Human Cytogenetics logo-FNB LMC 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. 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 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 Výsledek obrázku pro what is a chromosome Walther Flemming (1843 – 1905) published first illustrations of chromosomes in1882 „chromo-soma“ – 1888 Waldeyer -Hartz Chromozomes and DNA n n1924 - Feulgen and Rosenback ndemonstrated the presence of DNA in n chromosomes by histochemical staining n nthe first chromosome studies were ncarried out on insects (Drosophila) and nplants (maize, broad beans) n n1953 - Watson and Crick - deciphering the nstructure of DNA n nbut...how many chromosomes have humans? Joe Hin Tjio (1919 - 2001) Tjio, T.H., Levan, A.: The chromosome number of man. Hereditas 42:1, 1956 Albert Levan (1905 -1998) 1956 – the right number of human chromosomes Tjio0006 Humans have 23 pairs of chromosomes in the nucleus of each of their body cells, for a total of 46 chromosomes. Tjio0006 Tjio0003 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 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 Human Karyotype A B C D E F G Chromosomes and few numbers nGenomic DNA of eukaryotes - chromosomes – n linear double helix DNA 3 billion pb nThe human haploid genome contains: n1000 mm of DNA = 2 m of DNA in a diploid cell n = 23 molecules of DNA n na typical chromosome of a eukaryotic cell contains 1 to 20 cm of DNA nchromosome size in mitosis 1 to 10 um nsum of all chromosome sizes - 115 um Human chromosomes and genes Microscopic and submicroscopic structure of chromosomes 3T3-interphase zlomy 2 Interphase - chromatin a)Euchromatin (eu = true) b)Heterochromatin (hetero = different) Interphase - despiralization Mitosis, meiosis -spiralized form Characteristics of chromatin nEuchromatin ndespiralized in interphase nspiralized in mitosis ncontains structural genes (unique sequences) ngene expression nHeterochromatin n a) Constitutive repetitive sequences - satellite DNA, does not contain active genes (tandem repeats), somewhat spiralised in interphase – staining in humans especially in centromeres, Yq chromosome n nb) Facultative = structurally euchromatin, behaves like heterochromatin n inactive X = sex chromatin = Barr's body = X chromatin n one of the two X chromosomes in female mammals is inactive n Heterochromatin - later replication in S phase (inactive X at the end of S) n n Barr_Bodies Composition of chromatin nComposition of chromatin: •DNA, RNA (30 %) •Proteins (70%) n n nHistones = basic proteins (20-30% arginine and lysine): evolutionarily conserved, positively charged, important in interactions with DNA n H1, H2A, H2B, H3, H4 - pack chromosomes, regulate transcription, gene expression.... nNon-histone proteins = acidic proteins (e.g. HMG proteins) - regulation of gene expression n Obrázek1 Folding of DNA – from strand to chromosome Nucleosome Positioning and Its Role in Gene Regulation in Yeast | IntechOpen solenoid Chromosome morphology 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 Centromere (primary constriction) is an important part of chromosomes •holds together sister chromatids - cohesin proteins •essential for chromosome segregation • visible by C-banding ! •DNA of centromeres is composed of blocks of constitutive heterochromatin = (pericentric heterochromatin) repetitive sequences create satellite DNA e.g. in humans alpha satellite DNA - unit 171 pb - blocks of 300 to 5000 kb) + binding sites for centromeric CENP proteins Kinetochore - protein complex, microtubule binding with centormeres centromere disorders - nondisjunctions, acentric or dicentric chromosomes ! Centromere Centromere and kinetochore •zkracování či prodlužování vláken dělícího vřeténka – pohyb chromozomů Telomeres nphysical ends of eukaryotic chromosomes - they have unique properties ! nin humans, made up of proteins and tandem repeats of DNA (TTAGGG)n vs. (CCCTAA)n - 500 to 3000 repeats ! n accompanied by blocks of heterochromatin n nAbsence of telomeres → chromosome rearrangements - fusions („sticky ends“) leading to translocations, circular or unstable dicentric chromosomes n Significance of telomeres nprotection of chromosome ends against the action of exonucleases (ends of broken chromosomes) protection against fusion with other chromosomes – t-loop... nmaintain the architecture of the interphase nucleus npairing of homologous chromosomes in meiosis nsubtelomeric regions - most genes.... nallow replication of the ends of linear DNA - gap at the 5' end.. (ends are replicated by the enzyme telomerase ...absent in somatic cells...but active in sex cells, stem cells, tumors ...maintains telomere length) ncell aging - no telomerase - shortening telomere length during division Single strand 50 - 500 pb rich overlap at 3´ end rich in guanine - t-loop - end protection Antioxidants | Free Full-Text | Treating Cancer by Targeting Telomeres and Telomerase | HTML Telomeres shorten during each cell division of 50 - 200 bp nShort telomeres – blocking of cell cycle Shortenig of telomers and age Telomery3 n Progeria – rapid ageing short telomers The Hayflick limit determines the maximum number of divisions a cell undergoes before it dies. Human fibroblasts in culture can divide a maximum of 50 to 70 times, after which they age and die. ring dicent Consequences of telomere errors = chromosomal aberratios n Identification of chromosomes Chromosome staining nClassical staining nusing Giemsa Romanowski solution ngained chromosome aberration 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 Giemsa-Romanowski staining http://www.baso.com.cn/en/images_1/2016081039862569.jpg Characterization of G banding of human chromosomes •bands on chromosomes reflect the structure of the genome and its functional organization • •each band contains 5 to 10 Mb (resolution limit !!!) • •Giemsa positive bands - dark (G+, rich in AT pairs, late replicating chromosome regions •poor in genes • •Giemsa negative bands - light (G-, rich in GC pairs, early replicating chromosome regions, rich in genes G – banding (mitosis and descritption) G-banding classification pattern n Preprocessing G-banded metaphase: towards the design of automated karyotyping | SpringerLink c-pruhy C - banding detects regions of centromeric heterochromatin ! R-banding of human chromosomes Nonrobertsonian translocation t(6;11) is associated with infertility in an oligoazoospermic male - Fertility and Sterility R-banding karyotype of the KG-1a cell line as mentioned in Materials... | Download Scientific Diagram Clinical indications for investigation of karyotypes nearly growth and development problems (failure to thrive, delayed growth, short stature, bilateral genitalia, mental retardation, neurodevelopmental disorders) nstillbirth and neonatal death npartners treated for infertility and partners with repeated spontaneous abortions nsperm donors and egg donors nfamily history (known chromosomal abnormality in first-degree relatives) npregnancy in women of advanced age, pregnant women with pathological findings in biochemical screening or ultrasound findings npersons with prolonged exposure to environmental pollutants n 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 nthe images are captured by charge-coupled device (CCD) imaging and analyzed by using an interferometer attached to a epifluorescence microscope nImage processing software then assigns a pseudo color to each spectrally different combination, allowing the visualization of the individually colored chromosomes nmicroscope equipped with 2 fluorescent filters (SKY, DAPI) n fluorochromes (FITC, Rhodamin, TexasRed, Cy5, Cy5.5))scanned by one filter, based on a wave lenght each chromosome pair is colored n Resolution 15-20Mbs – complex chromosomal changes SKY (spectral karyotyping) Schematic representation of the steps involved in SKY analysis. (A) The... | Download Scientific Diagram Image Acquisition with SkyVision™ 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 M-FISH technique - (multicolor FISH) (24 whole chromosome DNA painting probes) sky •Analysis of complex chromosomal aberrations • •Each homologous pair of chromosomes is uniquely labeled with five fluorochromes set which are spectrally distinct in different combinations • •The images are captured by band-pass filter sets and defined emission spectra are measured by dedicated M-FISH software. •Resolution ~3–10 Mb in size • Advantages and disadvantages of SKY and M-FISH nadvantages ndetects balanced rearrangements ndetects aberrations in one step ncryptic translocations and insertions n marker chromosomes n redundant material with unknown origin n komplex rearrangements n ndisadvantages nneed of quality mitoses nsuccessful hybridization nexpensive methods 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 the chromosome from which the additional unknown material is derived nmap 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 https://www.researchgate.net/profile/Yi-Wei-Tang/publication/26888549/figure/fig5/AS:27701689822822 5@1443057383356/Agilent-oligonucleotide-microarray-A-Noncontact-inkjet-printing-technology-delivers -a_W640.jpg Agilent Sureprint Technology 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 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