Cytogenetics (Numerical Aberrations) PDF

CYTOGENETICS (NUMERICAL ABERRATIONS) Ms. Cherries Sanchez | Week Ewan INTRODUCTION CAUSES OF POLYPLOIDY TOPIC...

CYTOGENETICS (NUMERICAL ABERRATIONS) Ms. Cherries Sanchez | Week Ewan INTRODUCTION CAUSES OF POLYPLOIDY TOPIC 2 WAYS SUB-TOPIC 1. Nondisjunction Meiotic nondisjunction – failure of chromosomes to separate NUMERICAL ABNORMALITIES NUMERICAL ABNORMALITIES Mitotic nondisjunction – occurs in early embryo Defects involving abnormality in the number of chromosomes Subclassified as… 2. Genome duplication – inherits 2 copies of chromosomes 1. Aneuploidy – abnormal structure of chromosomes 2. Polyploidy – chromosome higher than 46, always an exact multiple of haploid HYPODIPLOID Hypodiploid EUPLOIDY Cell with fewer than 46 chromosomes Euploidy EU - true PLOIDY - number of sets of chromosomes NEAR-HAPLOID Normal number of structurally normal chromosomes Near Haploid Cells have 23 up to approximately 34 chromosomes ANEUPLOIDY Aneuploidy Any abnormal number of chromosomes that is not a multiple of the haploid number HYPERDIPLOID (23 chromosomes) Hyperdiploid Cells have more than 46 chromosomes Result of NONDISJUNCTION > failure of chromosomes to separate normally during cell division (meiosis/gametes) HIGH HYPERDIPLOIDY 1. TRISOMY – presence of an extra chromosomes (DOWN SYNDROME) High Hyperdiploidy 2. MONOSOMY – absence of a single chromosome (X - TURNER’s SYNDROME) Chromosome number of more than 50 DOWN SYNDROME Trisomy - 21 DISEASE ASSOCIATIONS Group G - 21 - Tripled Disease Associations 47 1. Infertility and Sterility – Unable to become pregnant/conceive a child The rest of the chromosomes/group is normal 2. Intersexes – person born with male and female biological traits TURNER SYNDROME Monosomy 45 - X - X0 3. Multiple congenital malformations – birth defects, undeveloped organs Total count of chromosomes is 45 Missing sex chromosomes - Y 4. Mental retardation – intellectual disabilities POLYPLOIDY Polyploidy Chromosome number is higher than 46 Always an exact multiple of haploid chromosome number of 23(n) Lethal = dead fetus 1. TRIPLOIDY (3n) – karyotype with 69 chromosomes 2. TETRAPLOIDY (4n) – karyotype with 92 chromosomes L.L.G.G | 1  TYPES OF ANEUPLOIDY TURNER SYNDROME – MONOSOMY Types of Aneuploidy Ullrich-Turner (XO) 1. Sex Chromosome Aneuploidies Gonadal Dysgenesis – defective development of reproductive 2. Autosomal Aneuploidies Monosomy X No Y chromosome 1. SEX CHROMOSOME ANEUPLOIDIES First described 1938 – Dr. Henry Turner Sex Chromosome Aneuploidies Occuring in 1 out of every 2,500 girls FEMALES - Turner Syndrome (XO) FEATURES: - Metafemale (XXX) 1. Short stature 2. Lymphedema (swelling) – hands and feet MALES 3. Rudimentary ovaries gonadal streak (underdeveloped gonadal structures) - Klinefelter Syndrome (XXY) 4. Shortened metacarpal IV (of hand) - Jacob Syndrome (XYY) 5. Small fingernails 6. Webbed neck TRIPLE X SYNDROME – TRISOMY Metafemale (47-XXX-aneuploidy) Presence of extra X chromosome in each cell of human female Unlike most other chromosomal conditions there is usually no distinguishable difference between women with triple X and the rest of the female population. FEATURES: 1. Menstrual Irregularities 2. Constriction of aorta 3. Poor breast development 4. Web of skin JACOB’S SYNDROME – TRISOMY 5. Increased risk of learning disabilities, XYY - Delayed speech, Phenotype is normal - deficient language skills - Delayed development of motor skills FEATURE： 1. Normal appearance > tall stature KLINEFELTER’S SYNDROME – TRISOMY (47-XXY-aneuploidy) 2. Increased vulnerability to ADHD (attention deficit hyperactivity disorder) In which males have an extra Y sex chromosomes 3. Learning disabilities Affected individuals have at least (2) X chromosomes and at least (1) chromosome 4. Autistic spectrum disorders gynecomastia. 