Cytogenetics & molecular cytogenetics Faculty of Medicine, Masaryk University University Hospital Brno Mgr. Marketa Wayhelova, PhD Mgr. Marta Navarikova WARNING •This presentation is intended exclusively for educational purposes • •Every form of misuse, including copying, distribution and sharing on public online platforms or social media (YouTube, Facebook etc.) is strictly prohibited and can be punished WHAT ARE WE GOING TO TALK ABOUT? 1.Department of Medical Genetics, genetic laboratories 2.What is cytogenetics 3.History 4.Chromosome morphology and aberrations 5.Molecular cytogenetics and its techniques 6.Case interpretation 7.Our laboratory and work 8. DEPARTMENT OF MEDICAL GENETICS •Medical specialists providing highly specialized and complex genetic counselling for different types of patients and diagnoses •infertility, recurrent abortions •preimplantation and prenatal diagnostics (abnormal screening) •developmental delay, intellectual disability, congenital somatic abnormalities, monogenic disorders.. (solitary or familial) •Familial segregation of various types of disorders (chromosomal abnormalities, monogenic disorders, etc.) •Familial occurrence of cancers •Oocyte and sperm donors •…… •Clinical diagnoses must by confirmed on the molecular level (chromosomes, DNA)! Services and types of analyses Ambulance of clinical genetics Laboratories of cytogenetics Laboratories of molecular diagnostics patient chromosomes DNA/RNA Molecular genetics analyses genealogy Cytogenetics analyses Classical and banding cytogenetics Molecular cytogenetics 1. WHAT IS CYTOGENETICS? •Cytogenetics 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. A BRIEF HISTORY OF (CYTO)GENETICS •1866 Gregor Johan Mendel – Experiment in Plant Hybridization •Father of genetics •Defined the basic principals of heredity (principle of segregation and combination) •During his life, his work was ignored • •Later, Mendel´s work was rediscovered •1910 Thomas Hunt Morgan proved that genes are located on chromosomes (using Drosophila) •1953 James Watson and Francis Crick determined DNA structure •1956 Tjio, Levan – Human chromosome number is 46 HISTORY OF HUMAN CYTOGENETICS •„Dark Ages“ - the development and improvement of tissue culture techniques •„Hypotonic Period“ • - hypotonization of cell samples (1951 - 0,075 m KCl) - utility of phytohaemagglutinin (PHA) - stimulation of peripheral blood lymphocytes - 1960 •„Trisomy Period“ - trisomy of chromosome 21-1959 •The first deletion syndrome - "Cri du chat" - 1963 •„Banding Area“ - chromosome banding techniques 1968 – 1970 •„Molecular Area“ • - in situ hybridization technique – 1970 •FISH – 1986 •Comparative genomic hybridization (CGH) – 1992 •spectral karyotyping (SKY), multicolor FISH (M-FISH) – 1996 •m-banding – 2001 •array-CGH (molecular karyotyping) 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 • (ISCN 1978; ISCN 2016 in present) http://cdn01.bookadda.com/bk_images/537/9783318022537.jpg short (p-) arm long (q-) arm centromere chromatid telomere telomere repetitive (alpha-satelite) sequence 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 *note: telocentric chromosomes are not present in human karyotype Rotation of karyotypy057 CHROMOSOME STAINING •Classical painting •using Giemsa Romanowski solution •Detection of acquired chromosome aberations • •G – bands •using trypsin, salty solution and Giemsa •each chromosome has characteristic pattern • (dark bands – A/T rich, light bands – G/C rich) •Detection of congenital chromosomes aberations (mostly > 10 Mb) • •R – bands •using salty solution of different • pH and temperature •reverse to G - bands • • chrom METHODOLOGY FOR CLASSICAL AND BANDING CYTOGENETICS •To drop processed cell suspension with mitoses on microscopic slides •Staining to obtain chromosomal bands •1) digestion the specimen in trypsin solution •2) staining in Giemsa-Romanovski dye •Evaluation in light microscope with CCD camera •-> to get karyotype (computational analysis of image) • přednáška 090 