNA1G Forward Primer TCATCCTGCTGCCCCTA (rs ) Reverse Primer GCTGAGAAGCTGACGTTC Extension Primer(F) CGACTGTAGGTGCGTAACTCAAGCAGGATTCCTCATTGACCTCTT CACNA1G Forward Primer CAATGGGACGCTCAGGG (rs ) Reverse Primer CTAAGTGGCAGTCGGCAT Extension Primer(R) CGTGCCGCTCGTGATAGAATGTCGAGGGTGGTGGCAGCATGAGCC CACNA1G Forward Primer CAGACTCCCTGACTCATTTTAC (rs198551) Reverse Primer GAACAGGAGAGTAGAGGTGAGA Extension Primer(F) ACTCATTTTACACATACTCASAGGGC CACNA1G Forward Primer CCATGGGCCTGAGCATCC (rs ) Reverse Primer GATCCAGTCCTCCAAACATG Extension Primer(R) GAGCGAGTGACGCATACTAACACCAGGGAGAGGAGGAGCAGGAA CACNA1G Forward Primer AGAGCAGAGCACCCCAC (rs ) 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 (rs ) Reverse Primer ACTCAGCACATCTTTCTTTGG Extension Primer(F) AgCgATCTgCgAgACCgTATGTATCTGCCTCCGGAGGAGGGCTCC

67 Gene Primer Type Primer sequence PPARD Forward Primer AAGGTGCGTGGTGAACA (rs ) Reverse Primer ATTCACATAACCCTCAAGTATCAG Extension Primer(R) AGGGTCTCTACGCTGACGATACATAACCCTCAAGTATCAGAGGAG PPARD Forward Primer TKACCTCTTCCTGTCTTCTCCT (rs ) Reverse Primer AGCTCTGGCATCGTCTGG Extension Primer(R) CGACTGTAGGTGCGTAACTCCTGAAYGCAGATGGACCTCTACAGG PPARD Forward Primer CCAGGCGTGGGGTAGTGT (rs ) Reverse Primer ATCTGAGACAGGCTTGAGTTTC (new102) Extension Primer(R) GACCTGGGTGTCGATACCTAATCAGTTCCCTTCTGGGCCAGAATG PPARD Forward Primer ATGCCGGAGGCTGAGAAG (rs ) Reverse Primer TTCTTTTTGGTCATGTTGAAGTT Extension Primer(F) GACCTGGGTGTCGATACCTAAGCTGGTGGCAGGGCTGACTGCAAA PPARD Forward Primer ATCTTCACACTGGTTTGCCA (rs ) Reverse Primer GAGGGAACCCTGCCTACTT Extension Primer(R) GCGGTAGGTTCCCGACATATTCCAAGCTGGGAGAGTGCCGTCCAG PPARD Forward Primer TTTMTCTAGCTGGCTCCACG (rs ) Reverse Primer CTGCAGGTAAAGTGATTTCAGTG Extension Primer(F) CGTGCCGCTCGTGATAGAATCCCCACTCCCCCTGAAGCTGCC PPARD Forward Primer CTGGTCCTCCCTCCCAAG (rs ) Reverse Primer GAATCCTGCCAGCAGAGAG Extension Primer(F) ACGCACGTCCACGGTGATTTTGGAAGTGCCCAGCCCCTGCCCCTA PPARD Forward Primer ATTTAAGAGCTGACTGGAAGC (rs760783) Reverse Primer AAGCCTCCTTTGCCCTC Extension Primer(F) AGGGTCTCTACGCTGACGATTTACTTGCATGTTTTTCCTGGGGCT PPARG Forward Primer CCAACCTAACAGCGTAAGTC (rs ) Reverse Primer CAGCTTATCCATCAATAGCAACT Extension Primer(F) AGCGATCTGCGAGACCGTATTYGTTTAAACATCAATTGATGTTCA PPARG Forward Primer GTTAGAAATCTCCAAGTCATCCAC (rs ) Reverse Primer GCCCTGAAAGATGCGGA Extension Primer(R) CCACCTCTTTGCTCTGCTCCTGCAG SNAP23 Forward Primer AGCTTTTCTACTAGAGTTATTAGTGTCATC (rs ) Reverse Primer ATGTTGTCTTATAAGCCTTGCC Extension Primer(F) CGTGCCGCTCGTGATAGAATTTGGTATCTCCCTACACTCCTGACT SNTG1 Forward Primer GGAAGATCCCCTACCTGGA (rs ) Reverse Primer AAGTCCTAGACGTTGTGCTTTC Extension Primer(R) GGATGGCGTTCCGTCCTATTCTTGCTTCCATCAAGGATTAGAGCG SNTG1 Forward Primer TCCCCTTAATTGGTAATCAAGAT (rs ) Reverse Primer TTCTTTGGAAAGCTCGTGG Extension Primer(R) GCGGTAGGTTCCCGACATATTCTTTAATTACATAAGATGAAAAGG SNTG1 Forward Primer TCTAGATAATTTACAGGGATTTGAGC (rs ) Reverse Primer AAATGTATCTACTTTACCCATGAGGA Extension Primer(F) GGCTATGATTCGCAATGCTTGTGGAAAATTGTGTATGATTTGAGT SNTG1 Forward Primer TGTATTAGTCTACACTTCTAAGASACAATTG (rs ) Reverse Primer TGCTTCCATAACAAGAGGTTTT Extension Primer(F) CGACTGTAGGTGCGTAACTCTCTTTGATATTAATCTTTTTGAAGC SNTG1 Forward Primer CGGCCCTGTACGAGAACC (rs ) Reverse Primer TGATGACAGCTAGGAGTCATAGAC Extension Primer(F) GCGGTAGGTTCCCGACATATGCCTGGGGTCCATGAACACATATTT SNTG1 Forward Primer GCATTAGTGGCTGAGTCTGC (rs ) Reverse Primer TAACTCATACCTGCTTTCAAGGG Extension Primer(R) GGATGGCGTTCCGTCCTATTTTCCCCTTCTGTGAGAGAAGTCCTC SNTG1 Forward Primer ATCTTTTAAGATGCTAACCATATAAGACA (rs ) Reverse Primer AAAGTTCCGCTGTGAATGAA Extension Primer(R) GCGGTAGGTTCCCGACATATAAGCACAATTGATAATGCATTGACT

