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VARIABILITY AND CHANGES OF GENETIC INFORMATION, MUTATIONS RNDr Z.POLÍVKOVÁ LECTURE NO 421– COURSE: HEREDITY Mutation = permanent heritable change of genetic material = change in nucleotide sequence or arrangement of DNA in the genome Genetic changes are frequent Most of genetic changes are functionally insignificant, some have minor effect on phenotype, some are deleterious (cause genetic disorder or miscarriages) Some genetic variants are called polymorphisms Mutations x polymorphisms Many genes have only one normal version = wild type allele Other genes exhibit polymorphism (many forms) in population Normal variants (alleles) are relatively common in population Variant allele found in more than 1% in population = polymorphism; this definition is independent of functional or pathogenetic relevance of alteration – most of common variants (polymorphisms) are without effect on human health, but some can modify the risk of common diseases (as tumors…) Alleles with frequencies of less than 1% are rare variants Mutant alleles are rare variants – identified through clinically significant disorder (disease-causing variants) More mutant alleles at same locus (each capable of producing an abnormal phenotype)= allelic heterogeneity But some of rare variants appear to have no deleterious effect, i.e. there is „grey zone“ between definitions used for mutation and polymorphism Types of polymorphism: Single nucleotide polymorphisms (SNPs) 90 % of all DNA variants are exchanges of single nucleotide bases - in coding or noncoding regions of human genome Frequent SNPs are mostly without effect or have subtle effect (alter disease susceptibility) Occurence 1/1000 base pairs Other variants: deletions, duplications, multiplications of several nucleotides or larger genome segments Different alleles are due to variable numbers of repetitions at particular location Effect: some are without effect to human health, some are pathogenetic, some can modify risk of disease Mutations: Mutations: spontaneous (errors in replication) induced (by mutagens) Mutations: somatic – consequences: tumors, ageing (accumulation of mutations) gametic – consequences in next generation: genetic disorder or carrier of mutation Mutations: • genome mutations – changes in chromosome number: a) euploid change = multiplication of haploid chromosome set (triploidy, tetraploidy) b) aneuploidy = additional chromosome (trisomy) or missing chromosome (monosomy) • chromosome mutations= structural aberrations of chromosomes =consequences of breaks and abnormal rearrangement of chromosomal segments – detected in light microscope submicroscopic deletions or duplications of large genomic segments = copy number variants CNVs (consequences:benign or pathogenic, or risk modifiers for common diseases) • gene mutations= qualitative or quantitative changes in DNA coding sequences point mutation affects one single base pair GENE mutations mutation without any change of amino acid (degeneration of genetic code) • MISSENSE mutation.........replacement of one amino acid by different amino acid in protein • NONSENSE mutation.........mutation generates one of three „stop“ codons → premature termination of translation • ELONGATION mutations.....change of stop codon to amino acid coding triplet • FRAMESHIFT mutations......insertions, deletions of coding nucleotides in a number not divisible by three Mutations in rRNA and tRNA genes - error in translation Mechanisms of mutations Single nucleotide SUBSTITUTION = base exchange – (transition = purine (A,G) for purine, pyrimidine (C,T) for pyrimidine or transversion = purine for pyrimidine or vice versa) e.g. base alkylation, oxidation, deamination leads to change of pairing properties and change of nucleotide during replication i.e. it alters triplet code → replacement of one amino acid by another in the gene product (missense mutation) consequences: enzyme inactivity or changed specificity altered properties or structure of protein → stop codon (nonsense mutation) – loss of protein function → elongation mutations - loss of protein function INSERTION DELETION - frame shift mutations (number of bases involved is not divisible by three) - alters translational reading frame→ premature stop codon Mutation in promotor region affect gene expression - impaired binding of transcription factors leads to reduced transcription Mutation on the boundary of exons and introns interfere with proces of splicing Mutations leading to loss of enzyme function mostly express itself as recessive Mutations leading to gain of abnormal function or to origin of abnormal structural protein – mostly express itself as dominant Examples of mutations: A) SUBSTITUTION (alkylation, methylation, hydroxylation→error in base pairing) nucleotide substitution = replacement