5. Eyes set slightly far apart FEATURES: 6. Large head 1. Small firm testicles > infertility 7. infertility 2. Low testosterone 3. Incomplete masculinization 4. Decrease libido 5. Enlarged breast tissue 6. Body shape patterns: Pear shaped, tall, abnormal proportions, teeth abnormality.  TYPES OF ANEUPLOIDY PATAU SYNDROME Types of Aneuploidy Trisomy 13-D 3. Sex Chromosome Aneuploidies XY Extra copy of chromosome 13 > causes numerous physical and mental abnormalities, especially heart 4. Autosomal Aneuploidies KARYOTYPING defects. 2. AUTOSOMAL ANEUPLOIDIES FEATURES: Autosomal Numeric Aberration 1. Small head size Down Syndrome (trisomy-21) 2. Extra toes/fingers Edward Syndrome (trisomy-18) 3. cleft lip or palate Patau Syndrome (trisomy-13) 4. HOLOPROSENCEPHALY – brain doesn’t divide into 2 halves; severe mental retardation Warkany Syndrome (trisomy-8) 5. Nasal malformation 6. HYPOTELORISM – reduced distance between the eyes or cyclops DOWN SYNDROME Trisomy 21-G WARKANY SYNDROME Presence of all or part of an extra 21st chromosome Trisomy 8-C Named after John Langdon Down – British doctor who described syndrome in 1866 Presence of an extra chromosome 8 FEATURES: FEATURES: 1. Microgenia (small chin) Elongation of the skull 2. Unusually round face Prominent forehead 3. Macroglossia (oversized tongue) Widely-spaced eyes 4. Almond shape to the eyes caused by an epithantic fold of the eyelid Deeply set eyes 5. Shorter limbs Broad upturned nose 6. Poor muscle tone Brain malformations 7. Larger than normal space between the big and second toes Highly arched or cleft palate Extra skin fields EDWARDS SYNDROME Long slim body with narrow chest, shoulders, pelvis Trisomy 18-E Kidney and cardiac abnormalities Extra 18th chromosome Incidence increases as the mother’s age increases Low rate of survival – resulting from - heart abnormalities - Kidney malformations, - Internal organ disorders FEATURES 1. Low birthweight 2. Small, abnormal shaped head 3. Small jaw and mouth 4. Long fingers that overlap 5. Underdeveloped thumbs 6. Clenched fists 7. Low set ears 8. Smooth feet with rounded soles 9. Cleft lip and palate  STRUCTURAL ABERRATIONS CYTOGENETIC NOTATION STRUCTURAL ABNORMALITIES Cytogenetic Notation Structural Abnormalities How to read and write cytogenetic notation Result from breakage of a chromosome region with loss or subsequent rejoining in an abnormal combination. INTERNATIONAL SYSTEM FOR HUMAN CYTOGENETIC NOMENCLATURE HAPPENS DUE TO: ISCN Each area of chromosome given number Errors during meiosis/gametes/sex cells Errors during mitosis/somatic/body cells Lowest number closest (PROXIMAL) to centromere Exposure to substances that cause birth defects (teratogens) Highest number at tips (DISTAL) 2 GENERAL TYPES: 1. Balanced Rearrangements – no loss or gain of genetic chromatin 2. Imbalanced Rearrangements – gain or loss of genetic material 1. BALANCED REARRANGEMENTS Balanced Rearrangement Change the chromosomal gene order but do not remove or duplicate any of the DNA of the chromosomes. CYTOGENETIC BANDING NOMENCLATURE Banding Nomenclature 2. IMBALANCED REARRANGEMENT Imbalanced Rearrangement Change the gene dosage of a part of the affected chromosomes.  