68 Gene Primer Type Primer sequence SNTG1 Forward Primer ATTAGGCAGATATGACATTTATCCA (rs ) Reverse Primer AATTCTTCAGAACACATTTCATCC Extension Primer(F) CGTGCCGCTCGTGATAGAATTATCAAATTTTCAAATGTTCTCTTA SNTG1 Forward Primer TTTAACACCCTTGAAAAGTCTGG (rs ) Reverse Primer ATAAACAAAATACACTGACATATGCTTG Extension Primer(R) AGATAGAGTCGATGCCAGCTAATTTGATCCATTATTTTATAGGTT SNTG1 Forward Primer ATATTATGCAAATCAAATACAGTGGTAA (rs ) Reverse Primer ATCTTGCACATAATCTCATAAACTGA Extension Primer(F) AGAGCGAGTGACGCATACTATAAAGGTGACCACCTGGGACTGGAC SNTG1 Forward Primer CGTCATGGCCAGAAGAATA (rs ) Reverse Primer AACTAAGAAAYGCAATTAGTTTTGTG Extension Primer(F) GGATGGCGTTCCGTCCTATTACTTACTTTAAGATTTTATTGGCAA SNTG1 Forward Primer AGGATGCACTTTAATTTGCTGAT (rs ) Reverse Primer TCCTTTGGAAGTTCTTCGGAA Extension Primer(F) GCGGTAGGTTCCCGACATATTGCCCTTGTGTTCTCATCCCATGAT STXBP3 Forward Primer GGCGGAAATTCCCTTGCT (rs ) Reverse Primer TACTTTGGCCGCAGAAGC Extension Primer(F) GGATGGCGTTCCGTCCTATTACGCGCGTGACGTCACCGGAGGGCG STXBP3 Forward Primer AAAGTAGGTTGGGAGTGGA (rs ) Reverse Primer ACGCTCTTTAGCCCCCTC Extension Primer(R) CTGCCACCGGCGGCGCCATCTTCCC STXBP3 Forward Primer TTGAGAGAGGGAAGGCCG (rs ) Reverse Primer TCCCAAAGCAACAAGTGC Extension Primer(F) GGCTATGATTCGCAATGCTTCGGGCCTCGTTTTGCAGCCCCAACC STXBP3 Forward Primer ACCTTAAAAAGCAGGTTCATCA (rs ) Reverse Primer TTTATGGCAATATTTTTCTACTAAAGTG Extension Primer(F) AGCGATCTGCGAGACCGTATTTTCACCATTGTTATGGTTTACTAA STXBP3 Forward Primer CAGGAAACAGCTATGACCcataagtcttggtggttcgt (NEW127) Reverse Primer TGTAAAACGACGGCCAGTacattttgggctgattttta STXBP3 Forward Primer TATATTTACTTCACTGACTGTAAGTCTTTTAA (rs ) Reverse Primer ACATTTTGGGCTGATTTTTAAA Extension Primer(F) GGATGGCGTTCCGTCCTATTAAGTTATTGCTTCATTGTTCAAAAT STXBP3 Forward Primer TGTTTGCTTCCTTAACTGTGT (rs ) Reverse Primer AWTGATAGTGAGCCTGGTATTTACTT Extension Primer(F) GGCTATGATTCGCAATGCTTTACAGTAGAGCTACATTTAAACACA STXBP3 Forward Primer TAATAGTGAAGGTGAATTTTAAAGTTTG (rs ) Reverse Primer TATTGAAACTAGGCAATGGGTAC Extension Primer(F) GTGATTCTGTACGTGTCGCCTTTTTATTTTTGGAAAGTTCTAACA STXBP3 Forward Primer AATGAATTTTGGTTATAGATCACTGAC (rs ) Reverse Primer TTTGTTGAGTAGAACTGGAAGGA Extension Primer(F) AATATTTTATTTTCATTAAGGACCT STXBP3 Forward Primer AGCAAATATTGGTGTAATAAGTAAATGA (rs ) Reverse Primer TTTGTTTGGCATAAAGGTCAA Extension Primer(F) GTGATTCTGTACGTGTCGCCAAGGTTTGTGAGTTTCAAAGAAAAG STXBP3 Forward Primer atattgttcccagtgtccag (NEW134) Reverse Primer ctgctatgtgaaggaagagg Extension Primer(F) CGACTGTAGGTGCGTAACTCAAAATCAATGAAATTTTCATTCTAC STXBP3 Forward Primer AATAAACCCAAGGATAAAGTCTCC (rs ) Reverse Primer CAACATTTTCTTTCTATTTTAGTTCAAC (new135) Extension Primer(R) CGTGCCGCTCGTGATAGAATAATCTCTAAACCCTCCAAAAAGAAA STXBP3 Forward Primer TTTTACTTGCCYTCATAGTGTTCT (rs ) Reverse Primer TATGTACCCAAACAACGCAG Extension Primer(F) GCGGTAGGTTCCCGACATATATGGCAGACCATAGTTTTATTGATT