přednáška 022 přednáška 065 přednáška 056 NORMAL MALE KARYOTYPE (46,XY) norm mužský HUMAN KARYOTYPE – 7 GROUPS OF CHROMOSOMES A B C D E F G HUMAN SOMATIC CELL CONTAINS: •23 pairs or 46 chromosomes •22 pairs of autosomes (1-22) •1 pair of gonosomes (XX or XY) •7 groups ordered according to chromosome size and morphology •A – large metacentric chromosomes •B – large submetacentric chromosomes •C – middle-sized submetacentric chromosomes and chromosome X •D – large acrocentric chromosomes •E – small meta- to submetacentric chromosomes •F – the smallest metacentric chromosomes („ribbons“) •G – small acrocentric chromosomes and chromosome Y KARYOTYPES OF ANIMALS (EXAMPLES) http://users.unimi.it/citozoo/cattle%20female.png Cattle (2n=60) http://users.unimi.it/citozoo/doga%20male.png Dog (2n=78) http://users.unimi.it/citozoo/T6%20cariotipo%20femmina%20cavalla%20female%20horse%20karyotype.jpg Horse (2n=64) CHROMOSOME ABERRATIONS •AUTOSOMES •1. Structural chromosomal aberrations •Polymorphisms •different lenght of chromosomes in homologous pair •no phenotype effect •Inversion •pericenric – including centromere •paracentric – does not include centromere •usually has no phenotype effect in its carrier •Ring chromosomes •breaks on both chromatides and their connection •loss of telomeric parts •intellectual and physical impairment •always newly created • ringchromosome http://ghr.nlm.nih.gov/handbook/illustrations/ringchromosome ring Heb 6 dup správné Dočkalová 06 CHROMOSOME ABERRATIONS •Deletion = loss of part of chromosome ->unbalanced karyotype •terminal – one break and loss of a terminal part •interstitial – two breaks and loss of a part between centromere and terminal part •deletion syndromes - examples: •Wolf-Hirschhorn syndrome (4p deletion), Cri-Du-Chat syndrome (5p deletion) •microdeletion syndromes - examples: •Prader-Willi syndrome; 15q11-q13 deletion •DiGeorge syndrome; 22q11.2 deletion •Angelman syndrome; 15q11-q13 deletion •Williams-Beuren syndrome; 7q11.23 deletion •Duplication = gain of chromosomal part -> unbalanced karyotype •Typically less harmful than deletions •Insertion •inserted part can be in the same or inverted position • • • • • •Translocation •reciprocal •mutual exchange between two or more nonhomologic chromosomes •balanced - no phenotype effect for carriers •genetic risk of unbalanced genomes in carrier‘s offspring • •Robertsonian •between two acrocentric chromosomes •breaks in the area of centromeres and deletion of short arms •centric fusion of the remaining arms •balanced – normal phenotype •genetic risk for offspring • •Simple •One break in the arm of one chromosome •Fusion of the broken part with another chromosome • • • • translocation%20-%20reciprocal http://www.larasig.com/node/3628 robertsonian-translocation-13045_3 http://drugline.org/medic/term/robertsonian-translocation/ •Marker chromosomes •Small supernumerical chromosomes (with centromere) •Often in mosaic form •Sometimes difficult to identify the origin • •Isochromosomes •Metacentric chromosomes – one arm is lost, the second one is duplicated • •Dicentric chromosomes •Breaks on two chromosomes •Fusion of parts with centromeres •Acentric fragment is lost • izoXq dic 13,14 bez šipky dic (13;14) M Moore, Charleen & Best, Robert. (2001). Chromosomal Genetic Disease: Structural Aberrations. 10.1038/npg.els.0001452. CHROMOSOMAL ABERRATIONS – OVERVIEW CHROMOSOME ABERRATIONS •2. Numerical •Trisomy •21 chromosome trisomy – Down syndrome •18 chromosome trisomy – Edwards syndrome •13 chromosome trisomy – Patau syndrome • •Triploidy •69 XXX, 69 XXY •nonviable •mosaic triploidy – mental retardation, syndactyly, abnormal genitals, lateral asymetry • • CHROMOSOME ABERRATIONS •GONOSOMES •Chromosome Y •structural aberrations – very rare •numerical aberrations •47, XYY – supermale syndrom •Chromosome X (males) •Numerical aberration •47, XXY – Klinefelter syndrom •Chromosome X (females) •numerical aberrations •45, X – Turner syndrom •47, XXX – XXX syndrom •Fragile X – FRA-X •the most