Kromozom bantları ve in situ hibridizasyon · PDF fileSpectral karyotyping and multifluor...
Transcript of Kromozom bantları ve in situ hibridizasyon · PDF fileSpectral karyotyping and multifluor...
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Kromozom bantları ve in situ hibridizasyon
Rasime Kalkan,PhD
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Yöntemler
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Konvasiyonel sitogenetik analiz
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Kromozomlar büyüklük, sentromer pozisyonu ve bantlama paternine göre ayrılmaktadır
1 kromozom bandı : 5-10 Mega baz çifti(Mbp).
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Visualizing Metaphase Chromosomes (Banding)
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Sayısal anomaliler
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Yapısal anomaliler
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High resolution karyotype
AvantajlarTüm genomu taramakRölatif olarak daha ucuzDezavantajları5 mb kadar anomali saptamaktadır
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Klasik sitogenetiğin limitleri
Kromozom bantlama tekniklerine bağlı olarak hassastır
İyi kromozom morfolojisi ve bölünen hücrelere ihtiyaç vardır
5 Mb rezolüsyon limiti
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Konvansiyonel sitogenetiğin avantaj ve dezavantajları
Avantajları
• 1 defada tüm genomun taranmasına imkan tanır.
• Spesifik anomali bilindiğinde saptanması ( ör KML de Philadelphia) ve ek kromozomal anomalilerin saptanması
Dezavantajları• Majör yapısal anomalilerin saptanması 1band = 5mb of
DNA ~ 150 gen ).
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Kromozomal anomali endikasyonları
• Kromozomal anomali süpesi• Mukonjenital anomali ve gelişim geriliği• Mental reterdasyon• Gonadal disgenezi• Infertilite• Düşükler• Ölü doğum öyküsü• Malignensiler
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Hangi durumlarda FISH önerilir?
• Metafaz kromozom elde edilememesi halinde – Başarısız kültür
– hastadan alınan dokunun kültüre edilmesinin uygun olmaması (preimplantation analysis, rapid prenatal examinations, examinations of solid tumors or autopsy material)
• Komplike kromozomal anomalilerin analizi
• Marker kromozomun tanımlanması
• Sub mikroskobik, kriptik anomalilerin saptanması– Mikrodelesyon sendromları
– Malignansilerde tümör baskılayıcı ve onkogenkerin amplifikasyonu
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• In situ hybridization spesifik nükleotid sekanslarının hücre süspansiyonu yada dokulardan saptanması. İlgili gen bölgesine spesifik prob un hücre yda doku süspansiyonuna hibridizasyonu prensibine dayanmaktadır.
Fluorescence in situ hybridization(FISH)
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Fluorescence in situ hybridization(FISH)
• In situ teknikler anomalinin görüntülenmesi için tanı amacı ile kullanılmaktadır veya hastalığa neden olan endojen veya ekzojen DNA veya RNA molekülünün saptanması için kullanılmaktadır.
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Moleküler sitogenetik:Fluorescence in situ Hybridization (FISH)
• RNA ve DNA yapılarının, sayı ve lokasyonlarının yerlerinin hücrede ve in situ olarak tanımlanmasına dayanmaktadır
• FISH :– Morfolojik olarak korunmus kromozom
preperatlarında kullanılmaktadır (Metafaz).
– Fikse hücre veya dokular (Interfaz)
• Spesifik kromozom anomaliler görüntülenmektedir– delesyon, amplifikasyon, translokasyon
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Fluorescence in situ hybridization(FISH)
Bölünen(metaphase) ve bölünmeyen (interphase nuclei) hücrelerdeki seçili genetik değişikliğin saptanması
ISH hücresel ve moleküler seviyede bilgi veren bir metoddur.
FISH hematolojik malignensilerin orjin ve progresyonunu belirlemek için kullanılabilmektedir
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FISH Analysis
AavantajlarıÇok specific (100 kb)Microdelesyon/Microduplikasyon
DezavantajlarıÇok specific500-600 proba ihtiyaç vardır tüm
genomu tarayabilmek için
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• Metafaz kromozomları
• Interfaz nukleusu
• Entire Cells/RNA
• Doku kesitleri
• FISH deneyleri yeterli uzunkluktaki ve ilgili DNA dizisine komplementer floresan işaretli DNA/RNA probları ile yapılmaktadır.