69 Gene Primer Type Primer sequence STXBP4 Forward Primer ATTCAGTTAATTTACGGAAGAATCTATG (rs ) Reverse Primer AGAGTCCCAGAGAAATTGAAGA Extension Primer(F) GCGGTAGGTTCCCGACATATGGATGATCCTCTTGATTTTAGTCTT STXBP4 Forward Primer AATCAGTTGCTGATGTGCTG (rs ) Reverse Primer AAGAAGCTGAAATTTAGGGCA Extension Primer(F) AGATAGAGTCGATGCCAGCTATATTACCATTATGTGTGTGCTCAG STXBP4 Forward Primer AAAGTGAGACAGTTTGTTACATTT (rs ) Reverse Primer TTATAGTCTACAGGATGGACCTTGA Extension Primer(F) GACCTGGGTGTCGATACCTAACATGTATCCATTTGTGAAATCATC STXBP4 Forward Primer AATCAAGGCACCTACTGTACCTC (NEW140) Reverse Primer TGTTTTTCTTCTCCTAACTAGAACTCAGG Extension Primer(R) GACCTGGGTGTCGATACCTATAGGYCTGGGTTCTCATTTGAAATT STXBP4 Forward Primer TATAACCTGAGTTCTAGTTAGGAGAAGAA (rs ) Reverse Primer ATCATTTAGTACAACTCTCTTCCT Extension Primer(F) ACGCACGTCCACGGTGATTTTTTGTGAGGGAAAACCACACAGTCT STXBP4 Forward Primer TTATGAAAAGGTGGTAAATCTCTGT (rs ) Reverse Primer TTCTTAAATGGCTTTCCTATGAAG Extension Primer(F) GTGATTCTGTACGTGTCGCCAATACAGTTTGTTTTAATTAAATGA STXBP4 Forward Primer AAGGAATCTATGATTGGTGTATCAT (rs ) Reverse Primer ATTCTAAATGACTCATTCTATCTCATGG Extension Primer(F) AGAGCGAGTGACGCATACTATGAAGAAGCAAAAAGCATAATTACC STXBP4 Forward Primer TTTAATAACATCTGAAACATTATCTTGG (rs ) Reverse Primer AGATTTTCTGGCTGAATGTTGT Extension Primer(R) AGCGATCTGCGAGACCGTATCTCCCAAGCAGATTCTAACCTAGTA STXBP4 Forward Primer TGGGGCTTAATTTTATTTTTAATG (rs ) Reverse Primer TTCACACTAAATGTTGCACAGATAT Extension Primer(F) ACGCACGTCCACGGTGATTTTATTCAGTTGGCCTTTAGAGAATTA STXBP4 Forward Primer GCCAGGGCAGAATTAGATG (rs ) Reverse Primer CATGAAAAAAAATGTATTTCCCA Extension Primer(R) CGACTGTAGGTGCGTAACTCCCCAAAGAACTCACAGGATCCAAAA UCP1 Forward Primer AATTTCCCAGTCAGTCAGGC (rs ) Reverse Primer AGGCAAATAAGTGGCATGC Extension Primer(F) GGATGGCGTTCCGTCCTATTAGAATGCATTCTATATACGCGACTT UCP1 Forward Primer ACCTTGTCCATTTCCCTCTT (rs ) Reverse Primer TTTCCTAGTTCGATCTTTGTGC Extension Primer(F) AGGGTCTCTACGCTGACGATCCTATTATTTCCCACTGTTTTTTTT UCP1 Forward Primer CTGCCCTGTCCTTTCCAC (NEW149) Reverse Primer AAATAGTACATTGCTAAAGCACCG Extension Primer(R) GACCTGGGTGTCGATACCTAGTACAAAATGAGATACCACCTGTTC UCP1 Forward Primer TTGACCATGACTACTACCAAATTCT (rs ) Reverse Primer TAGGATAGGACAGAACCCCC Extension Primer(R) GTGATTCTGTACGTGTCGCCGAGAGGCCAATATTCTCAGCTCTTA UCP1 Forward Primer AATGCCCGACGTCCAGTG (NEW151) Reverse Primer AGGCAGCCCGCTGTAGAG Extension Primer(R) AGATAGAGTCGATGCCAGCTATCCGCCCTTCTGTTTTTACCACAG UCP1 Forward Primer AAGTGTAACACACAGAGGAGT (rs ) Reverse Primer ACTTCCATGACTGGGTGAC Extension Primer(R) CGACTGTAGGTGCGTAACTCACTTTTTTCTATGAACAATGAGCTAAT UCP1 Forward Primer ATGTTGACCTGTTTCATCGATC (rs ) Reverse Primer AGAATTAATAAATCTGGTTTTTACTACATCC Extension Primer(R) AGAGCGAGTGACGCATACTATTTACTACATCCACCGGGGAGGACA UCP2 Forward Primer ACGTGTTTGTCCCGGCCA (rs659366) Reverse Primer AAAGGCGTCAGGAGATGG Extension Primer(F) GGGCCCAATTGTTGGCTGTTCACGC

