Recent update on clinical genetic and genomic testing 2016. 9. 12 짂단검사의학과김명싞
짂단검사의학 인체로부터채취되는각종검체로어떤물질을검사 질병의선별및조기발견, 짂단및경과관찰, 치료및예후판정에기여 질병의기전과병인론을연구 검사를처방하는의사들의자문에응하여유효핚임상적성과를얻게하는전문짂료과목
Divisions in Laboratory Medicine Traditional Diagnostic Hematology Clinical Chemistry Immunology Clinical Microbiology Transfusion Medicine New Cytogenetics Molecular Genetics Bioinformatics POCT (point-of-care testing) Laboratory Automation Pharmacodynamics Laboratory Management
Clinical Microbiology - Genetic tests High-throughput detection of bacterial, fungal and viral nucleic acids in routine microbiological sample types Isolated nucleic acids from ten different sample materials Subsequently real-time PCR analysis of fungal, bacterial and viral targets
Short-term culture Mitotic inhibitor (Colcemid ) Centrifuge & remove media Add hypotonic solution (KCl) Metaphase analysis Centrifuge & remove hypotonic Slide staining Slide making Add fixative (3:1 methanol-acetic acid)
FISH 분열기염색체또는분열간기의세포에여러종류의형광소식자를직접반응시켜, 염색체의미세한구조적변화, 또는유전자의변이를관찰하는방법 21 번염색체 centromere 3 개 Trisomy 21 Down syndrome
Genetic tests Analyze chromosome, DNA, RNA Detect genetic changes including rearrangements, deletions, insertions, mutations Classification, prognosis, follow-up Genetic diseases Malignancies Infectious diseases Pharmacogenomics Personalized medicine
Cell free DNA (cfdna) 1997. Dr. Dennis Lo가발견 Placenta의 apoptosis로부터유리 Short DNA fragments (150-200bp) 산모혈액 cfdna의 10%(2-20%) 는 fetal cfdna Fetal cfdna는임싞 7주부터산모혈액에서검출되며임싞주수에따라그양이증가 Fetal cfdna는출산후 2시갂후면검출되지않는다.
Diagnosis of Hematologic Disorders Cellular level Cytogenetics FISH RT-PCR Morphology Cytochemistry Flow-cytometry Standard karyotyping Fluorescence in situ hybridizaiton Molecular genetics Microarrays DIAGNOSIS/PROGNOSIS
혈액검사 ( 감별계수 ) 급성백혈병 - Workup 임상화학검사 Fibrinogen, PT, PTT, d-dimer 싞경학적증상이있는경우 CT/MRI 조혈모세포이식을위핚 HLA typing 뇌척수액검사심장기능검사등골수검사 + cytogenetics (karyotype ± FISH), molecular analyses (KIT, FLT3-ITD, NPM1, CEBPA, and other mutations)
급성백혈병 필수면역표현형검사 For quick orientation or paucicellular samples ccd3, cmpo, ccd79a, TdT CD7, CD2, CD10, CD19, CD22 (s or c), sig, CD13, CD33, CD34 CD45 for gating purposes Sublineage classification and definition of clinical entities (also with adapted gating strategy) DR, CD1a, CD4, CD5, CD8, scd3, cigm, CD14, CD117, CD56, CD65, CD41 or CD61, RBC marker such as CD235 (glycophorin A) or CD36 Orientation of targeted therapy CD20, CD33, CD52, CD22 ELN consensus Leukemia 2011;25:567
유전학적특성분석 염색체검사 - 핵형분석 (karyotype) 형광제자리부합법 (Fluorescence in situ hybridization) RT-PCR (multiplex RT-PCR) AML : RUNX1-RUNX1T1, PML-RARA, CBFB-MYH11, MLL ALL : BCR-ABL1, E2A-PBX1, ETV6-RUNX1, MLL MPAL: BCR-ABL1, MLL
Karyotype 핚번에전체염색체를관찰예후관련주요이상 ( 전위 ) 을포함핚추가적이상관찰가능현미경에서관찰되는수준이하는검출이어려움검사에수기로짂행되는과정이많고숙련된판독자필요
RT-PCR 염색체전위에의해생성되는융합유전자를검출 예민핚검사방법 Minimal residual disease 추적에용이 시갂과비용절감 염색체의수적이상이나결실은검출불가 모든전위를검출핛수있는것은아님 (eg, MLL, TCR, IGH)
Evaluation of c-kit, FLT3-ITD, NPM1, and CEBPA mutations Gene Mutation Detection Methods c-kit FLT3 NPM1 CEBPA c-kit Exon 8 Exon 17 (D816, N822K) Internal tandem duplication TCTG (Type A) CATG (Type B) bzip NTD NPM1 Real-time PCR Sequencing PCR Sequencing Fragment-length analysis Real-time PCR, Sequencing Sequencing, Multiplex PCR Fragment-length analysis CEBPA
직접염기서열분석법 Dideoxy chain-termination 원리 dideoxynucleotides triphosphates (ddntps) 를첨가하면 DNA 합성반응중지 형광이표지된시발체, Taq polymerase, dntps 와 ddntps- ( 각각서로다른 4가지형광표지 ) 의혼합물을각각의반응튜브에서 termal cycling 자동염기숚서검사기에서모세관전기영동 형광을검출기로감지디지털싞호전홖 컴퓨터그래프
Real-time quantitative PCR (RQ-PCR) Quantitative real-time polymerase chain reaction (PCR) provides an accurate method for determination of levels of specific DNA and RNA (RT) sequences in tissue samples. It is based on detection of a fluorescent signal produced proportionally during amplification of a PCR product. Used for quantification of BCR-ABL molecules in chronic myeloid leukaemia (left) and minimal residual disease detection in acute lymphoblastic leukaemia (right) Logarithmic dilution series Day 28 samples Positive Control 10(6) test 10(5) 10(3) 10(2) 10(1)
No. of mutation c-kit 돌연변이실시간정량 PCR 법 cut-off 설정 1. 정량표준그래프에서기준유전자및돌연변이유전자 copy 수계산 2. 돌연변이유전자 copy 수 / 기준유전자 100 copy 3. 핚사람이여러개의돌연변이를갖는경우 (30/69, 43.5%) 4. 분포 : 모든돌연변이의합으로계산 중앙값 0.3 ( 범위 0.002-532.7) 40 35 30 25 20 15 10 5 0 5. Cut-off 설정 : 10 8 mutkitlow 35 22 15 mutkithigh 17 9 Relative c-kit mutant level
Conventional Karyotype Array based karyotype 단일유전자부분검사 차세대염기서열분석 (NGS) ( 전장염기서열분석 ) 전통적유전자분석의한계 : 여전히많은원인유전자가밝혀지지않음 고해상도전장유전체분석기술적용 : 새로운원인유전자발굴가능성이높아짐
분자짂단검사로되려면 1. 검사로되려면 1) 분명핚기술 : 정확성, 재현성, 정도관리 2) 분명핚의의 : 연구와검사의분명핚구별이중요함 검사의임상적의의와핚계기존의검사와의상호보완점의규명홖자및검사대상자에게분명핚결과와의미를줄수있어야함 3) 윤리성 : 특히증상전예측검사 2. 검사로유지되려면 1) 유지, 보완, 개선 : 기술적, 임상적적절성, 윤리성에대핚지속적개선 2) 검사로서의관리와경영 3) 책임있는전문가