Hedef- Target
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FISH analizinde kullanılabilecek dokular
• Periferik kan
• Fibroblasts biyopsisi
• Epitel hücreler, buccal smear
• Kemik iliği (hemoblastosis)
• Solid tumor biyopsisi
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Interpfaz FISH’in avantajları
• Hücre kültürene ihtiyaç yoktur ve metafaz için hücrelerin bölünme stimüayonuna ihtiyaç yoktur– interphase FISH metafaza göre daha hızlıdır
– Fikse edilmiş hücrelerin ve bölünmeyen hücrelerin analizine izin verir.
• Mikroskop kullanılarak FISH ile 200–500 analiz edilebilir– Metafaz prosedürlerine göre daha hassasdır
• Kemik iliği transplantasyonunda rezidüel hastyakığın moniterize edilmesinde kullanılabilir.
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Kromozom tanımlama
Aneploidi saptama
Sentromer analizi
Marker kromozom kökenini belirleme
Tüm kromozom analizi, whole chromosome analysis
(chromosome painting)
Kromozom translokasyon analizi
Benzersiz –unique- sekansların analizi (single-copy sequence)
Mikrodelesyon analizleri
Gen amplifikasyon analizleri
Moleküler Sitogenetiğin Kullanımı
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FISH PROSEDÜRÜ
• Kromozom denatürasyonu
• Prob denatürasyonu
• Hibridizasyon
• Floresan boyma
• Slide ların analizi
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Fluorescent in situ Hybridization (FISH) kullanımı
Sayısal ve yapısal krozom anomlilerinin tanımlanması ve karekterizasyonu
Mikroskobda görülemeyen delesyonların saptanamsı
Sub relomerik anomalilerin saptanması.
Prenatal tanı,bilinen sayısal kromozomlar için(interphaseFISH).
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FISH limitleri
• Probun bağlandığı bölgeler dışındaki anomaliler saptanamaması
• İnterfaz fısh için hücre preparasyonu önemlidir– to permeabilize the cells for optimal probe target interaction
– to maintain cell morphology.
• Küçük mutasyonlar saptanamaz
• Uniparental dizomi saptanamaz.
• Inversiyonlar saptanamaz
• Tüm kromozomal bölgeler için problar tivcari olarak yoktur
• Pahalı bir yöntemdir
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Problar
• Hedef nükleik asit dizisinin komplamenteri
• Problar floresanboyalar ile işaretlenmektedir biotin, fluorescein, Digoxigenin
• Büyüklükleri20-40 bpto 1000bp
Fluorescein
Biotin
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• Indirect Probe labeling, antikorlar aracılığı ile FISH prosedürü tamamlanmaktadır Haptens---Biotin-dUTP, digoxigenin-dUTP
• Labeling-İşaretleme teknikleri: a) Nick translationb) Random primingc)PCR (Polymerase chain reaction)
• Direct Probe labeling, problar direkt olarak SpectralGreen veya SpectralOrange gibi florokromlar ile işaretlenmişlerdir One-step hybridization.
5‘ 3‘
3‘ 5‘
5‘ 3‘
3‘ 5‘+※++※+ ++※++++※++※
DNA Polymerase adds dNTP, labeled dUTP at 3’ and remove dNTP at 5’ DNase I makes nicks
Principle of Nick Translation
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Faktörler Seviye Stringency Sonuçlar
(if inappropriate)
Isı Yüksek
Düşük
Yüksek
Düşük
Düşük
verim
Yüksek
arkaplan
Tuz çözeltisi
(SSC) konsantrasyonu
Yüksek
Düşük
Düşük
Yüksek
Yüksek
arkaplan
Düşük
verim
Formamid Konsantrasyonu
Yüksek
Düşük
Yüksek
Düşük
Düşük
verim
Yüksek
arkaplan
Denaturation Stringency
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Prob Tipleri
Sentromer Probları• Alpha ve Satellite
III probes• Sentromer gibi
tekrarlayan dizilerde bulunmaktadırlar
• Sentromer bölgeleri parlak boyanmaktadır
Tüm Kromozom Probları• Tüm kromozom boyuna bağlanan prob kombinasyonlarının toplamıdır• Multiple probe labels are used• Tüm kromozom boyuna hibridiza olmaktadırlar
TelomerTelomer bölgelerine spesifiktirKromozom uçlarındaki 300 kb locus spesifiktir
LokusDelesyonTranslokasyon problarıGen saptanama & lokalizasyonGen amplifikasyon probları
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Denatürasyon & Hibridizasyon
Denatürasyon Hibridizasyon
Isı veya alkali metodu ile olabilir Prob ve hedef DNA denatürasyonu Hibridizasyon için ön koşuldur
İki komplementer nükleotid sekanısının dupleks oluşturmasıdır
• DNA-DNA• DNA-RNA• RNA-RNAArasında l olabilmektedir
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Detection & Visualization
Saptama-Detection Hibridizasyon
• Direct İşaterleme: Prob direkt floresan isaretlidir Daha az hassastır
• Indirect İşaretleme: Saptanması için ek basamaklara ihtiyaç vardır Alkalin fosfotaz gibi konjuge antiboylere ihtiyaç vardırSinyal amplifikasyonu ile saptanabilir
• Floresan prob hibridizasyon sırasında hedef sekansa bağlanmaktadır
• Bu uygun filtreler ile mikroskopta görüntülenebilmektedir.
