Chromosomes: Structure and Abnormalities - PDF

Chromosomes: structure and abnormalities MD210 – GGE – Flipped Lesson 2 1 Essential Learning Outcomes By the end of this lesson you should be able to: • Describe how DNA is packaged in chromosomes and understand the different states of chromatin condensation in chromosomes • Describe how gross ch...

Chromosomes: structure and abnormalities MD210 – GGE – Flipped Lesson 2 1 Essential Learning Outcomes By the end of this lesson you should be able to: • Describe how DNA is packaged in chromosomes and understand the different states of chromatin condensation in chromosomes • Describe how gross chromosomal abnormalities are named and how they can be identified using cytogenetics and FISH • Describe the different types of structural chromosome abnormalities • Understand the inheritance patterns of structural chromosome abnormalities and know how they can be identified clinically 2 chromosome DNA double helix chromatin Chromatin structure Euchromatin vs Heterochromatin Studying Chromosomes - Cytogenetics Microscopic Examination - routine diagnostics and research 1. Sample nucleated cells you can grow (easily) T lymphocytes /Skin Fibroblasts/ Bone Marrow/Chorionic villi or amniotic fluid (Antenatal) 2. Get chromosomes into a form in which they can be observed and differentiated from each other (Metaphase chromosomes - grow the cells to dividing stage) 3. Chemical preparation to arrest cells and lyse nuclei 4. Stain chromosomes (Giemsa) 6 Conventional Cytogenetics Identify Chromosomes by Banding Patterns Arrange Homologs Side By Side “Karyogram” Described as a Karyotype 46,XX Female 46, XY Male Limitation = resolution Detects gross chromosomal changes (>5 Mb) 7 International System for Human Cytogenetic Nomenclature (ISCN) p (petit) small centromere q (queue) tail Chromosome regions: counting out from centromere p1, p2, p3 etc. (p1 – near centromere ) q1, q2, q3 etc. (q 1 near centromere) Regions subdivided into Bands q11 ( q one one NOT q eleven) Bands divided into sub-bands q11.1 q11.2 Credit: NCBI 8 Molecular Cytogenetics Probe Hybridisation Visualisation of Location of Specific Nucleotide sequence e.g. to Find: You Use: AATTGCCCTAAGTC TTAACGGGATTCAG-FLUOROPHORE Uses: Is Sequence Present and If So On Which Chromosome? Are 2 Sequences close to each other or not? Or Preimplantation Genetic Screening 9 F.I.S.H. Fluorescent In Situ Hybridisation Metaphase FISH 10 Multiplex FISH (M-FISH) Metaphase chromosomes Detects small Chr rearrangements Uses “painting probes” Paint whole Chr 11 Structural Chromosome Abnormalities • Change in chromosome structure • Caused by chromosome breakage with subsequent abnormal realignment • 1 in every 375 births • 2 categories: • Balanced - no gain/loss of genetic content • Often not clinically relevant* • Unbalanced – gain or loss of genetic content • Clinically relevant Chronic Myeloid leukaemia (CML) By Paulo Henrique Orlandi Mourao CC BY-SA 3.0 • Structural chromosome abnormalities are heritable only if in germ line 12 Types of structural abnormalities - Deletion • Deletion • Part of a chromosome is deleted • Generally have severe effects, large deletions lethal • Large deletions (>5 Mb) are rare • Visible under light microscope – conventional cytogenetics • E.g. Cri-du-chat Syndrome del(5p) a b c d e a b c e • Smaller deletions (<2 Mb) more common • Microdeletions – not visible under light microscope • Some visible using Fluorescence In Situ Hybridisation (FISH) • Smaller ones using chromosome oligonucleotide arrays (Microarrays) • E.g. Smith-Magennis Syndrome del(17p11.2) A boy with Cri-du-chat Syndrome Types of structural abnormalities - Duplication • Duplication • Chromosome section repeated (often sequentially) • Usually less severe than deletion of the Chr section • E.g. Potocki-Lupski