Cytogenetics: Chromosomal Aberrations PDF

CYTOGENETICS Lesson #9: Chromosomal Aberrations 2 Major Categories of Chromosomal Abnormalities Major Categories of Chromosomal Abnormalities 1. Numerical Abnormalities 2. Structural Abnormalities...

CYTOGENETICS Lesson #9: Chromosomal Aberrations 2 Major Categories of Chromosomal Abnormalities Major Categories of Chromosomal Abnormalities 1. Numerical Abnormalities 2. Structural Abnormalities Ø Types of Aneuploidies Numerical Abnormalities Sex Chromosome Aneuploidies Definition Autosomal Aneuploidies Ø Defects involving an abnormality in the number of Polyploidy chromosomes. Ø Chromosome number is higher than 46 but is always Ø Subclassified as: an exact multiple of the haploid chromosome number Euploidy of 23. Aneuploidy Triploidy (3n) – a karyotype with 69 Polyploidy chromosomes Autoploidy Euploidy Ø Condition of having a normal number of structurally normal (46) chromosomes. Tetraploidy (4n) – a karyotype with 92 chromosomes o Most cases are on babies, wherein there Aneuploidy are very low to no chances of surviving. Ø Any abnormal number of chromosomes that is not a Autoploidy multiple of the haploid number (23 chromosomes) Ø 2 Ways Ø Result of nondisjunction → failure of chromosomes Nondisjunction to separate normally during cell division (meiosis) o Meiotic nondisjunction Trisomy – presence of an extra chromosome (47 o Mitotic nondisjunction chromosomes; e.g., Down Syndrome) (occur in early embryo) Monosomy – absence of a single chromosome Genome Duplication (45 chromosomes; e.g., Turner Syndrome – only have X chromosome) Huge damage of nondisjunction in Meiosis I compared to Meiosis II (that can lead to half normality and half abnormality.) Other Term for Numerical Abnormalities Ø Cause of Aneuploidy Ø Hypodiploid – cell fewer than 46 chromosomes Ø Near-haploid – cells have from 23 up to approximately 34 chromosomes. Ø Hyperdiploid – cells have more than 46 chromosomes. Ø High hyperdiploidy – chromosome number of more than 50 Disease Associations Ø Infertility and sterility – unable to get pregnant. Ø Intersexes – born with male and female traits. Ø Multiple congenital malformations – organs are not fully developed. Ø Mental retardation – intellectual disabilities. CYTOGENETICS  CYTOGENETICS Sex Chromosome Aneuploidies Triple X Syndrome (XXX) Sex Chromosome Aneuploidies Ø Triple-X, trisomy X, XXX syndrome, 47,XXX Ø Females: aneuploidy Turner Syndrome (XO) Ø Presence of an extra X Metafemale (Triple-X) chromosome in each Ø Males: cell of a human female. Klinefelter Syndrome (XXY) Ø Unlike most other Jacob Syndrome (XYY) chromosomal conditions there is usually no Ø Sex Chromosomal Aberrations distinguishable difference Aberrations = abnormalities / disorders between women with Defected sex chromosomes triple X and the rest of the female population. Monosomy Trisomy Ø Features: Turner Syndrome Klinefelter Syndrome Menstrual irregularities Metafemale Syndrome Increased risk of learning Jacob Syndrome disabilities, delayed speech, deficient language skills, and delayed development Turner Syndrome (XO) of motor skills. Ø Gonadal dysgenesis Ø Monosomy X Ø Ullrich-Turner Syndrome Ø First described in 1938 by Dr. Henry Turner Ø Occurring in 1 out of every 2,500 girls Ø Features: Short stature Klinefelter’s Syndrome (XXY) Lymphedema (swelling) of Ø 47, XXY, or XXY syndrome the hands and feet Ø A condition in which males Rudimentary ovaries have an extra X sex gonadal streak chromosome. (underdeveloped gonadal Ø Affected individuals have at structures) least two X chromosomes Shortened metacarpal IV and at least one Y chromosome. (of hand) Ø Features: Small fingernails Small Firm Testicles → Characteristic