Cytogenetics & Integrated laboratory of molecular cytogenetics, Brno logo-FNB LMC What are we going to talk about? n1. What is cytogenetics n n2. History n n3. Chromosome morphology and aberrations n n4. Molecular cytogenetics and its techniques n n5. Our laboratory and work n 1. What is cytogenetics? nCytogenetics is a branch of genetics focusing on the study of chromosome changes (number, morphology, numerical and structural abnormalities, segregation in normal and pathological conditions) and their correlation with phenotype. 2. Just a little history… n1866 Gregor Johan Mendel – Experiment in Plant Hybridization nFather of genetics nDefined the basic principals of heredity (principle of segregation and combination) nDuring his life, his work was ignored nLater, Mendel´s work was rediscovered n1910 Thomas Hunt Morgan proved that genes are located on chromosomes (using Drosophila) n1953 James Watson and Francis Crick determined DNA structure n1956 Tjio, Levan – Human chromosome number is 46 Development of human cytogenetics n„Dark Ages“ - the development and improvement of tissue culture techniques n n„Hypotonic Period“ n - hypotonization of cell samples (1951 - 0,075 m KCl) - using phytohaemagglutinin (PHA) - stimulation of peripheral blood lymphocytes - 1960 n„Trisomy Period“ - trisomy of chromosome 21-1959 nThe first deletion syndrome - "Cri du chat" - 1963 n„Banding Area“ - chromosome banding techniques 1968 – 1970 n„Molecular Area“ n - in situ hybridization technique – 1970 n - FISH – 1986 n - Comparative genomic hybridization (CGH) - 1992 - Spectral karyotyping (M-FISH, SKY) - 1996 - M - banding - 2001 - Array - CGH - molecular karyotyping „take home message“ Basic conditions for development of human cytogenetics nimproved techniques of cell cultivation in vitro nuse of hypotonic solution (0.075 M KCl) nestablishing squash techniques nuse of colchicine – arrest of mitotic division n1% orcein staining n Nomenclature of human chromosomes Ø1960: Denver Conference - sort of human chromosomes into groups according to size and shape Ø1963: London Conference - chromosomes are sorted into 7 groups A – G Ø1966: Chicago Conference - the description of chromosome changes Ø1971: Paris Conference - the identification and labeling of chromosomes using banding techniques ØAn International System for Human Cytogenetic Nomenclature n (ISCN 1978) http://cdn01.bookadda.com/bk_images/537/9783318022537.jpg short (p-) arm long (q-) arm centromere chromatid telomere telomere repetitiv (satelite) sekvence DNA repetitive sequence (TTAGGG)n repetitive sequence (TTAGGG)n DNA http://camelot3.lf2.cuni.cz/turnovec/ublg/vyuka/index.php?adresar=prf 3. Chromosome morphology Chromosome morphology Rotation of karyotypy057 Chromosome staining nClassical staining nusing Giemsa Romanowski solution ngained chromosome aberation detection n nG – bands nusing trypsin, salty solution and Giemsa neach chromosome has characteristic stripes ncongenital chromosomes aberation detection n nR – bands nusing salty solution of different pH and temperature nreverse to G - bands n n chrom Human caryotype A B C D E F G Caryotype of animals http://users.unimi.it/citozoo/cattle%20female.png cattle http://users.unimi.it/citozoo/doga%20male.png dog http://users.unimi.it/citozoo/T6%20cariotipo%20femmina%20cavalla%20female%20horse%20karyotype.jpg horse „take home message“ Human somatic cell contains n23 pairs or 46 chromosomes n22 autosomic pairs n1 gonozomic pair (XX or XY) n7 groups ordered according to chromosome size and morphology nA – large metacentric chromosomes nB – large submetacentric chromosomes nC – middle size submetacentric chromosomes, X nD – large acrocentric chromosomes nE – small meta- to submetacentric chromosomes nF – the smallest metacentric chromosomes „ribbons“ nG – small acrocentric chromosomes, Y Chromosome aberrations nAUTOSOMIC n1. Structural nPolymorphysmus ndifferent lenght of chromosomes in homologous pair nno phenotype effect n nInversion npericenric – including centromere nparacentric – does not include centromere nusually has no phenotype effect n nRing chromosomes nbreaks on both chromatides and their connection nmental and physical retardation nalways newly created nsometimes redundant n n ringchromosome http://ghr.nlm.nih.gov/handbook/illustrations/ringchromosome Chromosome aberrations nDeletion nterminal – one break nintersticial – two breaks ndeletion syndromes: §Wolf-hirschhorn syndrome; 4p deletion §Cri-Du-Chat syndrome; 5p deletion nmicrodeletion syndromes: §Prader-Willi syndrome; 15q11-12 deletion §DiGeorge syndrome; 22q13 deletion §Angelman syndrome; 15q11-13 deletion §Williams-Beuren syndrome; 7q11.23 deletion § nInsertion ninserted part can be in the same or inverted position n n n n n Chromosome aberrations nTranslocation nreciprocal nmutual exchange between two or more nonhomologic chromosomes nbalanced - no phenotype effect ngenetic risics of unbalanced genom gamets formation n nrobertsonian nbetween two acrocentric chromosomes nbreaks in the area of centromeres and deletion of short arms ncentric fusion of the remaining arms nbalanced – normal phenotype n ntandem ndeletion of part of an acrocentric chromosome nfusion of the remaining part with another chromosome n n n translocation%20-%20reciprocal http://www.larasig.com/node/3628 robertsonian-translocation-13045_3 http://drugline.org/medic/term/robertsonian-translocation/ n2. Numerical nTrisomy n21 chromosome trisomy – Down syndrome n18 chromosome trisomy – Edwards syndrome n13 chromosome trisomy – Patau syndrome n nTriploidy n69 XXX, 69 XXY nnonviable nmosaic triploidy – mental retardation, syndactyly, abnormal genitals, lateral asymetry n n Chromosome aberrations nGONOSOMIC nChromosome Y nstructural aberrations – very rare nnumerical aberrations n47, XYY – supermale syndrom nChromosome X (male) nNumerical aberration n47, XXY – Klinefelter syndrom nChromosome X (female) nnumerical aberrations n45, X – Turner syndrom n47, XXX – XXX syndrom nFragile X – fraX nthe most common cause of mental retardation nnonspecific phenotype n n n n Chromosome aberrations Down syndrome (47,XX or XY,+21) n1866 J.L.Down nIQ 25-50 nsmall dumpy figur nround face nshort neck nmongoloid eyes nepicanthic fold nwide nose root and flattened nose nsmall mouth, large tongue, small teeth nsingle transverze palmar crease nheart diseases n Down_Brushfield_spots Down_simian_crease +21a Edwards syndrome (47, XX or XY,+18) ngrowth retardation nmicrocephaly ndolichocephaly – elongate head ncleft palate nlow-set malformed ears nfinger holding nstructural heart defect at birth nsurvive only few months n Edwards peri215 +18 kar Patau syndrome (47,XX or XY,+13) nhard growth and mental retardation nmicrocephaly ntrigonocephaly ncutis aplasia ncongenital brain defects ncleft palate nhexadactily nkidney defects n domhoff4 +13 kar Wolf-hirschhorn syndrome (del 5p) nmicrocephaly nmicrognathia (small jaw) nocular hypertelorism ndysplastic ears ngrowth and mental retardation nmuscle hypotonia nseizures ncongenital heart defects Deletion_of_short_arm_of_the_chromosome_4_in_a_patient_with_Wolf-Hirschhorn_syndrome X2604-W-05 Cri-Du-Chat syndrome (del 5p) nlow birth weight, poor growth nhypotonia nsevere cognitive