syndrome dup(17p11.2) • Copy Number Variants (CNVs) • Deletions/duplications of genomic sequence • Range from 1 Kb to Mbs (variable number of repeats of a particular unit of sequence) • Major determinants of human genetic variation – about 5-10% of genome is CNVs • Can represent functional (disease causing) mutations as well as genomic polymorphisms of uncertain relevance Types of structural abnormalities - Inversion 1. a • Inversions • 2 breaks occur in chromosome • Part of chromosome in between is rotated 180° before re-joining • Result in looped Chr pairs in meiosis I • Can cause recombinant Chrs if cross over happens within loop • E.g. F8 inversions (intron 22) in haemophilia A 2. a b c d e a a b b c c f f 3. a b c c b d e d e Inversion of b and c e e d d 4. Looped chromosome pair a c b d e Types of structural abnormalities - Translocation • Non-homologous chromosomes exchange segments • Reciprocal translocation – 2 chromosomes swap nonhomologous sections • Chr number and genetic content unchanged • Common (1 in 600 births) • Robertsonian translocation – the break occurs at the short arm of the acrocentric chromosomes (13, 14, 15, 21, 22, Y) and results in a fusion chromosome • Change in Chr number (45) – short p arms lost • 4% of Down Syndrome is translocation (familial) DS – rob(14;21) or rob(21;21), +21 16 Balanced vs Unbalanced Translocations • • • • Balanced – No extra or missing material (reciprocal) DNA just packaged incorrectly Phenotype usually normal* Robertsonian translocations are also considered balanced • Unbalanced – Pathogenic extra or missing material • Usually result in miscarriage or baby with multiple congenital abnormalities • NB – Parent with balanced reciprocal translocations can generate gametes with balanced or unbalanced chromosome complement - Increased risk for all their children 17 Chronic Myeloid Leukaemia (CML) • 1st malignant disease in humans for which a specific chromosomal abnormality was detected bcr • Reciprocal translocation between q ends of Chr 9 and 22 t(9,22)(q34.1;q11.2) • Philadelphia chromosome is a somatic mutation – not inherited • BCR-ABL fusion protein with “always on” tyrosine kinase activity that promotes cellular proliferation bcr abl Philadelphia Chr abl Chr 9 Chr 22 Translocation t(9,22) FISH t(9,22) (yellow signal) • Gain-of-function mutation for haematopoietic stem cells 18 Acute Promyelocytic Leukaemia (APML) • Cancer of the bone marrow/white blood cells – abnormal accumulation of immature granulocytes (promyelocytes) in the bone marrow – shortage of normal white, red and platelets • The presence of promyelocytes containing multiple Auer rods on the peripheral blood smear is highly suggestive of APML • Characteristic cytogenetic abnormality in 95% • Reciprocal translocation between Chr 15 and Chr 17 t(15;17)q(24;21) • Somatic mutation (not inherited) RAR • Visible on Conventional Karyotype PML • Exchange of material between Long arm of Chr 15 (15q24) and long arm of Chr 17 (17q21) • Fusion protein = PML-RARα Chr 15 Chr 17 19 Things to Remember 1. Chromosomes are made of chromatin; a supercoiled DNA/protein complex resembling a string of beads 2. The basic unit of chromatin (the bead) is the nucleosome; an 146 base pair length of ds DNA wrapped around a histone octamer 3. Euchromatin is loosely packed and transcriptionally active; it contains unmethylated cytosines and acetylated histones 4. Heterochromatin is tightly packed and transcriptionally silent; it contains methylated cytosines and deacetylated histones 5. A parent with balanced reciprocal translocation can be phenotypically normal but will generate gametes with balanced and unbalanced chromosome complement 6. CML and APML are examples of genetic diseases that occur via translocation and are non-hereditary (somatic mutations) 21