facial Infertility features Low Testosterone Webbed neck Incomplete Masculinization Decreased Libido Enlarged breast tissue. Body shape patterns: o Pear shaped o Tall o Abnormal Proportions o Teeth Abnormality CYTOGENETICS  CYTOGENETICS Jacob’s Syndrome (XYY) Edwards Syndrome Ø Jacob’s syndrome Ø Trisomy 18 / Trisomy E or Edwards Syndrome Ø Phenotype is normal. Ø Presence of an extra 18th chromosome Ø Features: Ø Incidence increases as the mother’s age increases Normal Ø Very low rate of survival → resulting from heart Appearance → abnormalities, kidney malformations, and other tall stature internal organ disorders. Increased Ø Features: vulnerability to Low birthweight ADHD Small, abnormal shaped head (attention deficit Small jaw and mouth Hyperactivity Long fingers that overlap, with disorder) underdeveloped thumbs and Learning disabilities clenched fists Increased vulnerability Low-set ears to autistic spectrum Smooth feet with rounded soles disorders Cleft lip palate Eyes set slightly far apart. Large head infertility Autosomal Numeric Aberration Down Syndrome Ø Trisomy 21, Trisomy G Ø Caused by the presence of all or part of an extra 21st chromosome. Ø Named after John Langdon Down → British doctor who described the syndrome in 1866. Ø Lives the longest among the other syndrome, because Patau Syndrome of the 21st chromosome, the smallest. Ø Trisomy 13 Ø Features: Ø Presence of an extra copy of chromosome 13 → Microgenia (an abnormally small chin) causes numerous physical and mental abnormalities, Unusually round face especially heart defects. Macroglossia (protruding or oversized tongue) Ø Features: Almond shape to the eyes caused by an Small head size epicanthic fold of the eyelid. Extra toes or fingers Shorter limbs Cleft lip or palate Poor muscle tone Heart defects Larger than normal space Holoprosencephaly → the between the big and second brain doesn’t divide into toes two halves; Severe mental retardation. Nasal malformation Hypotelorism (reduced distance between the eyes) or cyclops CYTOGENETICS  CYTOGENETICS Warkany Syndrome Ø Trisomy 8 Ø Presence of extra chromosome 8 Ø Features: Characteristic facial Features: o Elongation of the skull (scaphocephaly) o Prominent forehead o Widely spaced eyes o Deeply set eyes o Broad upturned nose Brain malformations Highly arched or cleft palate Shortened neck with extra skin folds Long slim body with a narrow chest, shoulders, and pelvis Kidney and cardiac abnormalities CYTOGENETICS  CYTOGENETICS Lesson #10: Chromosomal Change the gene dosage of a part of the affected Aberrations Continuation chromosomes. Changing the dosage – lost or duplicated. Cytogenetic Notation International System for Human Cytogenomic Nomenclature (ISCN) Ø International System for Human Cytogenomic Nomenclature (ISCN) Ø Each are of chromosome given number. Lowest number closest (proximal) to centromere Highest number at tips (distal) to Centromere Cytogenetic Banding Nomenclature Structural Aberrations happen due to: Ø Errors during Meiosis Ø Errors during Mitosis Ø Exposure to substances that cause birth d efects (teratogens) Ø 3p22.1 3 – chromosome # Structural Rearrangements p – p or q arm Inversions 2 – region Ø A segment of a chromosome is reversed end to end. 2 – sub region Ø Occurs when a single chromosome..1 – sub-sub region undergoes breakage and Ø XX – female rearrangement within itself. Ø XY – male Ø Can be inherited or it may be a mutation that appears in a child Structural Abnormalities whose family has no history. Definition Ø Type of mutations where a Ø Result from breakage of a chromosome region with sequence of nucleotides in loss or subsequent rejoining in an abnormal the DNA is reversed or combination. inverted. Ø Proteins are the only defected. Ø Sometimes inversions are Ø There are some part/portions of the chromosomes visible in the structure of that may be defected. the chromosomes. 