speech and motor delays nbehavioral problems – hyperactivity, agression nsmall head and jaw nwide eyes nconstipation nabnormal larynx development ndifficulty of swallowing and sucking ndrooling ncat-like cry n cri-du-chat_diagram criduchat_person http://learn.genetics.utah.edu/content/disorders/whataregd/cdc/ Prader-Willi syndrome (del 15q11-q13) npaternal deletion nlow fetal activity nhypotonia nexcessive weight gain, hyperphagia nshort stature nhypogonadism nmental retardation nhypopigmentation nskeletal development delay (acromicria) Chaloupková Angelman syndrome (del 15q11-q13) nmaternal deletion nhard mental retardation nhypotonia nepilepsia, seizures nhypopigmentation nhyperactivity nspeech absence nprominent scull shape (mandibul, microcephaly, flat back of head..) n„happy character“ nmovement or balance disorder n Kubíková DiGeorge syndrome (del 22q11.2) nlow-set malformed ears nsmall mouth and lower jaw nnarrow eye-lits nsubmucosal or visible cleft palate nhypocalcemia ninterupted aortic arch ncardiac abnormality – tetralogy of Fallot nincomplete ventricular septum nright-to-left shunt of aorta nleft ventricle hypertrophy nlung stenosis http://diseasesforum.com/wp-content/uploads/2013/09/DiGeorge-Syndrome.jpg https://www.google.cz/search?q=digeorge+syndrome&espv=210&es_sm=93&source=lnms&tbm=isch&sa=X&ei=P9C FUo21HsqR7AbP1IBI&ved=0CAkQ_AUoAQ&biw=1920&bih=989#facrc=_&imgdii=_&imgrc=0EhFFG2IOAvB3M%3A%3BFG4R3 3YXExVhsM%3Bhttp%253A%252F%252Fd Williams Beuren syndrome (del 7q11) ndevelopmental delay nmental disability nfailure to thrive nheart defects (heart murmur, narrowing of main blood vessels) nflattened nasal bridge nwidely spaced teeth nhypercalcemia ngastrointestinal problems nurinary difficulties n n n http://www.thespeciallife.com/images/Williams-Syndrome.jpg http://geneticsf.labanca.net/wp-content/uploads/2010/11/Williams-syndrome.jpg https://www.google.cz/search?q=williams+beuren+syndrome&source=lnms&tbm=isch&sa=X&ei=MimGUvDpC4GctQ aJhYGwCg&ved=0CAcQ_AUoAQ&biw=1920&bih=989#facrc=_&imgdii=_&imgrc=HTjyFEuSnZo4JM%3A%3BkqK81uaGRtKPIM %3Bhttp%253A%252F%252Fgeneticsf.laba https://www.google.cz/search?q=williams+beuren+syndrome&source=lnms&tbm=isch&sa=X&ei=MimGUvDpC4GctQ aJhYGwCg&ved=0CAcQ_AUoAQ&biw=1920&bih=989#facrc=_&imgdii=_&imgrc=MdTDkcoWBwg-WM%3A%3BTKKZzTKDfniYlM %3Bhttp%253A%252F%252Fwww.thespeciallife.com%252Fimages%252F Supermale syndrome (47,XYY) nincreased growth velocity nno unusual physical features nnormal testosteron level, fertility and sexual development npossible learning disabilities ndelayed development of speech and language skills nbehavioral and emotional difficulties n XYY.jpg xyy1 Klinefelter syndrome (47 XXY) n48,XXYY; 48,XXXY; 49,XXXXY ntall figure nless facial and body hair nfemale distribution of body fat nhypogonadism (decreased testicular hormon function) ninfertility ngynecomastia (increased breast tissue) nlower intelect degree nvariations: 48, XXYY; 48, XXXY; 49,XXXXY n 3 klein Turner syndrome (45,X) nlower birth length and weight nlow hairline npterigya nbroad chest, widely spaced nipples nsmall growth ninfertility, absence of menstrual period ncoarctation of the aorta nwebbed neck nlymphederma n n turner Turner File:Puffy feet.JPG XXX syndrome (47,XXX) nmajority of triple X females are never diagnosed nnormal fertility ninactivated Barr body nmost often only mild effects ntall stature nsmall head nspeech, language and learning disabilities nweak muscle tone https://www.google.cz/search?q=xxx+syndrome&source=lnms&tbm=isch&sa=X&ei=xMiIUvlHgrG0BpOYgLgL&ved=0 CAcQ_AUoAQ&biw=1440&bih=783#facrc=_&imgdii=_&imgrc=Hz1JqrGzTyKpBM%3A%3BfZOiqHDJFB267M%3Bhttp%253A%2 