Molecular Diagnostics Lecture 5 Mutation and Cancer Genes PDF

MOLECULAR DIAGNOSTICS Lecture 5 Mutation and Cancer Genes MUTATION, MUTAGEN AND CARCINOGEN Heritable change in the length or base sequence of DNA caused by a mutagenic agent Gene Mutations Classification • Inherited (germ line) • mutations are inherited from a parent and are present throughout a...

MOLECULAR DIAGNOSTICS Lecture 5 Mutation and Cancer Genes MUTATION, MUTAGEN AND CARCINOGEN Heritable change in the length or base sequence of DNA caused by a mutagenic agent Gene Mutations Classification • Inherited (germ line) • mutations are inherited from a parent and are present throughout a person’s life in virtually every cell in the body. • These mutations are also called germline mutations because they are present in the parent’s egg or sperm cells, which are also called germ cells. • Acquired (somatic/sporatic) • mutations occur at some time during a person’s life • present only in certain cells, not in every cell in the body. • These changes can be caused by environmental factors such as ultraviolet radiation from the sun, or can occur if a mistake is made as DNA copies itself during cell division. • Acquired mutations in somatic cells (cells other than sperm and egg cells) cannot be passed on to the next generation. Mutations At Chromosomal Level • Deletions • Duplications • Inversions • Translocations t[#,#] Type of Chromosomal Structure Abnormality Examples Of Mutations At The Chromosomal Level • Acute Lymphoblastic Leukemia - d9p • Retinoblastoma - d13q14 • Rare form of cancer that rapidly develops from the immature cells of a retina, the light-detecting tissue of the eye. It is the most common malignant cancer of the eye in children. • Chronic Myelogenous Leukemia(CML): Philadelphia Chromosomet(9:22) • Burkitts Lymphoma- most common variant: t(8;14)(q24;q32) Chronic Myelogenous Leukemia (CML) Philadelphia Chromosome- T(9:22) • C-abl encodes a cytoplasmic tyrosine kinase • Bcr promotes oligomerization • Bcr-abl fusion promotes activation of abl by oligomerization induced autophosphorylation • Translocation resulting in fusion of 2 genes with alters structure of normal cabl protein • Philadelphia chromosome – translocation of chr 9 and 22 Burkitts Lymphoma-most Common Variant T(8;14)(q24;q32) • MYC is brought under the transcriptional control of the IG enhancer elements leading to its constitutive transcriptional deregulation. • Associated with the Epstein-Barr Virus (EBV) in nearly 100% of cases in endemic form in central Africa. • In the sporadic forms (occur in Western countries), EBV is present in approximately 30% of cases and in 40% of immunodeficiency-associated cases Mutations At The DNA Level • Base substitutions (Point Mutation) • transition (purine for purine, pyrimidine for pyrimidine) • transversion (purine for pyrimidine) • Frame shift mutation • Deletion • Insertion • SNP (when leads to risk factor and/or risk marker in disease) Consequences Of Point Mutations • Silent • Missense • hemaglobinopathies (sickle cell, T for A; glutamic acid to valine) • Nonsense-change to stop codon • Alpha thalassemias • decreased protein produced (alpha vs beta globin) • Constant Spring: from stop codon to glutamine @ 142 (TAA to CAA) in alpha-thalassemia Trinucleotide Repeat Diseases Fragile X: FMR-1(fragile -X mental retardation protein  Excessive amplification of a base triplet normally repeated only a few to 50 times, most commonly in the 5' untranslated region of FMR1 Fragile X: FMR-1(fragile -X mental retardation protein: > 200 repeats of CGG) Premutation alleles: generally considered to be between 55 and 200 repeats in length. Huntingtons Disease Caused by the length of a repeated section (CAG, codes for Gln) of a gene exceeding a normal range. The HTT gene is located at 4p16.3. >40 trinucleotide repeats – full penetrance ie. the proportion of individuals carrying a particular variant of a gene (allele or genotype) that also expresses an associated trait (phenotype) METHODS OF MUTATION