common cause of intellectual disability (excluding trisomy 21) •nonspecific phenotype (intellectual impairment, facial dysmorphology, ...) • • • • CHROMOSOMAL MOSAICISM •Chromosomal aberrations are mostly in all human cells • •Mosaicism = 2 or more cell lines with different karyotype in human body • •Nondisjunction in early post-zygotic mitotic division (prenatal period) •Numerical more frequent than structural • •Most often in gonosomal aneuploidies (-> spontaneous abortions, infertility) •Example: 45,X[6]/47,XXX[4]/46,XX[190] • •Autosomal aneuploidies •Example: mosaic form of Down syndrome • 47,XY,+21[172]/46,XY[28] DOWN SYNDROME (47,XX OR XY,+21) •1866 J.L.Down •IQ 25-50 •short stature •round face •short neck •mongoloid eyes •epicanthus •wide nose root and flattened nose •small mouth, large tongue, small teeth •single transverse palmar crease •heart diseases • Down_Brushfield_spots Down_simian_crease +21a EDWARDS SYNDROME (47, XX OR XY,+18) •growth retardation •microcephaly •dolichocephaly – elongated head •cleft palate •low-set malformed ears •specific finger holding •structural heart defect • at birth •survive only few months • Edwards peri215 +18 kar PATAU SYNDROME (47,XX OR XY,+13) •severe somatic retardation and neurodevelopmental disorders •microcephaly •trigonocephaly •cutis aplasia •congenital brain defects •cleft palate •hexadactyly •kidney defects • domhoff4 +13 kar PRADER-WILLI SYNDROME (DEL 15Q11-Q13) •deletion of paternal locus 15q11-q13 •low fetal activity •hypotonia •excessive weight gain, hyperphagia •short stature •hypogonadism •intellectual disability •hypopigmentation •skeletal development delay (acromicria) Chaloupková ANGELMAN SYNDROME (DEL 15Q11-Q13) •deletion of maternal locus15q11-q13 •severe intellectual impairment •hypotonia •epilepsy, seizures •hypopigmentation •hyperactivity •speech absence •prominent scull shape (mandibular shape, microcephaly, flat back of head..) •„happy puppet“ syndrome •movement/balance disorder • Kubíková DIGEORGE SYNDROME (DEL 22Q11.2) •low-set malformed ears •small mouth and lower jaw •narrow eyelids •submucosal or visible cleft palate •hypocalcemia •interrupted aortic arch •cardiac abnormality – tetralogy of Fallot •incomplete ventricular septum •right-to-left shunt of aorta •left ventricle hypertrophy •lung 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 7Q11.23) •developmental delay •mental disability •failure to thrive •heart defects (heart murmur, narrowing of main blood vessels) •flattened nasal bridge •widely spaced teeth •hypercalcemia •gastrointestinal problems •urinary difficulties • • • 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 KLINEFELTER SYNDROME (47,XXY) •tall stature •less facial and body hair •female distribution of body fat •hypogonadism (decreased testicular hormon function) •infertility •gynecomastia (increased breast tissue) •mild intellectual impairment •variations: 48, XXYY; 48, XXXY; 49,XXXXY • 3 klein TURNER SYNDROME (45,X) •lower birth length and weight •low hairline •pterigyum •broad chest, widely spaced nipples •small growth •infertility, amenorrhea •coarctation of the aorta •webbed neck •lymphedema • • turner Turner File:Puffy feet.JPG THE LINK BETWEEN KARYOTYPING (K) AND MOLECULAR CYTOGENETICS (MC) •Using MC methods to confirm and specify pathological chromosomal aberrations detected by G-banding K •Aneuploidies -> FISH •Structural chromosomal rearrangements -> FISH, array-CGH • •MC methods can detect very subtle chromosomal rearrangements, which escaped detection using G-banding K (due to its low resolution > 5-10 Mb) 4. MOLECULAR CYTOGENETICS •Interconnection and combination of approaches of classical cytogenetics and molecular biology • •Utility of the latest knowledge of molecular biology, microscopy and computer image analysis to study the structure and properties of chromosomal changes • •Ability to analyse both numerical and structural chromosomal imbalances unidentified classical cytogenetic techniques • •does not require the presence