Mutations: Molecular Biology and Diagnostics PDF

MOLECULAR BIOLOGY AND DIAGNOSTICS MBD_100 Mutations Michael O. Baclig, PhD, MSc, RMT, CBO Mutations Heritable changes in the nucleotide sequence of a given DNA Substitution, deletion or insertion of one or more nucleotides Could affect or not a given phenotype The...

MOLECULAR BIOLOGY AND DIAGNOSTICS MBD_100 Mutations Michael O. Baclig, PhD, MSc, RMT, CBO Mutations Heritable changes in the nucleotide sequence of a given DNA Substitution, deletion or insertion of one or more nucleotides Could affect or not a given phenotype The major basis of diversity among organisms The raw material of evolution Caused (mostly) by mutagens (e.g., base analogs: 5-bromouracil (5-BU), 2-amino-purine; alkylating agents: ethylmethane sulfonate (EMS); intercalating agents: proflavine, acridine orange, ethidium bromide) DNA damage: UV radiation, ionizing radiation (e.g., gamma rays, X-rays), byproducts (e.g., reactive oxygen species such as superoxides, hydroxyl radicals) Can be repaired by the body in normal conditions (e.g., base excision repair, nucleotide excision repair, mismatch-repair system) Classifications of Mutations Based on nature of occurrence Spontaneous - arise in the absence of known mutagen Induced - presence of mutagens or environmental agents Based on cell type where it occurs Somatic - originates in mitosis and affects subset of cells Gametic - originates in meiosis and affects all cells of an individual o Autosomal o X-linked Molecular Basis of Mutation Substitution: change in the sense of information (missense) Transition - purine to purine or pyrimidine to pyrimidine Transversion - purine to pyrimidine or vice versa Frameshift: deletion or insertion of one base Base substitution or point mutation GTT TTA GAT CCG TAC ATT TTA GAT CCG TAC | | | | | | | | | | val leu asp pro tyr Ile leu asp pro tyr Silent mutation changes 1 codon for an amino acid into another codon for that same amino acid ATC TTA GAT CCG TAC ATC TTG GAT CCG TAC | | | | | | | | | | Ile leu asp pro tyr Ile leu asp pro tyr Missense codon for 1 amino acid is replaced by a codon for another amino acid Missense Synonymous missense – codon specifies chemically similar amino acid AAA → AGA K R Basic Basic Does not alter protein function in many cases Missense Nonsynonymous missense - codon specifies chemically dissimilar amino acid UUU → UCU F S Phenylalanine (F): non-polar, hydrophobic Serine (S): polar, hydrophilic Point mutations and multiple variants in the “a determinant" region can destroy the antigenicity and immunogenicity of HBV Kyte-Doolittle scale is widely used for detecting hydrophobic regions in proteins Nonsense - codon for 1 amino acid is replaced by a translation termination codon Insertion Deletion Frameshift  Translocation A region from one chromosome is aberrantly attached to another chromosome (e.g., t (8;14) and t (9;22) Chromosomal Analysis AKA Karyotyping Giemsa G-banding: treat metaphase spread with trypsin that digests part of chromosomal protein. Stain with Giemsa and observe under light microscope. Quinacrine Q-banding: fluorescent staining method which uses quinacrine to identify chromosomes and their structural anomalies Reverse R-banding: involves denaturing in hot acidic saline followed by Giemsa staining. Centromere C-banding: used in identifying heterochromatin by denaturing chromosomes in a saturated alkaline solution followed by Giemsa staining. Chromosomal Aberrations Numerical chromosome Sex chromosome abnormalities Structural chromosome abnormalities abnormalities Trisomy 21 (47, + 21) Turner syndrome (45, X) Deletion Trisomy 18 (47, + 18) Klinefelter syndrome (47, XXY) Duplication Trisomy 13 (47, + 13) Translocation Inversion  Trisomy 21 Trisomy 18 Trisomy 13 Down Syndrome (47, + 21) Edward Syndrome (47, + 18) Patau Syndrome (47,+ 13) Down Syndrome Edward Syndrome Patau Syndrome Turner Syndrome Klinefelter Syndrome  Fluorescence in situ hybridization (FISH) A laboratory technique for detecting and locating a specific DNA sequence on a chromosome. The technique relies on exposing chromosomes to a small DNA sequence called a probe that has a fluorescent molecule attached to it. The probe sequence binds to its corresponding sequence on the chromosome. Useful in disorders with very small aberrations or samples with no metaphase cells available. Steps in FISH Denaturation of the sample Hybridization of probe to target cells Washing Detection (fluorescent microscopy) Applications Detection of BCR-ABL (Breakpoint Cluster Region-Abelson) gene fusion by FISH using bone marrow aspirate or peripheral blood Detection of AML-ETO (Acute Myeloid Leukemia) gene fusion by FISH using bone marrow aspirate or peripheral blood Detection of PML-RAR (ProMyelocytic Leukemia-Retinoic Acid Receptor) gene fusion by FISH using bone marrow aspirate Detection of Y-Chromosome for sex-mismatched bone marrow transplantation by FISH using bone marrow aspirate