2 General Types Ø Types of Inversions: 1. Balanced rearrangements Pericentric Inversions – chromosomal inversion No loss or gain of genetic chromatin. that involves the centromere. Change the chromosomal gene order but do not remove or duplicate any of the DNA of the chromosomes. Gene order changes – swapping the regions of the chromosomes. 2. Unbalanced rearrangement Gain or loss of genetic material. CYTOGENETICS  CYTOGENETICS Paracentric Inversions – chromosomal inversion Features: that does not include the o High-pitched, cat-like cry or weak cry centromere. o Low birth weight o Small head – microcephaly o Rounded face o Broad, flattened bridge of the nose o Eyes spaced wide apart o Folds of skin over the eyelids Ø Chromosome 9 Inversion o Abnormalities of the One of the most common structural balances palate chromosomal variants. o Small chin Some cases it is associated with: o Malformations of the o Congetial ears anomalies o Growth retardation o Infertility o Recurrent pregnancy loss o Cancer Ø Wolf-Hirschhorn Syndrome Deletions First described by Hirschhorn and Cooper 1961 Ø A part of a chromosome is missing Deletion of the distal short arm of chromosome 4 or deleted. Deleted genes. o NSD2 – distinctive facial appearance and developmental delay. o LETM1 – seizures Ø Types of Deletion: or other abnormal Interstitial Deletion – arise after activity in the brain. two breaks in the same o MSX1 – dental abnormalities and cleft chromosome arm and loss of the lip / or palate. segment between the breaks. Features: o Distinctive facial features - Broad, flat nasal bridge and a high forehead. - Greek warrior helmet o Delayed growth and development o Motor skills such as sitting, standing, and walking are significantly delayed. Terminal Deletion – loss of Ø Genomic Imprinting chromosomal material from the Normal form of gene regulation that causes a end of a chromosome. subset of genes to be expressed from one of the Ø Cri-du-chat Syndrome two parental chromosomes. Cat-cry syndrome, 5P Most genes – inherit working copies. minus syndrome and o One from mother, one from father Lejeune’s syndrome. Imprinting – inherit one working copy. First describe by o Either from paternal or maternal Jérôme Lejeune in 1963. o Epigenetically silenced Deletion of certain genes on chromosome 5 o Silencing occurring through addition of Deleted genes: methyl groups during egg/sperm o HTERT gene – DNA functioning formation → DNA Methylation o CTNND2 gene – Cell adhesion, Cell - DNA Methylation – adding methyl movement, Active in NS into the DNA. CYTOGENETICS  CYTOGENETICS For example: Point Mutation – there is Ø Prader-Willi Syndrome something wrong with the DNA Replication First described in 1956 by Swiss doctors (Molecular Biology) o Prof. A Prader, o Mutation – found in genes. Dr. A Labhart Maternal Chromosome – H19 gene is an and Dr H Willi imprinted gene expressed only from the maternal Occurs as the result of chromosome 11. absence of expression Paternal Chromosome – Igf2 (insulin-like growth of paternal genes from factor 2) is an imprinted gene, being expressed chromosome 15q11-q13. only from the paternal chromosome 11. Features: o Hypotonia o Hypogonadism o Obesity o CNS and endocrine Ø Angelman Syndrome gland dysfunction Mutation in UBE3A gene Ring Formations in maternal chromosome Ø Double strand breaks – result 15 (q12) from breakage & reunion of a o Ubiquitin protein single chromosome with loss of ligase chromosomal material outside Absence of chromosome the break points. region 15 (15q11-q13) Ø Telomere Dysfucntion – one Named after Harry Angelman who first described or both telomeres may join to the syndrome in 1965. form a ring chromosome. without significant loss of chromosomal material. Ø Ring chromosome 14 syndrome: r (14) r = ring Ø Uniparental Disomy 2 copies of a chromosome come from the same parent. Features: o Developmental delay o Movement or balance disorder o Behavioral uniqueness o Speech impairment CYTOGENETICS  CYTOGENETICS Duplications How a reciprocal translocation arises. Ø Partial trisomy for part of a chromosome. Ø This can result from an unbalanced insertion or unequal crossing over in meiosis or mitosis. Isochromosomes Ø Arise from either abnormal division of the centromeres. Ø Robertsonian Translocation Ø Horizontal division. Centric fusion (2 acrocentric are fused) Ø Each resulting daughter cell Translocation in which the centromeres of two has a chromosome in which acrocentric chromosomes fuse to generate one the short arm of the long arm larger chromosome. is duplicated. Ø Iso means equal (same) How a Robertsonian translocation arises Insertions Ø Involve movement of a segment of a chromosome from one location to another location of the same chromosome or to another chromosome. 1. Insertion – genetic material is added from another Translocations chromosome. Only 1 is giving Ø Breakage in two chromosomes and each of the an effort. broken pieces reunites with another chromosome. 2. Translocation – material is Ø Genetics / with existing case only swapped with another Ø Balanced Translocation – if chromatin is neither lost chromosome. Both parties are nor gained the exchange. giving an effort. Ø Unbalanced Translocation – loss or gain of chromatin material results in partial monosomy or trisomy for a segment of the chromosome. CYTOGENETICS  CYTOGENETICS Cytogenetic Notation Summary CYTOGENETICS  CYTOGENETICS Lesson #11: Cancer Cytogenetics Ø Bone Marrow Blast cells = young cells Cancer Cytogenetics Young cells are huge except for megakaryocyte. Overview When megakaryocyte is maturing, it grows until Ø Field that has been built upon discovery of it gets ruptured and become a platelet. nonrandom chromosome abnormalities in many types While the other blood cells are maturing, they of cancers. are getting smaller and smaller. Ø Most of the cancers can be associated with Blast cells are and can only be in bone marrow. cytogenetics and molecular. We can detect different Ø Blood mutations that occurs in cancers. Matured Cells o Basophil Cancer o Neutrophil Definition o Eosinophil Ø Multiple and sequential genetic mutations occurring o Monocyte in a somatic cell. o Lymphocyte Ø Uncontrolled proliferation – rapid increase in o Natural Killer (NK Cells) abnormal cell numbers. Ø Tissue In peripheral blood smear, you cannot distinguish the difference of B cell and T cell. (Small Lymphocyte) In peripheral blood smear, you cannot (hard to differentiate) distinguish the monocyte. Monocyte to: o Macrophage o Myeloid Dendritic Cell Small Lymphocyte to: (located in lymphatic organs) o B Lymphocyte (B cell) o T Lymphocyte (T cell) Ø Hematopoiesis – process of production of all blood cells including the formation, development, and Leukemia differentiation of blood cells. Definition Ø Uncontrolled proliferation of one or more of the Blood Cells: various hematopoietic cells. o White blood cells Ø Associated with many changes in the circulating cells o Red blood cells o Megakaryocyte = Platelet of the blood. Ø White blood cells, red blood cells and platelets can be Ø Hematopoietic Stem Cells – a cell in bone marrow. affected in leukemia. But mainly affects the white It gives rise to other blood cells. blood cells. Ancestor of