STRUCTURAL REARRANGEMENT CRI-DU-CHAT SYNDROME 1. INVERSION Cri-Du-Chat Reversed or inverted Cat-cry syndrome Sometimes visible in the structure of the chromosomes 5p minus syndrome Segment of a chromosome is reversed end to end Jerome lejeune – 1963 Occurs when a single chromosome undergoes breakage and rearrangement within itself Can be inherited or it may be a mutation that appears in a child whose family has no history. ➔ DELETION of certain genes on CHROMOSOME 5 TYPES OF INVERSION: ➔ Deleted genes: 1. Pericentric – involves the centromere HTERT gene – DNA functioning 2. Paracentric – does not include the centromere CTNND2 gene – cell adhesion, movement, active in NS CHROMOSOME 9 INVERSION – one of the most common structural balanced chromosomal variants, FEATURES: some cases it is associated with… 1. High pitched, cat cry, weak cry - Congenital anomalies 2. Low birth weight - Growth retardation 3. Small head – microcephaly - Infertility 4. Rounded face - Recurrent pregnancy loss 5. Broad, flattened bridge nose - cancer 6. Eyes spaced wide 7. Folds skin over the eyelids STRUCTURAL REARRANGEMENT 8. Abnormalities of palate 2. DELETIONS 9. Small chin Part of a chromosome is missing or deleted 10. Malformation of ears 1. INTERSTITIAL DELETION – arise after 2 breaks in the same chromosome arm and loss of the segment WOLF-HIRSCHHORN SYNDROME between the breaks Wolf Hirschhorn 2. TERMINAL DELETION – loss of chromosomal material from the end of a chromosome Hirschhorn and Cooper (1961) DELETION of distal short arm CHROMOSOME 4 ➔ Deleted Genes: NSD2 – distinctive facial appearance and developmental delay LETM1 – seizures or abnormal electrical act in the brain MSX1 – dental abnormalities, cleft lip, palate FEATURES: 1. Broad, nasal flat bridge 2. High forehead, greek warrior helmet 3. Delayed growth and development 4. Motor skills are delayed (sitting, standing, walking)  GENOMIC IMPRINTING 3 RING CHROMOSOMES Genomic Imprint 3 Ring Chromosomes Normal form of gene regulation Causes subset of genes to be expressed from 1 or 2 parental chromosomes. DOUBLE STRAND BREAKS – results from breakage & reunion of single chromosome with loss of chromosomal material outside the break points. ➔ MOST GENES – inherit working copies One from mother, other from father (X,Y) TELOMERE DYSFUNCTION – one or both telomeres may join to form a ring chromosome without significant loss of chromosomal material. ➔ IMPRINTING – inherit one working copy Either paternal or maternal (XX, XY) Epigenetically silenced STRUCTURAL REARRANGEMENT 4. Duplication Silencing occurring through addition of methyl groups during egg/sperm form > DNA Methylation. Partial trisomy for part of chromosome This can result from unbalanced insertion or unequal crossing over (meiosis or mitosis). ANGELMAN SYNDROME Angelman ISOCHROMOSOME Isochromosome Mutation in UBE3A gene in maternal chromosome 15 (q12) - Ubiquitin protein ligase Arise from either abnormal division of the centromere Horizontal division Absence of chromosome region 15 (15q11-q13) Each resulting daughter cell has a chromosome in which the short arm or the long arm is duplicated Harry Angelman - 1965 STRUCTURAL REARRANGEMENT 6. Insertions FEATURES: Involve movement of a segment of a chromosome from one location to another location of the same 1. Developmental delay chromosome or to another chromosome 2. Balance disorder 3. Behavior uniqueness STRUCTURAL REARRANGEMENT 4. Speech impairment 7. Translocations Breakage in 2 chromosomes and each of the broken pieces reunites with another chromosome UNIPARENTAL DISOMY Uniparental Disomy 1. BALANCED TRANSLOCATION – if chromatic is neither lost nor gained the exchange 2. UNBALANCED