of mitosis for most applications • •sources of material for cytogenetic investigation •peripheral blood •samples from different tissues •amniotic fluid cells, chorionic villi, placenta umbilical cord blood •bone marrow •samples of solid tumors • • nádory 003 dřeň 002 peripheral blood solid tumor bone marrow MOLECULAR CYTOGENETICS •The key interest of the laboratory of molecular cytogenetics is the identification and analysis of chromosomal aberrations using molecular cytogenetic approaches • fish FISH FLUORESCENT IN SITU HYBRIDIZATION • • obr1 FISH •detection of the fluorescent signals through microscope equipped with specific fluorescent filters •material •cultivated peripheral blood •cultivated bone marrow •cultivated amniotic fluid cells •uncultivated amniocytes •tumor and bone marrow prints •we determine: 1.presence of signals 2.number of signals 3.position of signals •the use of FISH • - clinical cytogenetics • - oncocytogenetics • - human genome mapping • typy sond typy sond delece exonu počet signálů translokace METHODS § Fluorescence in situ hybridization (FISH) § Spectral karyotyping (SKY) or M-FISH § Comparative genomic hybridization (CGH) or High resolution-CGH (in past) §array-CGH § MLPA pokus88d Obr 493-04 t(11-14)fotka1 2Lfe-screen-agilent THE EQUIPMENT •Classical Cytogenetics, FISH, CGH/HR-CGH •Microscopes – Olympus BX61 •CCD cameras Voskuhler •Digital Image Analysis System (LUCIA, LIM Ltd.) •LUCIA-KARYO •LUCIA-FISH •LUCIA-CGH/CGH Advanced Statistics • •System for SKY (SKY View – Applied Spectral Imaging Ltd., Israel) •System for array-CGH: SureScan Microarray Scanner (Agilent Tech.) •System for MLPA: capillary electrophoresis (Beckman Coulter) P1010088 image002 P1040907 MOLECULAR CYTOGENETIC INVESTIGATIONS (LABORATORIES OF CYTOGENOMICS) APPLICATIONS OF FISH • •Prenatal cytogenetic diagnosis • •Postnatal cytogenetic analyses • •Cancer cytogenetic analyses PRENATAL CYTOGENETIC ANALYSES •FAST FISH Prenatal Enumeration Probe Kit (Cytocell) •Mix1: •CEP 18 Sp. Aqua •CEP X Sp. Green •CEP Y Sp. Orange •Mix 2: •LSI 21 Sp. Orange •LSI 13 Sp. Green • •Microdeletion syndromes (DiGeorge, Prader-Willi, Angelman, Williams-Beuren, ...) Aneuvision amniocenteza 2 § Uncultered and cultured amniotic cells, fetal blood cells, chorionic villi cells § interphase FISH (I-FISH) for aneuploidies § metaphase FISH (in specific cases) Prenatal cytogenetic analyses PV 1 PV 2 PV 6 18a t(13;15) trisomy of chr. 18 Normal cells Example: FISH on uncultured and cultured cells/mitoses POSTNATAL CYTOGENETIC ANALYSES § material: peripheral lymphocytes, buccal swab § methods: FISH, array-CGH, MLPA § Microdeletion syndromes – FISH probes, MLPA kits P245, P297 (targeted detection) •DiGeorge syndrome •Prader-Willi/Angelman syndrome •Williams-Beuren syndrome •1p36 microdeletion syndrome, etc. § Subtelomeric screening – MLPA kits P036, P070 (MRC-Holland), FISH ToTel Vysion kit (Vysis) § Origin of marker chromosomes – array-CGH, WCP FISH probes § Identification and specification of numerical and structural aberrations – array-CGH (in specific cases SKY) § Detection of gonosomal mosaicism – FISH (X/Y probes) in infertile couples or gonosomal syndromes P1040911 ToTel Vysion Kit, Abbott-Vysis DiGeorge Taborska Postnatal cytogenetic analyses SKY: identification of marker chromosome (chr. 11) FISH: deletion of (22)(q11.2) (DiGeorge syndrome) HERM FISH: 46,XX/46,XY (mosaicism) Cultivated and uncultivated solid tumors (tumour prints) • FISH, M-FISH/SKY, CGH Children solid tumours •FISH: targeted analysis (panel testing) •Neuroblastoma –MYCN amplification, 1p36 deletion, gain 17q, 11q deletion; •Medulloblastoma – MYCN, MYCC amplification •array-CGH: whole genome screening of unbalanced chromosomal aberrations (and losses of heterozygosity) • 4g N-myc CANCER CYTOGENETIC ANALYSES – SOLID TUMOURS ADVANTAGES AND DISADVANAGES OF FISH •advantages •does not require the presence of mitoses (for most applications) •quick assessment of big amount of cells • •disadvantages •does not provide