70 Gene Primer Type Primer sequence UCP2 Forward Primer TGTAACCAATCGACAGCGA (rs ) 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 (rs ) Reverse Primer TCCTTGTCATTCACTGTTGTCT Extension Primer(F) AGCGATCTGCGAGACCGTATTCTAGGATAAGGTTTCAGGTCAGCC UCP3 Forward Primer CCTGCAGCGCCAGATGAG (NEW159) Reverse Primer TCACTGTCCGCGCCTTTG Extension Primer(F) GACCTGGGTGTCGATACCTATCGCCTCCATCCGCATCGGCCTCTA UCP3 Forward Primer AAAGTGTGTGGGTCAAGAGAC (rs ) Reverse Primer TTGATRTCCACTCAGAGCCTC Extension Primer(R) CGACTGTAGGTGCGTAACTCAGCGTCCGGTACCAGCTTCCCCCCG UCP3 Forward Primer TATGAGGAGGCTCTGAGTGG (rs ) Reverse Primer ATCCGGGTAGTGAGGCTG Extension Primer(R) GACCTGGGTGTCGATACCTAGGGCCAGTGTCCCCTACTAAGCAGC UCP3 Forward Primer ATGAGGACAGCAGAGGAGATAA (rs ) Reverse Primer TTAGGTGTCACCTTCTCTGCA Extension Primer(R) GTGATTCTGTACGTGTCGCCGAGCTCCACCTCTGGGGCAACCCCT UCP3 Forward Primer CAGCMAGGGCATCCATTT (rs ) Reverse Primer CAAAGTTCCTGTTAGGAAGGC Extension Primer(F) CCCATTTCCCATTCCTCCCTCCCCA UCP3 Forward Primer TGAGGTGGTGACCTACGAC (rs ) Reverse Primer AGAGCTGCCTGCCTGGAG Extension Primer(F) AGCGATCTGCGAGACCGTATTCCTCAAGGAGAAGCTGCTGGACTA UCP3 Forward Primer AGATAATGAATGATAAGCAGGAAGG (rs647126) Reverse Primer CATGTGTGATGGTGCAGC Extension Primer(F) CGTGCCGCTCGTGATAGAATAGATCAGCCACGGAGCCTGGAGGGA UCP3 Forward Primer TACCAGAGAAGTGGGAATAACAAT (rs ) Reverse Primer TTAAGCCTCCCAGGTAGCA Extension Primer(F) GGCTATGATTCGCAATGCTTCTGCTTCACAGGCTTGTTGTGAGGG VAMP2 Forward Primer AGTGATTTGCGCCATCAT (NEW167) Reverse Primer TCCACTGGGGAAACCTCT Extension Primer(R) AGGGTCTCTACGCTGACGATCCAGGGAAAGGGCCCCGGCCATTCT VAMP2 Forward Primer AACCCTCAGCCGTTCCTC (rs ) Reverse Primer ACAGCTGGCTATTTACAGGG Extension Primer(F) AGATAGAGTCGATGCCAGCTCTCTCAGCCATATCTTTCAGCCCCC VAMP2 Forward Primer CAATTCCTGCCTGAAATCTCTG (NEW169) Reverse Primer AGGAGGGTTTTCAGTCCAAC Extension Primer(F) ACGCACGTCCACGGTGATTTTTCCTGCCTGAAATCTCTGGCCTCA VAMP2 Forward Primer TACTAGTCACTGCCAATGTGTGTAT (rs ) 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