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Kromozomal sekansların sınıflandırılması
• Beta satellite
• Alpha satellite
• Classical satellite
• Telomeric sequences
• Unique gene sequences
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Spesifik kromozom yapılarına özel Problar
• Kromozom spesifik sentromer probları(CEP)– Sentromer bölgelerine hibridize olmaktadır – İnterfaz ve metafazda anoploidilerin saptanması için kullanılmaktadır
• Chromosome painting probes (WCP)– Tüm kromozoma hibridize olan problardır– Metafaz üzerinde yapısal kromozom anomalilerin saptanması için kullanılmaktadır
• Unique DNA sequence probes (LSI)– Benzersiz DNA dizilerine hibridize olmaktadır – Gen yeniden düzenlenmeleri, delesyon ve gen amplifikasyonlarının saptanması için
kullanılmaktadır
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• Telomere-specific probes (TEL)– Subtelomerik bölgelere hibridize
olmaktadır
– Subtelomerik delesyon veya yenidendüzenlenmelerin saptanması için kullanılmaktadır
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α-satellite DNA – centromeres
Sayısal anomalilerin saptanması, marker kromozomun sentromer orjininin belirlenemsi, kemik iliği transplantasyonu sonrası hücre görüntülemesi (opposite sex of donor and recipient)
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Locus specific DNA probes:
Kromozom üzerindeki genlerin görüntülenmesi ve yapısal kromozom anomalilerinin saptanması (translocations, deletions)
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trizomi-21 kız fetus
chromosomes 18 (aqua), X (green), and Y (red).
chromosomes 13 (green), and 21 (red)
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X–ckromozomu için Satellite (centromeric) probe
45,X or 46,XYKaryotip?
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X- and Y-centromeric probes
46,XYDetermine probable karyotype.
Green = X
Red = Y
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Interfaz FISH ile yapısal anomaliler LSI
Probe (Fusion Probe) ile saptanmaktadır
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Interfaz FISH ile yapısal anomaliler LSI Probe (Break Apart Probe)
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Dual color break-apart probes
• Translokasyon kırık noktalarına spesifik olarak tasarlanmıştır.
• Traslokasyon olması halinde prob birbirinden ayrılmakta ve sinyal vermektedir
0.6–1.5 Mb
Translokasyonu saptamaktadır
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FISH kullanılan mikrodelesyonsendromları
Syndrome Chromosome Location
Probe/Gene Locus
DiGeorge 22q11.2 D22S75
Velocardiofacial 22q11.2 D22S76
Miller-Dieker 17p13.3 D17S379
Smith-Magenis 17p11.2 D17S29
Prader-Willi 15q11.2 SNRPN
Angelman 15q11.12 D15S10
Williams 7q11.23 Elastin
Cri du chat 5p15.2 D5S23
Wolf-Hirschhorn 4p16.3 D4S96
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Microdeletion konfirmasyonu(1 kırımızı sinyalin kaybı)
Red signal –TUPLE1 (HIRA)
locus
Green signal –ARSA locus
(control probe)
Deleted chromosome – red signal absent
normal chromosome –red signal on HIRA locus is
present
Microdeletion 22q11.2 is associated with
DiGeorge syndrome.
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Chromosome painting probes:
İlgili kromozom bölgesi veya tüm kromozama komplementer sekans hey contain sequences from whole chromosomes or chromosomal parts (partial probes) yapısal yenidendüzenlenmelerin saptanması (translocations and deletions of large extent),veya marker kromozomun kökeninin belirlenmesi için kullanılmaktadır
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der 5
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der 5
SpectrumGoldWCP 5
+SpectrumRed
WCP 9
95
9
9
der 5
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Multicolor FISH - mFISH
her kromozm çifti için farklı florersan kombinkeri kullanılmakta ve her kromozm çifti farklı floresanvermektedir
Hematolojik malignensilerde kompleks kromozom anomalilerin saptanmasına imkan tanımaktadır
kompleks yapısal anomaliler saptanabilmektedir
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Spectral karyotyping and multifluor FISH paint each human chromosome in one of 24 colors (SKY)
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Multicolor banding with high resolution - mBAND
Klasik bant tekniklerine oranla daha yüksek çözünürlükte kromozom kırık noktaları saptanmaktadır
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Kromozom 4 de küçük bir terminal delesyon var mı?