52F%252Fworms.zoology.wisc.edu%252Fzooweb%252FPhelps%252FZWK01047k.jpg%3Bhttp%253A%252F%252Fwww.zap pa.com%252Fmessageboard%252Fviewtopic.php%253Ff%253D5%2526t%253D7057%3B768%3B576 Triple%2BX%2BSyndrome ZWK01047k http://pics2.this-pic.com/image/triple%20x%20syndrome 4. Molecular cytogenetics npresents the connections between classical cytogenetics and molecular biology n nutilizes the latest knowledge of molecular biology, microscopy and computer image analysis to study the structure and properties of chromosomal changes n nallows the analysis of numerical and structural chromosomal imbalances unidentified classical cytogenetic techniques n ndoes not require the presence of mitosis n nsources of material for cytogenetic investigation nperipheral blood nsamples from different tissues namniotic fluid cells, chorionic villi, placenta umbilical cord blood nbone marrow nsamples of solid tumors n n nádory 003 dřeň 002 peripheral blood solid tumor bone marrow FISH fluorescent in situ hybridization n n obr1 FISH ndetection of the fluorescent signals through microscope equipped with specific fluorescent filters n nmaterial ncultivated peripheral blood ncultivated bone marrow ncultivated amniotic fluid cells nuncultivated amniocytes ntumor and bone marrow prints n nwe determine: 1.presence of signals 2.number of signals 3.position of signals n nthe use of FISH n - clinical cytogenetics n - onco cytogenetics n - human genom mapping n typy sond typy sond delece exonu počet signálů translokace Advantages and disadvanages of FISH nadvantages ndoes not require the presence of mitoses (mostly) nquick assessment of big amount of cells n n ndisadvantages ndoes not provide whole genomic view n nmikroskop equipped with 2 fluorescent filters (SKY, DAPI) n nfluorochromes (FITC, Rhodamin, TexasRed, Cy5, Cy5.5))scanned by one filter, based on a wave lenght each chromosome pair is coloured pseudocoloures n Image Acquisition with SkyVision™ SKY spectral caryotyping Display Image Picture analyse using SkyView Classified Image The objective of the SkyView spectral karyotyping software is to automatically classify and karyotype chromosomes in the Display image, thereby overcoming the ambiguity inherent in the display colors. 7 7 12 12 7 7 12 12 n specdapi2 Advantages and disadvantages of SKY nadvantages ndetects balanced rearrangements ndetects aberations in one step nkryptic translocations and insertions n marker chromosomes n redundant material with unknown origin n komplex rearrangements n ndisadvantages nneed of quality mitoses nsuccesful hybridisation nexpensive method n n CGH comparative genomic hybridization na modification of FISH technique to measure DNA gains or losses throughout the entire genome n nenables detection of unbalanced chromosomal changes (gains or losses) throughout an entire genome in one hybridization reaction n nis based on comparison of two genomes n Conventional FISH normal DNA → select DNA → make probe → label abnormal target → abnormal target identified Comparative genomic hybridization normal DNA → no DNA selection → make probe (entire genome) → quantify on normal target → abnormal genome quantified n Materials : • Good quality DNA isolated from •peripheral blood •bone marrow •solid tumour •amniocytes •Fluorescent miroscope (filters DAPI, SpGreen, SpRed) • Sensitive CCD kamera • Computer with software for CGH analysis and data interpretation (LUCIA CGH Advanced Statistics, Laboratory Imaging Ltd., Prague, Czech Republic) • CGH requirements P1040823 P1040826 P1040833 P1040842 Equipment : CGH principle www.abbottmoleculars.com