whole genomic view •can detect only a specified locus or a limited number of loci (using fluorescent-labeled DNA probes) • • •Microscope equipped with 2 fluorescent filters (SKY, DAPI) • •fluorochromes (FITC, Rhodamin, TexasRed, Cy5, Cy5.5))scanned by one filter, based on a wave lenght each chromosome pair is coloured pseudocolours • SKY SPECTRAL KARYOTYPING Image Acquisition with SkyVision™ Display Image Image analysis using SkyView Classified Image The SkyView spectral karyotyping software is able 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 • specdapi2 ADVANTAGES AND DISADVANTAGES OF SKY •advantages •detects balanced rearrangements •detects aberations in one step •cryptic translocations and insertions • marker chromosomes • redundant material with unknown origin • complex rearrangements • •disadvantages •need of quality mitoses •succesful hybridisation •expensive method • • CGH - COMPARATIVE GENOMIC HYBRIDIZATION HR-CGH – HIGH-RESOLUTION CGH ARRAY-CGH •improvement of FISH technique to measure DNA gains or losses throughout the entire genome • •detection of unbalanced chromosomal changes (gains or losses) throughout an entire genome in one hybridization reaction • •is based on comparison of two genomes • FISH normal DNA → select DNA → make probe (limited number of targets in one hybridization) → label abnormal target → abnormal target identified CGH/HR-CGH/array-CGH normal DNA → no DNA selection → make probe (entire genome) → quantify on normal target → abnormal genome quantified • • Good quality DNA isolated from • peripheral blood • bone marrow • solid tumour • amniocytes, CVS, ... Equipment: •Fluorescent microscope (filters DAPI, Spectrum Green, Spectrum Red) • Sensitive CCD kamera • Computer with software for CGH analysis and data interpretation, (LIM, Czech Republic) – CGH/HR-CGH • •Hybridization oven, microarray scanner •Computer with software for array-CGH analysis and data interpretation – array-CGH • • Requirements P1040823 P1040826 P1040842 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 At least 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. • ADVANTAGES OF CGH •detects and quantifies DNA copy number gains and losses throughout an entire genome in a single analysis • •does not require cell culturing and metaphases from test tissue • •is 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 • •in 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 •low sensitivity: about 10 Mb for single copy changes •solution: array-CGH • •does not detect balanced rearrangements (inversions, balanced translocations) •solution: mFISH • •cannot detect overall ploidy changes, e.g. tetraploid tumor •solution: use in conjunction with regular FISH • •requires minimally 50 % aberrant cells for reliable results •solution: HR-CGH, array-CGH • 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 analytical software § genome resolution is about 4 Mb § abnormal cell detection limit is about 30 % MODIFICATIONS OF CGH Array-based comparative genomic hybridization (array-CGH) •Solinas-Toldo et al. 1997 •based on principle of CGH •current routine approach of whole-genome screening of unbalanced chromosomal aberrations (including those of submicroscopic size < 5-10 Mb) •nowadays fully replaces classical CGH and HR-CGH analysis •the chromosomes on slide (CGH) are replaced by separated clones (array-CGH) (BAC – bacterial artificial chromosome, PAC – phage artificial chromosome, nowadays in most designs – oligonucleotides (miniaturized array) CGH array-CGH evolution The origin of clones BAC, PAC, c-DNA clones (in past), oligonucleotides sciFLEXARRAYE1 GENERAL PRINCIPLE OF ARRAY-CGH •Effective approach of the whole-genome screening of unbalanced chromosomal rearrangements in one hybridization reaction •Co-hybridization of differently labeled DNA samples (patient‘s DNA and reference DNA) on DNA microarray covered by a large amount of oligonucleotide fragments (representing whole genome) •Losses and gains of genetic material in patient‘s DNA