TRANSLOCATION – loss or gain of chromatin material results in partial 2 copies of chromosome come from same parent monosomy/trisomy for segments of the chromosome. PRADER-WILLI SYNDROME ROBERTSONIAN TRANSLOCATION Prader Willi Robertsonian Translocation Swiss doctors: Centric fusion 1. Prof. A Prader, 2. Dr. A Labhart Translocation in which the centromeres of 2 acrocentric chromosomes fuse to generate one large 3. Dr H Willi chromosome. Occurs as the result of absence of expression of paternal genes from chromosome 15q11-q13 FEATURES: 1. Hypotonia 2. Hypogonadism 3. Obesity 4. CNS and endocrine gland dysfunction CANCER CYTOGENETICS TERMS: HEMATOPOIETIC STEM CELL – gives rise to the blood cells CANCER It can develop into: Cancer MYELOBLAST – myeloid lineage Multiple and sequential genetic mutations - Granulocytes (granulopoiesis) Occuring in a somatic cell - Monocytes Uncontrolled proliferation - rapid increase in cell number - RBC / erythrocytes - Megakaryocytes / platelets HEMATOPOIESIS Hematopoiesis Production of all blood cells LYMPHOBLAST – lymphoid lineage formation, development, differentiation of blood cells - Lymphocytes Main focus – wbcs - B BONE MARROW – Hematopoietic stem cell – gives rise to blood cells, ninuno - T LEUKEMIA - etc Leukemia Uncontrolled proliferation of 1 or more HEMATOPOIETIC CELLS Associated with many changes in the circulating cells of the blood ➔ (2) main classification of leukemia: 1. LYMPHOCYTIC - lymphoid - Acute Lymphoblastic Leukemia (ALL) - Chronic Lymphocytic Leukemia (CLL) 2. MYELOCYTIC - myeloid - Acute Myeloid Leukemia (AML) - Chronic Myelogenous Leukemia (CML) ACUTE vs CHRONIC Acute VS Chronic (mabilis, BLAST cells) (mature, matagal.CYTES)  ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) - Lymphocytic ACUTE MYELOID LEUKEMIA (AML) - Myelocytic classification classification M0: minimally differentiated L1 – ALL, children M1: Myeloblastic Leukemia WITHOUT MATURATION M2: Myeloblastic Leukemia WITH MATURATION t(8;21)-(q22;q22), t(6;9) M3: Hypergranular Promyelocytic Leukemia t(15;17) M4: Myelomonocytic Leukemia inv(16) (p13;q;22), del(16q) M4Eo: variant, INCREASE in MARROW EOSINOPHILS inv(16), t(16;16) M5: Monocytic Leukemia del(11q), t(9;11), t(11;19) L2 – ALL, adult M6: Erythroleukemia M7: Megakaryoblastic leukemia t(1;22) L3 – ALL, (burkitt’s lymphoma)  CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) - Lymphocytic CHRONIC MYELOGENOUS LEUKEMIA (CML) - Myelocytic classification classification TRISOMY OF CHROMOSOME 12 FIRST MALIGNANCY to be associated with a specific chromosome defect t(14;18) (q32;q21) PHILADELPHIA CHROMOSOME t(9;22) - Peter Nowell & David Hungerford Proliferation of mature granulocytes FOUND MAINLY IN ADULTS 45 years of age and older Blood findings include mild anemia an WBS markedly increased, may have a few circulating blasts 95% of patients – Philadelphia chromosome translocation t(9;22)(q34;q11.2) ABL1 gene – abelson murine leukemia viral oncogene homolog 1 (chromosome 9) BCR gene – breakpoint cluster region (chromosome 22) ABL & BCR FUSED – Activates tyrosine kinase – signal to drive proliferation of the cell  MYELODYSPLASTIC SYNDROME (pre-leukemia) MOLECULAR ALTERATIONS MDS Myelodysplastic Syndrome Molecular Alterations Myelodysplastic Syndrome Acquired clonal disorder affecting stem cells. Genetic Defects STEM CELL DISORDER with INEFFECTIVE HEMATOPOIESIS ➔ THE MOST COMMON MUTATIONS: DEFECTS IN MATURATION OF ALL CELL LINES OF MYELOID LINEAGE CHROMOSOME 17 p arm Of all MDS CASES, 30% to 40% end in an AML TP53 gene (Tumor protein 53) – regulates