71 Gene Primer Type Primer sequence VAMP3 Forward Primer GCCCTCAAGTTCCCAAGT (NEW173) Reverse Primer AGAGAAGCGAAGTTGGGAC Extension Primer(R) AGGCCAGGGAGATGGGTGGGACGGC VAMP3 Forward Primer AGTCATCAGCCCCTCCCA (rs ) Reverse Primer ATTCACAGAACTACGAGAAAACTGA Extension Primer(F) CTCTACCCCAGCAGGGCCCCAAACA VAMP3 Forward Primer AAGCAACAATTCTGTAGCCTCA (NEW175) Reverse Primer ATAATTTAGAAAACTGATTTAGGAAGCA Extension Primer(F) GGCTATGATTCGCAATGCTTTCTGGCCCAAATATGAAGATAAACT INSR Forward Primer TGAATGTTCTTCTTGCTTGG (rs ) Reverse Primer TCACTTCTATAATCAGATAAGAACTAAA Extension Primer(F) ACGCACgTCCACggTgATTTGGGAGAGGAAAAGGAGTGGAAAACT INSR Forward Primer AGAGGAGAAGGAACGTGGG (NEW202) Reverse Primer AAAACAGGGTGCAAAAGTAGC Extension Primer(F) TGTGACGATGCCGCTTGTTAAAACT INSR Forward Primer CTGAAGACCAATGGGGAC (rs ) Reverse Primer CACCAGTCCATGGAAAAAC Extension Primer(F) TCACTGGTCCCCAACCTTTTTGGCA INSR Forward Primer TCAGTCTTACAAGTCCATATGCG (rs ) Reverse Primer AGTCAACTGAATAAGCGCAGTC Extension Primer(F) CGTGCCGCTCGTGATAGAATTCACCCTTGGAAATCCCTTCCTCCA IRS1 Forward Primer ATCAGCACCTCCGCCTT (rs ) Reverse Primer ATGTGGAAGGCTGGGCT Extension Primer(F) AGCGATCTGCGAGACCGTATGTCGCCTTCTCTATGCTGCAACAGC IRS1 Forward Primer TGAGGAGTACATGAAGATGGAC (rs ) Reverse Primer TCATCTGCATGGTCATGTAGTC Extension Primer(F) GGGCAGACTGGGCCCTGCACCTCCC IRS2 Forward Primer TCTCTGCTAACTTCCCTCCT (rs ) Reverse Primer TTCCAAACACAGTCATTGCTC Extension Primer(F) GCGGTAGGTTCCCGACATATTCACCAGGATGTCACATGTCAGAGA IRS2 Forward Primer ATATAATGTTAGCTGGAAGGTCAAT (NEW208) Reverse Primer ACCCATTGATGCCTATTCC Extension Primer(R) CAAAACTAAATAAAAACTTCAGGAT CBL Forward Primer TCCCCAAGACCATATCAAAG (rs ) Reverse Primer CTATAAAGAATATACTAAAGCTTGTGTCCA Extension Primer(F) GACCCAGGTGAGTTTTGTTTCACAT RAPGEF1 Forward Primer AAAGACTTGAGAATGAGATTCCTC (rs ) Reverse Primer CGAGTCACTGGCTGAAAGG Extension Primer(F) GGCTATGATTCGCAATGCTTGAGGCTGCCTGGGTTCTGCCGCTGG RAPGEF1 Forward Primer ATATTTCACACAGGCTTGAAGTATT (rs ) Reverse Primer CTTTAAAAGTTCTTTGTCATTATGAAAC Extension Primer(F) AGGGTCTCTACGCTGACGATGTTTCCAAGGATCTTCTGTGAGCGC RAPGEF1 Forward Primer AAAGAAACCCATGGTCTTCAG (rs ) Reverse Primer GAACTTGGACCGTGTCCTG Extension Primer(F) GTGATTCTGTACGTGTCGCCTGGTGTTTGTCACATCGAGGAGTGC RAPGEF1 Forward Primer TTGCTGGAGGACTACTCGG (rs ) Reverse Primer TATACCTCCATGAGGAGCTTGT Extension Primer(F) GACCTGGGTGTCGATACCTACGCAGCCCTCTATGTTCTACCAGAC RAPGEF1 Forward Primer CTTGGCCTCCAGGCATC (rs ) Reverse Primer CTGGACATAATTTCGTTGTGGT Extension Primer(F) AGTCGGCTCAGTCGGA RAPGEF1 Forward Primer AACAGACCCTGTGCCTGG (rs943851) Reverse Primer TCATGTGCTGCCTGGGCC Extension Primer(F) GCCCAGGCAATTCCCCAGGCCCTCA

72 Gene Primer Type Primer sequence PIK3CA Forward Primer AGGTAAAGTCAAATGCTGATGCT (rs ) 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