are assessed from spots with abnormal ratios of signal intensities • •Who are the most suitable patients? •Individuals with neurodevelopmental disorders (intellectual disability, autism spectrum disorders), multiple congenital abnormalities, facial dysmorphology, … •Prenatal samples – abnormal pregnancy (prenatal screening) •Fetal tissue (abortions) •Tumor tissues (solid tumors, bone marrow, …) lsca_104b_aCGH Brief schema of sample processing using array-CGH Types of DNA microarrays (Agilent Technologies) ü1X1M ü2X400K ü4X180K ü8X60K (parametres on www.agilent.com) Agilent Feature Extraction for Cytogenomics – microarray scan 4X180K CGH+SNP Agilent Feature Extraction for Cytogenomics – microarray scan 8X60K CGH Agilent Feature Extraction – Microarray scan and quality control (QC Report) AGILENT GENOMIC WORKBENCH – COPY-NUMBER VARIATION EVALUATION (GLOBAL VIEW ON CHROMOSOMES –> CHROMOSOME 17 –> DETAIL OF 17Q11.2 MICRODELETION) THE INTERPRETAION OF COPY-NUMBER VARIATIONS MUST BE PERFORMED IN THE CONTEXT OF: 1.Patient‘s phenotype 2.Analysis of parental genomes -> to assess CNVs origin (de novo or inherited CNV from parent with normal/abnormal phenotype) 3.Information in databases of genetic variants (UCSC, DECIPHER, DGV...) and about genes in CNVs (database OMIM) 4.Information in relevant scientific and clinical literature (Pubmed…) • • • • • • • UCSC GENOME BROWSER CNV genomic position Patients in DECIPHER database (click to open) OMIM genes (click to open) DECIPHER HTTPS://DECIPHER.SANGER.AC.UK/ CNV details Genes within the CNV OMIM (WWW.OMIM.ORG) WRITE GENE NAME External databases (click to open) Associated pathologies Details about the gene DETAIL ABOUT THE GENE (jump on chapters directly) ADVANTAGES AND DISADVANTAGES OF ARRAY-CGH •advantages •detects and quantifies DNA copy number gains and losses throughout an entire genome in a single analysis •resolution depends on microarray platform of the choice (from ~50 kb) •ability to detect mosaicism < 30% •precise aberration location (based on aberrant clone positions) • •disadvantages •unable to detect balanced rearrangements (translocation, inversion) •unable to detect ploidy changes •very expensive method •highly qualified and experienced laboratory stuff • • • MLPA MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION •sensitive method able to detect differences up to nucleotide level •detects changes of copy number in 45 sequences in one reaction •simple – all the reaction takes place in one test tube •relatively cheap method PCR primer Y Hybridization sequence PCR primer X inserted sequence (specific for each probe) Hybridization sequence Synthetic oligonucleotide 50-60 bp M13-derivated oligonucleotide 60-450 bp MRC Holland, www.mlpa.com MLPA PRINCIPLE (IN BRIEF) Patient Reference ADVANTAGES AND DISADVANTAGES OF MLPA •advantages •sensitive •specific •multiplex •simple •cheap • •disadvantages •highly sensitive to contamination •time difficulty •the aberrations have to occur in 50% of cells (mosaicism) •mutations or polymorphisms can lead to false results 5. CASE INTERPRETATION Zemanovafoto sejmout0002 46,XX,add(1) § girl, born in 2002 § dg: stigmatization – mongoloid eye position, hyperplastic gingival mucose membrane, atypical chest and tummy § mother 46,XX, inv(9), father 46,XY,add(1)[87]/46,XY[13] CASE INTERPRETATION 1 CGH: rev ish enh (11p15-pter) – unbalanced translocation ZemanovaWCP FISH: der(1)t(1;11) Gazda_Patrik_04_1 Gazda2 45,XY,-22,der(14) Vasickova 46,XX,der(14)t(14;22)(q32.3;q11.2) CASE INTERPRETATION 2 •boy, born in 2004 •heart defect, facial dysmorphology mother_wcp t(14 22) mother_DiG son_DiG mother_del tel14 45,XY,der(14)t(14;22)(q32.3;q11.2) DiGeorge sy 46,XX,der(14)t(14;22)(q32.3;q11.2) CASE INTERPRETATION 3 •boy, born in 2018 •severe lissencephaly, global developmental delay 17p13.3 microdeletion (~280 kb) GENOTYPE-PHENOTYPE CORRELATION (OMIM DATABASE) GENOTYPE-PHENOTYPE CORRELATION (OMIM DATABASE) GENOTYPE-PHENOTYPE CORRELATION (PUBMED) THANK YOU FOR YOUR ATTENTION