CGH principle www.abbottmoleculars.com Reference Cot–1 Test Unique sequences are labeled by in situ hybridization Cot–1 suppresses hybridization of repeat sequences Relative brightness depends on amount of labeled DNA with appropriate complementary sequences, i.e. on the DNA copy number at this locus CGH modra CGH cervena CGH zelena CGH slozeny obrazek Mitoses scanning, CCD camera filters for B, G, R heterochromatin treshold 0.8 treshold 1.2 chromosome number number of chromosomes in analysis gain loss fluorescent ratio profile Identification of aberrations Minimaly 10 metaphases should be processed. Florescent ratio profile is compared to the fixed tresholds (15-20% from ratio 1). The ratio profile that deviates 15 % - 20 % from ratio 1.0 is typically regarded as aberrant. n Advantages of CGH ndetects and quantifies DNA copy number gains and losses throughout an entire genome in a single analysis n ndoes not require cell culturing and metaphases from test tissue n nis able to identify not only the chromosome from which the additional unknown material is derived, but also to map the region involved to specific bands on the source chromosome n nin combination with whole-genome PCR, can analyze DNA from a single or very few cells (Nacheva et al., 1998, Levy and Hirschhorn, 2002) Disadvantages of CGH nlow genomic sensitivity: about 10 Mbp for single copy changes nsolution: microarrays n ndoes not detect balanced rearrangements (inversions, balanced translocations) nsolution: mFISH n ncannot detect overall ploidy changes, e.g. tetraploid tumor nsolution: use in conjunction with regular FISH n nrequires minimally 50 % aberrant cells for reliable results nsolution: HR-CGH, microarrrays n High Resolution Comparative Genomic hybridization (HR-CGH) § Kirchhoff et al., 1997 § the same principles and laboratory processing as CGH § different data interpretation based on dynamic standard reference intervals – special software § genome resolution is about 4 Mbp § abnormal cell detection limit is about 30 % Modifications of CGH Array-CGH § Solinas-Toldo et al. 1997 § based on principle of CGH § the chromosomes (CGH) are replaced by separated clones (array-CGH) § miniaturized array of DNA (genetic material) Modifications of CGH CGH array-CGH evolution The origin of clones BAC, PAC, c-DNA clones, oligonucleotides sciFLEXARRAYE1 lsca_104b_aCGH Array-CGH Advantages and disadvantages of array-CGH nadvantages ndetects and quantifies DNA copy number gains and losses throughout an entire genome in a single analysis nprecise aberration locating n ndisadvantages ndoes not detect balanced rearrangements (translocation, inversion) ndoes not detect ploidy changes nvery expensive method n n n n MLPA Multiplex Ligation-dependend Probe Amplification nsensitive method able to detect differences in one nucleotide ndetects changes of copy number in 45 sequences in one reaction nsimple – all the reaction takes place in one test tube nrelatively cheap method PCR primer Y Hybridization sequence PCR primer X inserted sequence (specific for each probe) Hybridization sequence Syntetic oligonukleotide 50-60 bp M13-derivated oligonukleotide 60-450 bp MRC Holland, www.mlpa.com MLPA principle n Pacient Refferent Advantages and disadvantages of MLPA nadvantages nsensitive nspecific nmultiplex nsimple ncheap n ndisadvantages nhigly sensitive to contamination ntime difficulty nthe aberation have to occur in 50% of cells nsome mutations or polymorphismus can lead to false results 5. Integrated laboratory of molecular cytogenetics, Brno logo-FNB LMC http://www.cba.muni.cz/cytogenlab City of Brno