all blood cells. Ø Broad term for blood cancer. It can develop into: Ø 2 Main Classifications of Leukemia: o Myeloblast – myeloid lineage Lymphocytic (only affects lymphoid lineage) o Lymphoblast – lymphoid lineage o ALL – Acute Lymphoblastic Leukemia o CLL – Chronic Lymphocytic Leukemia Ø Myeloblast – myeloid lineage Myelocytic (only affects myeloid lineage) Granulocytes o AML – Acute Myeloid Leukemia Basophil CML – Chronic Myelogenous Neutrophil Leukemia Eosinophil Monocyte Red blood cells Platelets Ø Lymphoblast – lymphoid lineage Prolymphocyte (in Bone Marrow) CYTOGENETICS  CYTOGENETICS Acute VS Chronic Acute Lymphoblastic Leukemia (ALL) Acute L1 – All, child Ø Children & Young Adults Ø ALL-pre B (precursor of B cells) Ø Sudden onset Ø t(1;19) Ø Weeks to months Ø Mostly in children Ø Blast Cells: Ø Characteristics: AML – myeloblast Small blasts, uniform size ALL – lymphoblast Scanty cytoplasm Chronic Round nucleus Ø Middle Age & Elderly Small nucleolus Ø Insidious onset Ø Years Ø Mature Cells CML – granulocytes CLL – lymphocytes L2 – All, adult Ø ALL-T (T lymphocyte) Acute Myeloid Leukemias Ø t(11;14) (AML) Classification – FAB Ø Mostly in young adults Acute Myeloid Leukemias Ø Characteristics: Ø M0 – minimally differentiated Large blasts, irregular size Ø M1 – myeloblastic leukemia without maturation More cytoplasm Ø M2 – myeloblastic leukemia with maturation Irregular nucleus Ø M3 – hypergranular promyelocytic leukemia Prominent nucleolus Ø M4 – myelomonocytic leukemia Ø M4Eo – variant, increase in marrow eosinophils Ø M5 – monocytic leukemia Ø M6 – erythroleukemia L3 – All, (Burkitt) Ø M7 – megakaryoblastic leukemia Ø t(2;8), t(8;14), t(8;22) Ø Burkitt Lymphoma Ø FAB – French, American, British Classification Ø Characteristics: Large blasts, uniform size Abundant cytoplasm Vacuoles Round nucleus Prominent nucleolus Chronic Myelogenous Leukemia Definition Ø First malignancy to be associated with a specific chromosome defect. Ø 95% of patients – Philadelphia chromosome translocation – t(9;22)(q34;q11.2) Ø ABL1 gene – Abelson murine leukemia viral oncogene homolog 1 (Chromosome 9) Ø BCR gene – breakpoint cluster M0, M1, and M6 – none region (Chromosome 22) M4 – pericentric inversion CYTOGENETICS  CYTOGENETICS Ø Activates tyrosine kinase – signal to drive Ø Characteristic karyotype proliferation of the cell. anomalies involve mainly Philadelphia Chromosome t(9;22) chromosomes 5, 7, and 8. Ø Proliferation of mature The most frequent granulocytes. abnormalities: Ø Found mainly in adults o del 5q 45 years of age and older. o Monosomy 7 Ø Blood findings include o Trisomy 8 mild anemia an WBC Ø Unbalanced translocations are relatively frequent. markedly increased, may For example: have a few circulating blasts. o Unbalanced t(5;17) and t(7;17) Ø Peter Nowell and David Hungerford discovered - Translocations lead to 17p deletion. Philadelphia Chromosome. They are cancer Molecular Alterations MDS researchers who are based in Philadelphia. Ø Genetic Defects Ø When these genes have mutations, it could lead to MDS. Since, these genes prevents the cell from growing and dividing in an controlled way. They are the regulators. Ø The most common mutations: Chromosome 17 p arm o TP53 gene (Tumor Protein 53) – regulates the cell cycle. - It functions as the tumor suppressor. They suppress the cells in order to avoid them in becoming cancer cells. Chromosome 21 q arm Chronic Lymphocytic Leukemia (CLL) o RUNX1 (Runt related transcription Definition factor 1) – control the development of Ø Trisomy of Chromosome 12 blood cells. Ø t(14;18)(q32;q21) Chromosome 4 q arm o TET2 gene (Tet methylcytosine dioxygenase 2) – regulating the process of transcription. - Tumor