DETECTION GENES AND CANCER Modern Knowledge About Cancer Causes • Viral and chemical carcinogens, in late 1970 • Cancer control gene modification (mutation) 1990 and after • Oncogenes • Tumor suppressor genes • DNA repair genes • Causes of genetic modification/mutation may lead to cancer: • Physical/chemical • Viral infection • Genetic alterations that stimulate the rate of mitosis • Oncogene or tumor suppressor mutation/amplification/translocations Mutagen And Carcinogen • A mutagen is something that causes a mutation in an organism's genetic code (DNA). Mutations can be neutral, beneficial, or harmful (although they are rarely beneficial.) • Carcinogen is a type of mutagen that specifically leads to the development of cancer. • metabolism transforms procarcinogen into carcinogen • benzopyrenes of smoke • Aflatoxin of peanuts (most important mycotoxins produced from Aspergillus Physical-Chemical Carcinogens • Spontaneous (deamination C > U; uracil-DNA-glycosidase) • Chemicals • • • • Non-alkylating (nitrous acid, formaldehyde) Alkylating-DMS, methylnitrosourea) benzopyrenes of smoke Aflatoxin of peanuts (most important mycotoxins produced from Aspergillus) • UV light-thymine dimers • Ionizing Radiation-strand breaks Viral Carcinogens Viruses- introduction of foreign DNA (transformation) Epstein-Barr virus, one of the herpes viruses Burkitt’s Lymphoma-involves c-myc and IGH@. Causes infectious mononucleosis and linked to non-Hodgkin lymphomas and nasopharyngeal cancer. Human T-cell Leukemia (HTLV-1) The cancer is thought to be due to the pro-oncogenic effect of viral DNA incorporated into host lymphocyte DNA HIV/ Kaposi’s Sarcoma and non-Hodgkin lymphoma. The Kaposi's sarcoma-associated herpesvirus (KSHV), also called HHV Long-standing infection with the hepatitis B or C viruses can lead to cancer of the liver Certain Human Papillomavirus Types May Cause Cancer Early genes of HPV E1: viral replication E2: transcriptional regulation E4: disrupts cytokeratins E5: interacts with growth factor receptors E6: transforming protein; causes p53 degradation E7: transforming protein; interfers with Rb binding E7 being largely responsible for driving cell proliferation and E6 for enhancing cell survival The combination of E6 and E7 is a very potent inducer of keratinocyte immortalization Cancer Is Caused By Nonlethal Genetic Mutations Affecting Certain Genes • Oncogenes: as proto-oncogenes, normally promote cell division or cell survival. • Oncogene mutations are usually a gain of function and dominant. • Tumor suppressors: genes normally arrest cell division. • Tumor suppressor gene mutations are usually a loss of function and recessive Oncogenes • Dominantly acting gene involved in up-regulated cell growth and proliferation responsible for tumor development • Derived from normal cellular genes (by viral enhancement, mutation, inappropriate overexpression) that function in cell growth and division. Tumor Suppresser Genes (Anti-oncogenes) Code for proteins that regulate cell-cycle progression, and hold cells in quiescence or induce apoptosis if conditions are unfavorable for cell cycle progression Prevent cells from becoming cancerous Retinoblastoma (Rb) p53 Proto-oncogene and Tumor Suppressor Gene Products Regulate Cell Proliferation Regulated by tumor suppressor genes Proto-oncogene products Cell growth and proliferation Loss or mutation of tumor suppressor genes Oncogenes Normal Abnormal cell growth and proliferation Cancer Neoplasia Designations • Proto-oncogenes and oncogenes have italicized three letter designations, such as ras. • An oncogene that occurs within a virus has the prefix “v”. v-ras • An oncogene that occurs within a cell has the prefix “c”. c-ras • A protein that a c-proto-oncogene or c-oncogene encodes has the same three letter designation as the v-proto-oncogene or v-oncogene. However, the term is not italicized, and the first letter is capitalized. Ras Human Oncogenes Types Of Proteins Encodes By Oncogenes Oncogene by function: • Growth