the cell cycle CHROMOSOME 21 q arm RUNX1 (Runt related transcription factor 1 – control the development of blood cells CHROMOSOME 4 q arm TET2 gene (Tet methylcytosine dioxygenase 2) – regulating the process of transcription Characteristic karyotype anomalies involve mainly CHROMOSOMES 5, 7, 8 ➔ The most frequent abnormalities: Del 5q Monosomy 7 Trisomy 8 Unbalanced translocation are relatively frequent ➔ For example: Unbalanced t(5; 17) and t(7; 17) Translocations lead to 17p deletion  SOLID TUMORS 1. Breast Cancer HER2 / ERBB2 Human epidermal growth factor receptor 2 Erythroblastic oncogene B2 BRCA genes Chromosome 17 Q arm Breast cancer gene HER2 genes – (HER2 / neu proteins) Human tumor suppressor gene Proteins are receptors on breast cells Controls growth, division, repair of breast cells Responsible for repairing DNA: MUTATION – HER2 gene amplification ➔ BRCA 1– chromosome 17 ➔ BRCA 2 – chromosome 13 MOSTLY BEGINS AT DUCTS & LOBULES PALB2 gene Partner and localizer of breast cancer 2 (BRCA2) Chromosome 16 p arm DNA damage repair  SOLID TUMORS TARGET GENES FOR (2) COMMON CHROMOSOMAL ABERRATIONS IN PROSTATE CANCER 2. Prostate Cancer Common Chromosomal Aberrations in Prostate Cancer CHROMOSOMAL DELETION: AR gene (androgen receptor) – 5q ❖ Xq12 6q 8p TMPRSS2 & ERG fusion – 21q 10q 13q ❖ Transmembrane protease serine 2 16q ❖ Erythroblast transformation specific-regulated gene (TRANSCRIPTIONAL REGULATOR) 17p 18q CHROMOSOMAL INSERTION: 7p/q 8q 9p Xq CHROMOSOMAL REARRANGEMENT – 11/21q DIAGNOSTIC CYTOGENETICS IMPORTANCE OF CYTOGENETIC ANALYSIS KARYOGRAM Importance of Cytogenetic Analysis Karyogram 1. Prenatal diagnosis Graphical representation of karyotype 2. Detection of carrier status 3. Useful in the study and treatment of patient with malignancies and hematologic disorders Cytogenetic Analysis KARYOTYPING Karyotyping ➔ Technique that allows geneticists to visualize chromosomes under a microscope Proper extraction and staining techniques Cells are arrested during metaphase SPECTRAL KARYOTYPING Spectral Karyotyping Multi-fluorochrome FISH ALL CHROMOSOME PAIRS are simultaneously visualized in different colors in a single hybridization  PROCEDURE KARYOTYPING REAGENTS Procedure Karyotyping Reagents Collection of sample Phytohemagglutinin Cell culture stimulates the metabolic activity of the cell Stopping the cell division at metaphase Hypotonic treatment - to burst the chromosome of cell Colcemid / colchicine Fixation prevents the formation of spindle fiber Slide preparation (it arrests the cells in metaphase so we can harvest the chromosomes) Slide dehydration Treatment with enzyme Potassium Chloride Solution Staining Hypotonic solution It bursts the cell TYPE OF SAMPLE Type of Sample Methyl Alcohol and Acetic Acid 1. Peripheral Blood Smear Fixative the sample 2. Bone Marrow Blood – aspirate, young cell samples 3. Amniotic Fluid – prenatal testing Giemsa Stain Solution 4. Chorionic Villus – prenatal testing 5. Fibroblasts from skin bx 6. Epithelial cells from buccal smear Chromosomes are photographed THROUGH MICROSCOPE PHOTOGRAPH OF CHROMOSOMES – cut up and arranged to form karyotype diagram.  