Suppressor as well. Solid Tumors Breast Cancer Myelodysplastic Syndrome (MDS) Definition Ø Myelo – Myeloid Dysplastic – abnormal Ø Also called as pre-leukemia. It could lead to AML (Leukemia – AML) Ø Acquired clonal disorder affecting stem cells. Ø Stem cell disorder with ineffective hematopoiesis. Ø Defects in maturation of all cell lines of myeloid lineage. Ø Difference of MDS to AML Of all MDS, 30% to 40% end in an AML CYTOGENETICS  CYTOGENETICS Ø It is a disease in which cells in breasts grow out of BRCA 1 – Chromosome 17 control. BRCA 2 – Chromosome 13 Ø The breast is made of 3 main parts: Lobules Ø PALB2 Ducts Partner and Localizer Connective Tissue of Breast Cancer 2 (BRCA2) Ø The common breast cancer mostly begins in lobules Chromosome 16 p arm or ducts. DNA damage repair Ø Progression of Breast Cancer Benign – non-invasive Malignant – invasive Ø HER2 / ERBB2 Human epidermal growth factor receptor 2 (HER2) Erythroblastic oncogene B 2 (ERBB2) Chromosome 17 q arm HER2 genes → HER2 proteins (HER2/neu proteins) o HER2 proteins are receptors on breast cells. o Control the growth, division, and repair of breast cells. Mutation → HER2 gene amplification o When HER2 gene amplification occurs the grown, division and repair will be uncontrollable because of the HER2 proteins will gain more receptors. Prostate Cancer Ø Prostate cancer – enlarged. Ø Chromosomal Deletion 5q, 6q, 8p, 10q, 13q, 16q, 17p, and 18q Ø Chromosomal Insertion 7p/q, 8q, 9p, and Xq Ø Chromosomal Rearrangement 21q Ø BRCA genes Ø Target Genes for 2 Common Chromosomal Breast cancer gene Aberrations in Prostate Cancer Human tumor suppressor gene AR gene (androgen receptor) o Tumor Suppressor = caretaker gene, to o Xq12 avoid the cells in becoming cancer cells. Responsible in repairing the DNA. CYTOGENETICS  CYTOGENETICS TMPRSS2 and ERG fusion o 21q o Transmembrane protease serine 2 (TMPRSS2) o Erythroblast transformation specific- Regulated Gene (ERG) → Transcriptional regulator CYTOGENETICS  CYTOGENETICS Lesson #12: Diagnostic Cytogenetics Types of Sample Ø Peripheral blood smear Cytogenetic Analysis Ø Bone marrow blood Importance Ø Amniotic Fluid – for prenatal testing Ø Prenatal Diagnosis (can detect chromosomal Ø Chorionic Villus – for prenatal testing abnormalities in prenatal diagnosis) Ø Fibroblasts from skin biopsy Ø Detection of carrier status (for a certain genetic or Ø Epithelial cells from buccal smear trait and for general diagnostic purposes) Karyotyping Reagents Ø Useful in the study and treatment of patients with 1. Phytohemagglutinin (PHA) malignancies (cancers) and hematologic (leukemia) It stimulates the metabolic activity of the cell. disorders. It continues the mitotic division of the cell. Cytogenetic Analysis 2. Colcemid / Colchicine Ø Karyotyping It prevents the formation of the spindle fibers. Ø Chromosome Banding There is no Anaphase since there are no spindle Ø Molecular fibers to pull. FISH – Fluorescent In situ Hybridization The cells will get arrested in metaphase during CMA – Chromosomal Microarray Analysis the cell division, thereby allow the cell to harvest NGS – Next Generation Sequencing at metaphase for karyotyping. 3. Potassium Chloride Solution Karyotyping Hypotonic Solution Definition It bursts the cell. Ø A technique that allows geneticists to visualize 4. Methylalcohol and acetic acid chromosomes under a microscope. For fixative. Ø Proper extraction and staining techniques. 5. Giemsa Stain Solution Ø Cells are arrested during metaphase. In metaphase, the chromosomes are clearly visible. Karyogram Ø Graphical representation of karyotype. Ø Trypsin is an enzyme that partially digests some of the chromosomal proteins. Thereby, relaxing the chromatin structure and allowing the giemsa stain to Procedure of Karyotyping access the DNA. 1. Collection of Sample 2. Cell culture 3. Stopping the cell division at Metaphase 4. Hypotonic treatment 5. Fixation 6. Slide preparation 7. Slide dehydration 8. Treatment with enzyme. 