Brno, the cradle of genetics opatství mendel88 Augustinian monastery in Brno – place of G. J. MENDEL´s work Who are we? n nDept. of Genetics and Molecular Biology, Inst. of Experimental Biology, Faculty of Science, Masaryk University n n Dept. of Medical Genetics, University Hospital Brno n nUniversity Research Centre – Czech Myeloma Group Brno logo_fnb logo_sci Integrated laboratory of molecular cytognetics is an integrated clinical and research centre, which is a result of co-operation among: Department of Medical Genetics, University Hospital Brno: the centre for genetic investigation for South Moravia region dnbrno Praque Brno Genetic consulting Laboratory of DNA diagnosis Cytogenetic laboratories prenatal oncogenetic molecular cytogenetic postnatal vyzkum-2 karyotyp-klasicka-cytogenetika lmc-olg-small laborator_molgen1 What is our interest? nThe main interest of the Integrated laboratory is the research of chromosomal aberrations using molecular cytogenetic techniques. n fish Methods § Fluorescence in situ hybridization (FISH) § Spectral karyotyping (SKY) § Comparative genomic hybridization (CGH) § High resolution CGH (HR-CGH) § Array-CGH (Agilent) § MLPA pokus88d Obr 493-04 t(11-14)fotka1 2Lfe-screen-agilent The equipment nClassical Cytogenetics, FISH, CGH/HR-CGH nMicroscopes – Olympus BX61 nCCD cameras Voskuhler nDigital Image Analysis System (LUCIA, LIM Ltd.): nLUCIA-KARYO nLUCIA-FISH nLUCIA-CGH/CGH Advanced Statistics n nSystem for SKY (SKY View – Applied Spectral Imaging Ltd, Israel) nSystem for array-CGH: Agilent Scanner nSystem for MLPA: capillary electrophoresis nBeckman Coulter P1010088 image002 P1040907 Molecular cytogenetic investigations at Department. Of Medical Genetics n nPrenatal cytogenetic diagnosis n nPostnatal cytogenetic analyses n nCancer cytogenetic analyses Prenatal cytogenetic analyses nAneuVysion Assay Kit (Abbott Vysis) nMix1: nCEP 18 Sp. Aqua nCEP X Sp. Green nCEP Y Sp. Orange nMix 2: nLSI 21 Sp. Orange nLSI 13 Sp. Green n nMicrodeletion syndromes (DiGeorge) amniocenteza 2 § Uncultered and cultured amniotic cells, fetal blood, chorion villi § I-FISH Aneuvision Prenatal cytogenetic analyses PV 1 PV 2 PV 6 18a t(13;15) trizomy of chr. 18 Normal cells Postnatal cytogenetic analyses § Peripheral lymhocytes, buccal swab § FISH, CGH, HR-CGH, array-CGH, MLPA, SKY § Microdeletion syndromes – FISH probes, MLPA kits P245, P297 DiGeorge syndrome Prader-Willi/Angelman syndrome Williams-Beuren syndrome 1p36 microdeletion syndrome § Subtelomeric screening – MLPA kits P036, P070 (MRC-Holland), ToTel Vysion kit (Vysis) § Origin of marker chromosomes – CGH, SKY, WCP FISH probes § Identification and specification of numerical and structural aberrations – CGH, SKY § Detection of gonosomal mozaics – FISH (X/Y probes) in infertile couples or gonosomal syndromes P1040911 ToTel Vysion Kit, Abbott-Vysis DiGeorge Taborska Postnatal cytogenetic analyses SKY: marker chromosome identification (chr. 11) FISH: deletion of (22)(q11.2) (DiGeorge syndrome) HERM http://mesta.obce.cz/VismoOnline_ActionScripts/Image.aspx?id_org=15614&id_obrazky=4514 How do we proceed? normal MLPA array-CGH FISH FISH specific syndrome aberation Karyotype G-banding normal confirm Case interpretation Patient : del(4p)dup(8p) nBorn in 2001 nClinical symptoms: n nhard PMR nfacial dysmorphy nstigmata nhypertelorism nhemangiom on right eye lid n Examination nkaryotype: 46,XY,der(4) nHR – CGH: negative nMLPA: P036B del(4p)dup(8p) n P070 del(4p)dup(8p) n Deletion confirmation by the FISH method Array CGH – confirmation of the aberation dup(8)(p23.2pter) ~ 6.5 Mb del(4)(p16.2pter) ~3.7 Mb