AMNIOCENTESIS CHORIONIC VILLUS SAMPLING Amniotic Fluid Chorionic Villus Sampling Prenatal diagnostic test Prenatal genetic testing Determines any genetic abnormality To confirm / rule out certain abnormalities 15-20th weeks of pregnancy 10-13th weeks of pregnancy Small amount of amniotic fluid is removed PROCEDURE: Determines any genetic abnormality 1. Transcervical – CATHETER 2. Transabdominal – NEEDLE / ASPIRATE  CHROMOSOMAL BANDING EUCHROMATIN Chromosomal Banding Euchromatin Staining technique for chromosome Lightly packed chromatin Comprised of alternating light and dark bands OPEN chromatin conformation Appear along its length after being stained with a dye Enriched in genes Active transcription TYPES OF CHROMOSOMAL BANDING HETEROCHROMATIN Types Heterochromatin Tightly packed chromatin 1. G – GIEMSA STAIN CLOSED chromatin conformation Dark bands = A-T [heterochromatic] repression Light bands = G-C [euchromatic] Low gene density 2. R – REVERSE PATTERN OF G Constitutive heterochromatin Staining with giemsa dye Facultative heterochromatin Dark bands = G-C [rich euchromatic] Light bands = A-T [rich heterochromatic] 3. Q - QUINACRINE STAIN Fluorescent pattern Dark bands = A-T Light bands = G-C 4. C – CONSTITUTIVE HETEROCHROMATIN Centromeric Dark Bands = C heterochromatin Centromere & telomere staine 5. T - TELOMERE Staining telomeric regions Giemsa stain Acridine orange 6. NOR – NUCLEOLAR ORGANIZING REGION Silver staining method Identified genes for rRNA that were active in a previous cell cycle Located on the short arms of ACROCENTRIC chromosomes (13-15, 21-22)  IN SITU HYBRIDIZATION (ISH) DENATURATION In Situ Hybridization Denaturation Either by heat or alkaline method Method of detecting specific nucleotide sequences in preserved tissue/cell preparation Prerequisite for the hybridization of probe and target Hybridizing the complementary strand of a nucleotide probe against the sequence of interest HYBRIDIZATION Hybridization FLUORESCENT IN SITU HYBRIDIZATION (FISH) Formation of duplex between 2 complementary nucleotide sequences Fluorescent In Situ Hybridization Can be between.. Molecular technique commonly used in cytogenetic laboratories 1. DNA-DNA – 2. DNA-RNA DETECT PRESENCE OR ABSENCE OF SPECIFIC DNA SEQUENCES ON CHROMOSOMES 3. RNA-RNA Fluorescence-labeled nucleic acid – probes hybridize to selected DNA/RNA sequences Metaphase – interphase usement 3 MAIN /REQUIREMENTS FOR FISH : Sample Fluorescent probe Fluorescence microscope PROTOCOL OUTLINE Protocol Outline 1. Preparation of the fluorescent probes & samples 2. Denaturation of the probe and target 3. Hybridization of probe and target 4. Detection & visualization SPECIMENS Specimen Bone marrow aspirate Peripheral blood smear Fixed and sectioned tissue WHAT KIND OF PROBE CAN BE USED? What kind of probe Complementary sequences of target nucleic acid Designed against the sequence of interest Biotin, fluorescein, digoxigenin 1. Whole chromosome 2. Centromere probes 3. telomere 4. Locus  SPECTRAL KARYOTYPING NEXT GENERATION SEQUENCING Sprectral Karyotyping Next Generation Sequencing Multi-fluorochrome FISH Powerful platform that has enabled the sequencing of thousands – millions of DNA molecules All chromosome pairs are simultaneously visualized in different colors in a single hybridization SIMULTANEOUSLY. NGS APPROACHES: CHROMOSOMAL MICROARRAY ANALYSIS Whole genome sequencing Chromosomal Microarray Analysis Transcriptome sequencing Stephen Fodor & colleagues (1991) Whole-exome sequencing MICROARRAY CHIP – device containing probes ➔ Detection of whole genome sequence TYPES OF CMA Comparative Genomic Hybridization Array (aCGH) 1. BAC ARRAY & OLIGOARRAY 1 mb genomic intervals – BAC array (bacterial artificial chromosome) 100 kb – OLIGOarray Control DNA sequences – labeled with specific dye (red) Patient’s sample DNA – labeled with different fluorescent colored (green) 2. SNP ARRAY ( single nucleotide polymorphism) SNP > DNA sequence variation Change affect only one single nucleotide base