9. Staining Ø Limitations in Karyotyping: low diagnostic yield, long turnaround time (1-2 weeks), prone to human errors (possibility of not noticing the small changes and abnormalities such as deletions and insertions.) CYTOGENETICS  CYTOGENETICS Ø Chromosomes are photographed through microscope. Ø Photograph of chromosomes is cut up and arranged to form karyotype diagram. Chorionic Villus Sampling Ø Prenatal genetic testing Ø 10th and 13th week of pregnancy Ø To confirm or rule out certain abnormalities. Procedure: Transcervical or Transabdominal Peripheral Blood Smear Bone Marrow Blood Chromosomal Banding Ø Samples are Definition commonly young Ø Staining technique for chromosomes. cells. Ø Comprised of alternating light and dark stripes (bands) Ø Appear along its length after being stained with a dye. Amniocentesis Ø Amniotic Fluid Ø Prenatal diagnostic test Ø Small amount of amniotic fluid is removed. Ø 15th to 20th week of pregnancy. Ø Determine any genetic abnormality. CYTOGENETICS  CYTOGENETICS Types of Chromosomal Banding Ø NOR banding Ø G-banding Identifies genes for ribosomal RNA (rRNA) that Giemsa stain were active in a previous cell cycle. o Dark bands = A-T Nucleolar Organizing Region (heterochromatic) Silver staining method o Light bands = G-C NORs are located on the short arms of the (euchromatic) acrocentric chromosomes 13, 14, 15, 21, and 22. o Ø R-banding Reverse pattern of G bands Staining with Giemsa dye o Dark bands = G-C (rich euchromatic) o Light bands = A-T (rich heterochromatic) Euchromatin Ø Q-banding Ø Lightly packed chromatin. Quinacrine stain Ø Enriched in genes. Fluorescent pattern Ø Active transcription. o Dark bands = A-T o Light bands = G-C Heterochromatin Ø Tightly packed chromatin. Ø Low gene density. Ø Constitutive heterochromatin It cannot be fully expressed. It occurs around the chromosomes centromere Ø C-banding and near the telomeres. Dark bands – constitutive Strictly heterochromatin. heterochromatin Ø Facultative heterochromatin Centromeric Flexible, reversible. Can be euchromatin. FISH In situ Hybridization (ISH) Ø Method of localizing and detecting specific nucleotide sequences in preserved tissue or cell preparations. Ø Hybridizing the complementary strand of a Ø T-banding nucleotide probe against the sequence of interest. Staining telomeric regions. Ø In situ – in place Giemsa stain or acridine. Hybridization – binding of complementary sequence CYTOGENETICS  CYTOGENETICS Fluorescent In situ Hybridization Probes Ø A molecular technique commonly used in cytogenetic Ø Complementary sequences of target nucleic acids. laboratories. Ø Designed against the sequence of interest. Ø Used to detect and localize the Ø Probes are tagged with fluorescent dyes. presence or absence of specific Biotin DNA sequences on chromosomes. Fluorescein Ø Fluorescence-labeled nucleic acid Digoxigenin probes hybridize to selected DNA or RNA sequences. FISH Technique Different Kinds of Probes 1. Whole chromosome 2. Centromere probes 3. Telomere 4. Locus Requirement for FISH Ø 3 Main Components for FISH Sample – target DNA Fluorescent Probe – small fragment of nucleic acid that is complementary to the part of the DNA or RNA of the organism that we are searching for. Fluorescence Microscope Protocol Outline 1. Preparation of the fluorescent probes & sample 2. Denaturation of the probe and the target 3. Hybridization of the probe and the target 4. Detection & Visualization