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Hematolojide sitogenetik ve moleküler sitogenetiğin önemi
Diyagnoz
karyot,ip hakkında bilgi
* Tanının spesifikleşmesi
*prognoz hakkında bilgi
* Tedavinin izlenmesi
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Sık kullanılan florokromların Absorption Spectrası
Fluorochrome Absorption Emission
Cascade Blue 400 420Fluorescein 494 518Rhodamine 570 590Texas Red 595 615Cy5 650 670
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Diagnostic Potential For Karyotype, FISH, and Chromosomal Micro- array Analysis (CMA) For Selected Disorders
CMATelomere FISHDisease
specific FISH
KaryotypeLocus studied
Condition
~100%Detected by karyotype
Not detected~100%variousAneuploidy
Karyotype better for
present
Detected by karyotype
Not detected~100%variousLarge deletions, large dupllications, translocation of large segments
~100% for unbalanced
~100%Not detectedNot
detected
variousCryptic
Rearrangements of telomeres
~99%>95%~99%Few1p36.31p36 deletion
~99%>95%~99%Most4p16.3Wolf-Hirschhorn
~99%>95%~99%Most5p15.2Cri-du-chat
~99%Not detected~99%Almost none
7q11.2Williams-Beuren
~70%Not detected~70%Unreliable15q11-q13Prader-Willi
~70%Not detected~70%Unreliable15q11-q13Angelman
>90%Some detected>90%Few17p13.3Miller-Dieker lissencephaly
>95%Not detected>95%Some17p11.2Smith-Magenis
>95%Not detected>95%Rarely22q11.2Velocardiofacial/DiGeorage 1
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FISH vs. Karyotyping
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Case 1
• 2 yaşında, MR erkek
• Inborn cardiac defect –supravalvular aortic stenosis.
Abnormal fenotipik özellikler mevcut
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irides stellataehypertelorism
open mouth, thick lip
abnormal teeth
„elfin face“
low set ears
Case 1
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Case 2• Williams-Beuren syndrome çağrıştırmakta
• Microdeletion sendromu, 7q11.23
• %95 microdeletion FISH ile tanımlanabilmektedir
• Öncelikle konvansiyonel sitogenetik önerilmiştir
Kromozom 7 microdeletion tanımlamak için hangi prob kullanılmalıdır?
Case 1
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Case 1 - karyotype
Normal sonuç:
46,XY
Microdeletion FISH analizi ile konfirme edilmelidir
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7q11.23 MICRODELETION MOLECULAR SİTOGENETİK ANALİZ
LOCUS SPECIFIC PROBE FOR THE CRITICAL REGION ELN/LIMK/D7S613– (labeled with the Spectrum
Orange, red signal)
CONTROL PROBE D7S522 – (labeled with the Spectrum
Green, green signal)
ELN LIMK1 D7S613
CENTROMERE TELOMERE
180 kb
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Case 1 –molecular cytogeneticardından sonuç
• 7q11.23
microdelesyon
doğrulandı
• Tanı: Williams-
Beuren syndrome
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Case 2
• Turner syndrome?
• 11 yaşında, 128 cm-kısa boy, zayıf okul performansı
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• Doğumda :900 gr
• Hormon seviyeleri:
*FSH :106,1 uU/mL
*LH: 21.38 uU/mL
*UE: <5,00
*G6PD: 0,4u/g Hb.
• 128 cm
• 35,5 kg.
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Fenotipik Değerlendirme :
• Dismorfik özellikler:
*simian çizgisi-her iki elde mevcut
*klinodaktili-sağ elde
*kısa ve hipoplastik tırnaklar
*düşük ense saç çizgisi
*düşük yerleşimli gözler
* hipertelorizm
*basık burun
*uzun filtrum
• Abdominal USG yumurtalıklar gözlenmedi
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Testler• Karyotip analiz;
*G/LTB
*C-Bantlama
*NOR Bantlama
• Karyotip:
45,X [95]/46,X,+mar[5] (5%)
• Moleculer Cytogenetic Tests:– Multicolor FISH
– LSI AR and LSI SHOX and XIST
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Androgen Receptor / SHOX gene/ XIST
AR, Xq11.2-
Xq12SHOX:sp red
Cep X: Sp aqua
SHOX:sp red
Cep X: Sp aqua XIST, Xq13.2
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