of one amino acid by another → MISSENSE MUTATION a) Change inside coding sequences • in sickle cell disease G A G → G T G in β-globin gene–replacement of amino acid glu → val HbA → HbS b) Mutation outside coding sequences • in hemophilia B: change A → G in promotor of gene for antihemophylic factor IX = prevention of transcription factor binding → decrease in the amount of product → NONSENSE MUTATION - generates stop codon → abnormal product • in neurofibromatosis - NF1 gene C GA → T G A arg → stop NF1 = tumor supressor gene premature termination of translation RNA SPLICING mutation – on boundary between exon and intron in Tay-Sachs disease mutation in hexosamidase A gene - intron between 12. and 3. exon is not removed Defect of hexosamidase A enzyme B) DELETION, INSERTION (deletion of 1 or more base-pairs, deletion of a part of gene, deletion of whole gene, or deletion of several genes = microdeletion syndromes) a) small number of base-pairs (not divisible by three) frameshift mutation • in ABO blood groups deletion G T G → single base-pair deletion at the ABO locus alters reading frame (allele A → allele O) • in Tay – Sachs disease 4 base-pairs insertion → frameshift leading to the origin of premature stop codon = deficiency of enzyme hexosaminidase A b) 3 or a multiple of 3 bases • in cystic fibrosis the most frequent mutation = 3 base-pair deletion → 1 amino acid is missing (delta F 508 = fenylalanin is missing) c) Total gene deletion • in X- linked ichtyosis deletion of steroid sulphatase gene d) Large deletion within gene • in: Duchenne muscular dystrophy large deletion within dystrophin gene (in 60 % of cases) Origin of large deletion and insertions: •Unequal crossing over or exchange between misaligned sister chromatids or homologous chromosomes (aberrant recombination) • deletion of -globin gene in -thalasemia • deletions of pigment genes in X-linked defect in green and red color perception • deletion of retinoblastoma gene (Rb1) • Error in replication Mutagens Physical: radiation • UV (ultraviolet radiation) → T-T, C-C, T-C dimers (covalent bonds) → error in replication and transcription • ionizing (rtg, gamma) direct effect → DNA breaks indirect effect = ionization of molecules → DNA breaks Chemical – alkylating agents – adducts – base substitution - base analogs – error in base pairing - acridine dyes – insertions – frame shift mutations - nitric acid – base deamination – error in base pairing direct mutagens indirect mutagens – reactive product arises after metabolic activation (cytochrom dependent oxygenases) Biological –viruses - viral nucleic acid integrates into the genome of host cell Dynamic mutations – gradual origin = amplification of triplet repeats - in fragile X syndrome, Huntington disease… Origin through premutation in previous generation This type of mutation is not caused by the environmental mutagens ! Genetics of cancers Forms: sarcomas – mesenchymal tissue carcinomas – epithelial tissue hematopoetic and lymphoid malignancies (leukemias, lymphomas) Uncontrolled growth – invasivity, metastases Tumor cells in tissue culture: • loss of contact inhibition • changes in surface antigens • chromosomal changes • unlimited number of cell generations Genetic nature of cancers 5% familiar (AD with reduced penetrance) multifactorial All cancers – mutations of specific genes in somatic cells (growth controlling genes): 1. protooncogenes 2. tumor suppressor genes 3. mutator genes = genes involved in reparation→ increased frequency of mutations CARCINOGENESIS =multistep process – genetic + environmental factors Multiple mutations (growth controlling genes) Multiple causes and mechanisms Environmental factors: • chemical carcinogens • UV, ionizing radiation • tumor viruses – RNA, DNA viruses Mutations – role in iniciation of carcinogenesis Clonal nature of tumors – origin from single cell Genetic change in one cell and division of cell Protooncogenes: control of cell proliferation, differentiation Protooncogenes products: role in cell communications in transport of signal from cell surfice to the genes which regulate cell cycle Protooncogenes: signal molecules, their receptors, tyrosin kinases, transcription factors, cell cycle regulation proteins… Change of protooncogenes to oncogenes → abnormal cell division Mechanisms: 1.gene mutation 2. translocation 3. retroviral insertion 4. amplification – double minutes, homogenously staining regions = amplified copies of protooncogenes 5. error in gene methylation (gene expression) = epigenetic changes Consequences of change of protooncogene to oncogene • synthesis of abnormal product • increased synthesis of normal product Dominant character of mutation of protooncogene (change in one allele) Examples of chromosomal translocations involving protooncogenes: CML = chronic myelogenous leukemia Ph1 