73 부록 2. CYP11B2 유전자 Blast 결과 CYP11B2 Original Sequence ctgcatcctttggaggatggggaaggagtgcagcacatgctggtctgtgg <<<<<<<<< <<<<<<<<< ctgcatcctttggaagatggggaaggagtgcagcacgtgctggtctgtgg CYP11B1 Sequence tgctgccagggcaggggatagtgcagagaaaaccccagctcactgcagag <<<<<<<<< <<<<<<<<< cgctgccagggcaggggatggtgcagagcaaatcccagctcgctgcagag agggcaggactcagaagcactaaagttgaaaggttccagggagccagcag <<<<<<<<< <<<<<<<<< agggcaggactcagaggcactgaagttaagaggttccgggcagtcagcaa gagggctttagctgtgaagccgctaatccaggagcagggagggtggacag <<<<<<<<< <<<<<<<<< gagggctttagctgtgaagccgctaatccaggagaggggagggtggacag Primer gagacactttggattgggactgcagggtggggccacgagggacatgaccc <<<<<<<<< <<<<<<<<< gagacactttggattgggactgcagggtggggctagcggggacatggtcc rs Ctccagcagggcctcctgcttggccccacaggtacaacttgggaggacc <<<<<<<<< <<<<<<<<< catccagcacggcctcgtgcttggccccacaggtacgacttgggaggagc Primer acgcatggtgtgtgtgatgctgccggaggatgtggagaagctgcaacagg <<<<<<<<< <<<<<<<<< aggcatggtgtgtgtgatgctgccggaggacgtggagaagctgcaacagg tggacagcctgcatccctgcaggatgatcctggagccctgggtggcctac <<<<<<<<< <<<<<<<<<

74 tggacagcctgcatccccacaggatgagcctggagccctgggtggcctac agacaacatcgtgggcacaaatgtggcgtgttcttgttgtaagcggcgag <<<<<<<<< <<<<<<<<< agacaacatcgtgggcacaaatgtggcgtgttcttgctgtaagcggc ttgggagctgagagctgggagcagggtgggcagcctgggtgtagggggga <<<<<<<<< <<<<<<<<< gagctgagagctgggagcagggtgggcagcctgggtgtagggggga g <<<<<<<<< <<<<<<<<< g #21 rs : chr8: ~ chr8: 를포함한 800bp 이상에서 homology 보임 gggacatgaccccgtccagcagggcctcctgcttggccccacaggtacaa <<<<<<<<< <<<<<<<<< gggacatggtcccatccagcacggcctcgtgcttggccccacaggtacga cttgggaggaccacgcatggtgtgtgtgatgctgccggaggatgtggaga <<<<<<<<< <<<<<<<<< cttgggaggagcaggcatggtgtgtgtgatgctgccggaggacgtggaga agctgcaacaggtggacagcctgcatccctgcaggatgatcctggagccc <<<<<<<<< <<<<<<<<< agctgcaacaggtggacagcctgcatccccacaggatgagcctggagccc Primer tgggtggcctacagacaacatcgtgggcacaaatgtggcgtgttcttgtt <<<<<<<<< <<<<<<<<< tgggtggcctacagacaacatcgtgggcacaaatgtggcgtgttcttgct rs

75 gtaagcggcgagttgggagctgagagctgggagcagggtgggcagcctgg <<<<<<<<< <<<<<<<<< gtaagcggc...gagctgagagctgggagcagggtgggcagcctgg Primer gtgtaggggggaggcgagagaggtaggacccaaaagcacatctgccctgg <<<<<<<<< <<<<<<<<< gtgtaggggggaggcgagagaggcaggacccaaaagcacatctgccctgg gcccctgtggtgggcagtgagggtgagcacccggcccagaggacggccat <<<<<<<<< <<<<<<<<< gcccctgtggtgggcagtgagggtgagcacccgacccagaggacggccat cctgtggggtcgcgtctgcactgtgggttggggaagcagggcggtggtgg <<<<<<<<< <<<<<<<<< tccgtggggtcgtgtctgccctgtgggttggggaagcagggcggtggtgg agaaatgggcacgggcacctctgcagagaagacgcagagcaatgagccct <<<<<<<<< <<<<<<<<< agaaatgggcacgggcacctctgcagagaagatgcagagcaatgagccct tctgtgtagtgagaacccgctctgcaccaacctcggcggctgctttctc <<<<<<<<< <<<<<<<<< tctgtgtagtgagaacccgctctgcaccaaccttggccgatgctttctc #22 rs chr8: agctgagagctgggagcagggtgggcagcctgggtgtaggggggaggcga <<<<<<<<< <<<<<<<<< agctgagagctgggagcagggtgggcagcctgggtgtaggggggaggcga gagaggtaggacccaaaagcacatctgccctgggcccctgtggtgggcag <<<<<<<<< <<<<<<<<< gagaggcaggacccaaaagcacatctgccctgggcccctgtggtgggcag