Specimens Ø Bone Marrow Aspirate Ø Peripheral Blood Smear Ø Fixed and sectioned tissue Ø Advantage of FISH is that Metaphase and Interphase can be used. In Metaphase, it needs culturing and treatment with colchicine in order to stop during Denaturation metaphase of cell division. While in Interphase, there Ø Either by heat or alkaline method. is no need to treat it with colchicine. Ø A prerequisite for the hybridization of probe and target. CYTOGENETICS  CYTOGENETICS Hybridization Chronic Myelogenous Leukemia Ø Formation of duplex between two complementary nucleotide sequences. Ø Can be between: DNA-DNA RNA-RNA DNA-RNA Solid Tumors Detection & Visualization Ø Amplification of the gene HER2 on chromosome 17 Ø Detection is associated with an aggressive form of invasive Direct Labelling breast cancer. o Label is bound to the probe. Indirect Labelling o Require an additional step before detection. o Probe detected using antibodies conjugated to labels. o Results in amplification of signal. Ø Visualization Fluorescent probe attaches to the target sequence during hybridization. This is visualized through a fluorescence Advantages of FISH Ø Detection of genetic changes that are too small to be microscope. seen under a microscope. Ø Cytogenetic data can be obtained from non-dividing or terminating differentiated cells. Ø Highly sensitive & specific. Limitations of FISH Ø Restricted to those abnormalities that can be detected with currently available probe. Ø Due to failure to detect signal FISH is higher sensitive for trisomy but less sensitive for detecting chromosome loss or deletion. Ø Cytogenetic data can be obtained only for the target chromosome thus FISH is not a good screening tool for cytogenetically heterogeneous disease. Ø Requires fluorescence microscopy and an image analysis system. Spectral Karyotyping (SKY) Ø Multi-fluorochrome FISH Ø All chromosome pairs are simultaneously visualized in different colors in a single hybridization. CYTOGENETICS  CYTOGENETICS Copy Number Variation Ø Copy number change Ø Gain or loss of DNA segments Ø Known to play a role in shaping a wide range of phenotypes. Ø DNA segments of 1Kb or larger. Chromosomal Microarray Analysis (CMA) Single Nucleotide Polymorphism Definition array (SNP array) Ø Invented by Ø Single Nucleotide Polymorphism (SNP) array Stephen Fodor and Ø SNP → DNA sequencing variation his colleagues in 1991 Ø Change affects only one single nucleotide base Ø Microarray chip → device containing probes. Ø Detection of whole genome sequence. Types of CMA Ø Comparative Genome Hybridization Array (aCGH) BAC array Oligoarray Ø SNP array (aSNP) BAC array and Oligoarray Ø BAC array – Bacterial Artificial Chromosome 1Mb Genomic intervals (million base pairs) Ø Oligoarray 100 Kb (kilo base pairs) Ø Control DNA sequences Labeled with a specific dye (e.g., Red dye) Ø Patient’s Sample DNA Labeled with a different fluorescent colored dye (e.g., Green dye) CYTOGENETICS  CYTOGENETICS Limitations of CMA/CGH Ø Arrays cannot currently detect balanced rearrangements. Ø Some aneuploidies may also be missed. Ø Some gains may also be missed depending on the size and DNA composition and array coverage of the chromosomal region present. Ø Interpretation of arrays may also be difficult to access. Next Generation Sequencing Technology (NGS) Definition Ø Powerful platform that has enabled the sequencing of thousands to millions of DNA molecules simultaneously. Ø NGS – effective at all size scales NGS Approaches Ø Whole-genome sequencing Whole genome, all genes. Ø Transcriptome sequencing Collection of RNA molecules derived from those genes, including introns and exons. Ø Whole-exome sequencing Coding sections, exons. CYTOGENETICS