factors • Growth factor receptors • G proteins and Intracellular kinases • Cell cycle control proteins • Apoptosis control protein • Transcription factors Oncogenes By Location Secreted c-sis wnt1 int1 c-erbB neu kit mas gsp Membrane gip associated ras src Cytoplasmic Nuclear myc myb fos jun rel erbA abl fps raf mos Vav AKT Transmembrane The EGFR Gene Family Human Chromosome Locations Of Proto-oncogenes Tumor Suppressor Genes Knudson Hypothesis • Also known as the two-hit hypothesis or multiple-hit hypothesis • cancer is the result of accumulated mutations to a cell's DNA • Knudson's hypothesis refers specifically to the heterozygosity of tumor suppressor genes. A mutation in both alleles is required, as a single functional TSG is usually sufficient. • Carcinogenesis (the development of cancer) depended both on the activation of proto-oncogenes (genes that stimulate cell proliferation) and on the deactivation of tumor suppressor genes (TSG), which are genes that keep proliferation in check. LOSS OF A TUMOR SUPPRESSOR GENE LOCUS (ACCORDING TO THE TWO-HIT MODEL) Lost of Heterogeneity (LOH) Retinoblastoma (Rb) • Deletion in long arm (q) of chromosome 13 (d13q14) • Retinoblastoma protein (RB) is a tumor suppresser that controls the cell cycle • Autosomal recessive inheritance Inherited form: • one chromosome has deletion • second copy lost by somatic mutation • Sporadic form: both copies lost by individual somatic mutations 2nd mutation Normal Functions Of TP53 Protein G1 growth arrest Induces apoptosis following DNA damage Inhibits tumor cell growth Inhibits S phase Represses transcription factors for growth and replication Cancer may involve deletion, mutation, or functional inactivation of TP53 protein Several Components Concerned With Cell Cycle Control Are Found As Tumor Suppressors Rb is phosphorylated to pRb by certain cyclin-dependent kinases (CDKs). pRb is described as being hyperphosphorylated and when in this state, it is unable to complex E2F and therefore, unable to restrict progression from the G1 phase to the S phase of the cell cycle. During the M-to-G1 transition, pRb is progressively dephosphorylated by PP1, returning to its growth-suppressive hypophosphorylated state Rb Mutation In P53 Gene/TP53 Protein - In humans, a common polymorphism involves the substitution of an arginine for a proline at codon position 72. - A 2011 study found that TP53 codon 72 polymorphism associated with an increased risk of lung cancer - Another 2011 study found the p53 homozygous (Pro/Pro) genotype was associated with a significantly increased risk for renal cell carcinoma Summary Cancer Genes’ Abnormalities Point mutation within the proto-oncogene/ oncogene (RAS-GTP) Genetic rearrangements within the coding sequence of the proto-oncogene (c-abl/bcr in CML) Genetic rearrangements outside the coding sequence of the protooncogene (c-myc Burkitt’s Lymphoma) Amplification and/or over expression of the proto-oncogene  insertion of a strong viral promotor gene copy number variation Deletion of a tumor suppresser gene Other Cause Of Cancer Epigenetic overexpression of genes Mutations outside of the structural gene may cause over expression (high enhancer binding, decreased repressor binding) Angiogenesis-The formation of new blood vessels  . Tumors need blood vessels to grow and spread.  The physiological process through which new blood vessels form from pre-existing vessels.  Different from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors  VEGF, VEGF-R, angiopoietin-2, Tie1, Tie2 etc  Angiogenesis inhibitors for cell migration, proliferation, adhesion: TSP-1, TSP-2 and Angiostatin Multistep Model Of Colon Carcinogenesis • Initiation • 1st mutation • Increased proliferation of a single cell • Progression • Additional mutations MCC = Mutation of colon cancer genes DCC = Deletion of colon cancer genes • Selection for more aggressive cells- CLONAL SELECTION Multi-step Model for Colon Cancer MCC = Mutation of colon cancer genes (APC) DCC = Deletion of colon cancer genes