76 tgagggtgagcacccggcccagaggacggccatcctgtggggtcgcgtct <<<<<<<<< <<<<<<<<< tgagggtgagcacccgacccagaggacggccattccgtggggtcgtgtct gcactgtgggttggggaagcagggcggtggtggagaaatgggcacgggca <<<<<<<<< <<<<<<<<< gccctgtgggttggggaagcagggcggtggtggagaaatgggcacgggca Primer cctctgcagagaagacgcagagcaatgagcccttctgtgtagtgagaacc <<<<<<<<< <<<<<<<<< cctctgcagagaagatgcagagcaatgagcccttctgtgtagtgagaacc rs Primer Cgctctgcaccaacctcggcggctgctttctcttgcggtctggggactgt <<<<<<<<< <<<<<<<<< cgctctgcaccaaccttggccgatgctttctcttgcggtctggggactgt ccttcccataggtcagaaaactgaggccctgagaaggggacttccactgg <<<<<<<<< <<<<<<<<< ccttcccataggtcagaaaactgaggccctgagaagggtactcccactgg cccaggtcacaggctgagtgctgagcctggtgttcgccggggccacagcc <<<<<<<<< <<<<<<<<< cccaggtcacaggctgagtactgagcctggtgttcgccggggccgcagcc tccctcagggcgctcagggtccctgcagtcctggcaaaccttcctgatgg <<<<<<<<< <<<<<<<<< tccctcagggcgctcagggtccctgcagtcctggcaaaccttcctgatgg ggacagtccggggcaggaggcaggtggggacgcaggtggctggtgg <<<<<<<<< <<<<<<<<< ggacagtccggggcaggaggcaggtggggatgcaggtggctggtgg

77 #30 New30 chr8: tctgcctgaccccaggccacctgtcttctctcccacgtgcacagcttcct <<<<<<<<< <<<<<<<<< tctgcctgaccccaggccacccctcttctctcccacatgcacagcttcct gagtcacccctctgtccagccagctcctgcacaaatggaactccccaggg <<<<<<<<< <<<<<<<<< gagtcacccctctgtctaaccagccccagcacaaatggaactcccgaggg cctccaggactggggcttgccaggct <<<<<<<<< <<<<<<<<< cctctaggaccagggtttgccaggct agcaaggccagggcacagctggagacgatcttgctg <<<<<<<<< <<<<<<<<< agcaatgccagggcacagctggggaagatcttgctg primer ccttgtccccagctccacctggccccttctccagcaagcagtgccctctg <<<<<<<<< <<<<<<<<< ccttgtccccagccccacctggccctttctccagcaagcactgtcctctg g <<<<<<<<< <<<<<<<<< g new ctcccagcgctggctccaggctcctcatgaggccatgcaagggtgctgtg <<<<<<<<< <<<<<<<<< ctcccagtgctggctccaggctcctcgtgtggccatacaagggtgctgtg primer

78 attttgtcccttgccttcctgcctagtctcacatgtccctgtccctctcg <<<<<<<<< <<<<<<<<< gttttgtcccttgccttcctgcctagtctcacatgtccctgttcctcttc ccctggccagggcctctgtgcagacagtgtcagagtcattaagcgggatc <<<<<<<<< <<<<<<<<< ccctggccagggcccctgcgcagac..tgtcagagtcattaagcgggatc ccagcatctcagagtccagtcaagttccctcctgcagcctgacccc.agg <<<<<<<<< <<<<<<<<< ccagcatctcagagtccagtcaagttccctcctgcagcctgacccctagg cagctcgagcatgccctgagctctctgaaagttgtcaccctggaata <<<<<<<<< <<<<<<<<< cagctcgagcatgccctgagctctctgaaagttgtcaccctggaata #32 New32 chr8: ccaggcagctcgagcatgccctgagctctctgaaagttgtcaccctggaa <<<<<<<<< <<<<<<<<< ctaggcagctcgagcatgccctgagctctctgaaagttgtcaccctggaa tacgatcctgcagggtagactaaaaaggcccctgtggtcacttatcctga <<<<<<<<< <<<<<<<<< tagggtcctgcagggtagaataaaaaggcccctgtggtcacttgtcctga ca...cattttcaagtgatacaactgagtctcgaggggcgtgtgttccc <<<<<<<<< <<<<<<<<< catccccattttcaagtgatacaactgagtctcgagggacgtgtgttccc cagctgatcatgtcagcctcatgccccaggcctcgtctttcatggaccag <<<<<<<<< <<<<<<<<<