chromosome = t(9q;22q) = translocation of protooncogene c-abl from 9q to 22q near to protooncogene bcr → fused gene bcr/abl →abnormal protein with stable tyrosinkinase activity = abnormal stimulation of cell division BL = Burkitt lymphoma – t (8q;14q) Protooncogene c-myc transfered from 8q to 14q near to immunoglobuline genes (IgH) → abnormal transcriptional activity of protooncogene in a new position → increased synthesis of normal product Cme.medscape.com Fused gene bcr/abl in CML detected by locus specific probe (FISH) Wysis 1996/97 Fused gene bcr/abl in CML Translocation 8q/14q in Burkitt lymphoma ncbi.nlm.nih.gov Cytogenetic manifestation of amplification: • „double minutes“ – free copies of oncogene • HSR=homogenously staining regions= copies tandemly integrated to chromosome • or copies inserted to different sites of chromosome Tumor suppressor genes Products - suppress cell division and abnormal proliferation loss of function of both alleles→ malignant transformation = recessive character of mutation Example: RB – retinoblastoma – two-step origin of cancer a) Hereditary tumor: bilateral 1st step = germline mutation (or deletion) of one allele of Rb1 gene = heritable or „de novo“ origin in one germ cell of parent (mutation in all cells of body = individual is heterozygote) 2nd step: somatic mutation of the 2nd allele in one cell of retina = loss of heterozygosity (LOH) b) Sporadic form : unilateral mutation of both alleles are somatic - in one cell of retina tumor suppressor gene Rb1 gene on chromosome No 13 Wilms tumor: embryonal tumor of kidney – tumor suppressor gene on 11p Tumor suppressor gene TP53 – protein p53 Manager of genes involved in DNA reparation and apoptosis • blocks cell cycle and starts reparation in G1 or G2 (cell cycle checkpoints) • if DNA damage is unrepaired it starts apoptosis Mutation of TP53 in many tumors Li Fraumeni syndrome = heritable mutation of TP53 = tumor families = tumors in young people in family Mutator genes Genes responsible for DNA repair - Mutations have recessive character Example: heritable nonpolyposis colon cancer Role of viruses in tumorigenesis Neoplastic proliferation: 1. Integration of viral promoters („enhancers“) to the host genome near the cell protooncogenes → increased expression of the cell protooncogenes = latent tumor viruses 2. Insertion of viral oncogenes = acute tumor viruses DNA viruses - oncogene = viral oncogene RNA viruses – transmit cell protooncogene Retroviruses = RNA tumor viruses Their oncogenes – homologous to cell protooncogenes viral oncogenes – without introns Probable origin = from cell protooncogenes = Integration of virus (DNA after reverse transcription) to host genome, replication and transcription with host genome mRNA protooncogene transcript after introns splicing is „picked up“ by virus altogether with viral genome and transfered to other cell (e.g. Rous sarcoma virus) Viral infection: Viral RNA → DNA (by reverse transcriptase) integration to the host genome replication, transcription with the host genome translation – complete viral particules oncogene product → cell transformation Other factors of carcinogenesis Different ability of metabolisation of mutagenic and carcinogenic compounds Example: enzyme aryl hydrocarbone hydroxylase (family of cytochrome P450 genes) genetic polymorphisms in drug metabolisation polycyclic hydrocarbons (from cigarette smoke) are converted to epoxydes (carcinogenic metabolites) Individuals with high-inducible allele and smokers = great risk of lung cancer Recessive homozygotes – resistant Individuals with variant alleles – different activity of enzyme DNA reparation – gene polymorphisms – differenet ability to repair DNA damage Immunity T lymphocytes – cell immunity – cytotoxic effect defect in immunity, inborn or acquired(AIDS)→risk of tumors Chemical carcinogens Radiation Viruses Mutagenic, carcinogenic, immunosuppressive efects Complex origin of tumors Multistep origin of colon cancer - multiple genetic changes Mutation/deletion tu su gene MCC 5q 1.st step also heritable change– mutation on 5q –in polyposis coli, Gardner sy Increased proliferation Adenoma III Mutation K-ras oncogene 12p Adenoma I Mutation/deletion chrom.loss tu su gene p53 on 17p Mutation/deletion, chrom.loss tu su gene DCC 18q Normal cell Adenoma II DNA hypomethylation Carcinoma metastasis Genotoxic effects: • mutagenic • carcinogenic • teratogenic – affects embryonal development • immunosuppressive • allergenic Each mutagen = possible carcinogen But not all carcinogens are mutagenic (nongenotoxic carcinogens) Thompson &Thompson: Genetics in medicine,7th ed. Chapter 9: Genetic variation in individuals and population: Mutation and polymorphism (till page 184) Chapter 16: Cancer genetics and genomics + informations from presentation http://dl1.cuni.cz/course/view.php?id=324