79 cagctgatcgtgtcagcctcatgccccaggcctcatctttcatggaccag primer gtcttgttcaagcagcgagtgttgggtcctctgcttcctgagctgtcccc <<<<<<<<< <<<<<<<<< gccttgttccaggagtgggtgttgggtcctctgcttcctgtgctgtcccc new 32 primer tggagaaggtcccgaggatgctgtcaggagatggaagagtcatgtggggc <<<<<<<<< <<<<<<<<< tggggaaggtcccgaggatgctgtcaggagatggaagagtcatgtggggt gggaacctggggtgtggttccagaaatgtttttggcaacaggagagacag <<<<<<<<< <<<<<<<<< gggaacctggggtgtggttccagaaatgtttttggcaacaggagagacag gattgggccaacaaggactcagatgagttttattgactcattcctctggt <<<<<<<<< <<<<<<<<< gattgggccaacaaggactcagacgagttttattgactcatttctctggt tgatacggagccatgtcatgtgccacgacctggggtgggcacagggaggc <<<<<<<<< <<<<<<<<< tgatacggagccatgtcatgtgccacgacctggggtgggcacagggaggc tgcagttccctacgtgaacctgccttgggcctcatctgctcctagcccag <<<<<<<<< <<<<<<<<< tgcagtttcctgcgtgaacctgccttgggcctcatctgctcctagcccag cagag <<<<<<<<< <<<<<<<<< cagag #33 rs chr8:

80 ggctgcagttccctacgtgaacctgccttgggcctcatctgctcctagcc <<<<<<<<< <<<<<<<<< ggctgcagtttcctgcgtgaacctgccttgggcctcatctgctcctagcc cagcagagagagttgacccctcctgaactggccactccccagtgctcctg <<<<<<<<< <<<<<<<<< cagcagagagagttgacccctcctgaactggccactccccagtgctcccg tgcagggataggaag <<<<<<<<< <<<<<<<<< tgcagggagaggaag cccagccacatcatctttatctcctgggattttcattagggcaaagatct <<<<<<<<< <<<<<<<<< cccagccagactgtctttatctccatggattttcatcagggcaaagatca primer cagcagccagctcctggtagctgatgaggatcagagcgtttgttccccca <<<<<<<<< <<<<<<<<< cagcagccagctcctggtggctgatgaggatcagagcatttgttccccca rs primer tgaaaggggaaatacttaggtaatcattccaggtgtgttcagtagttcca <<<<<<<<< <<<<<<<<< tgaaaggggaaatccctatggcatcattccagtggtggtcagtagatcca ggac <<<<<<<<< <<<<<<<<< ggac ggactcag <<<<<<<<< <<<<<<<<< ggactcag

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

81 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

82 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 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 μl ᄂ Thermal Cycling Step Temp Time : : : :00 5 Go to Step 2 for 34 Times 6 4 forever

83 2 Clean-up (384 plate 1개기준 ) Manual Automatic Number of plates 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 min/ 96 10min/ hold at 4 / store at 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 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 : : :11 4 Go to Step 2 for 45 Times 5 4 forever

84 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 Xhybridization 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

85 - 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

86 < QC Review >

2) SNP-IT assay using SNPstreamR 25K system - PCR 이후의모든과정이완전자동화된 system으로하루 25,000여 genotyping이가능한 high-throughput SNP scoring system임 ( 아래그림참조 ).

87 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

88 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 >

89 - 실험방법 1 PCR amplification 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

90 <SNPstream 25K SOP> Date: Exonuclease Treatment TargEx (ul of 50U/ul) 472.5ul Lot # TargEx Dilution Buffer ml 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 ml 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 ml Lot #

91 3 결과분석 : QC review program - SNPstreamR 전용분석 program인 QC review program으로각각의 genotyping 실험결과를 confirm. - 아래그림의각점은개개의샘플을표현. 한 plate에서분석된샘플의전체적인경향을볼수있고 system에의한 genotype call을사용자가확인하고필요에따라서는정정할수있음. < QC review window >

92 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

reaction temp. reaction time 96 10sec 50 5sec 60 30sec 25cycles ( 이반응을통해 labeling되지않은 primer가 template에붙어 SNP 부위바로앞까지 anneal되고 SNP에해당하는부위에는 dideoxynucleotide가들어가합성되므로 SNP 부위까지만합성된 product가반복되어만들어짐.

93 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

94 - 92 -

95 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 )

96 Liquid handler를이용한 PCR master mix의분주 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

5) PCR Clean Up PCR 이후에과잉으로남아있는 primer와 dntp 등의불순물을 magnetic bead를이용한방식으로제거함. magnetic bead kit는 Agencourt사의 AMPure 를이용하며, automation 장비는 Beckman Coulter사의 Biomek FX를사용.

97 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

<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.

98 <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

99 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

<Deck Layout> - CleanSEQ Startup Dialog 창을띄워 reaction format, number of plates, quadrants, sequencing reaction volume, elution time, ethanol concentration 등의 parameter 에대한

( 용액을옮기고 pipette mix를하는작업들은모두 96 head의 Biomek FX 장비를이용하며, tip을사용하는각단계가끝날때마다 wash station에서 tip wash.) - 85% 에탄올 42μl를각 well에넣고 14회 pipette mix 한다.

100 <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

101 - 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

102 <Polyphred : SNP 발굴> - 발견된 SNP 위치에서 각 샘플의 genotype 결과(wild, hetero, homo mutant)를 얻음. <Wild> <Hetero> <Homo mutant>

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