Role of Genetic PolymorphismsRole of Genetic Polymorphismsin Responses to Toxic Agentsin Responses to Toxic Agents• Definitions• “Forward genetics” and toxicology• “Reverse genetics” and toxicology• Genetic markers• SNPs and their use in toxicology• Ethical, Legal and Social Issues (ELSI)“Toxicology is concerned with the interaction between xenobiotics and biological molecules directly or indirectly coded in the DNA, and can be regarded as a branch of GENETICS.”Michael F.W. Festing (2001)Gregor Mendel (1822 – 1884)TERMINOLOGYTERMINOLOGYGene: A sequence of DNA bases that encodes a proteinAllele: A sequence of DNA basesLocus: Physical location of an allele on a chromosomeLinkage: Proximity of two alleles on a chromosomeMarker: An allele of known position on a chromosomeDistance: Number of base-pairs between two allelescentiMorgan: Probabilistic distance of two allelesPhenotype: An outward, observable character (trait)Genotype: The internally coded, inheritable informationPenetrance: No. with phenotype / No. with alleleModified from M.F. Ramoni, Harvard Medical SchoolThe 80s Revolution and the Human Genome ProjectGenetic Polymorphisms: naturally occurring DNA markers that identify regions of the genome and vary among individualsThe intuition that polymorphisms could be used as markers sparkled the revolutionOn February 12, 2001 the Human GenomeProject announced the completion of a firstdraft of the human genome and declared:“A SNP map promises to revolutionize both mapping diseases and tracing human history”SNP are Single Nucleotide Polymorphisms – subtlevariations of the human genome across individualsModified from M.F. Ramoni, Harvard Medical SchoolDISTANCES ON A GENETIC MAPDISTANCES ON A GENETIC MAP• Physical distances between alleles are base-pairs• But the recombination frequency is not constant• A useful measure of distance is based on the probability of recombination: the Morgan• A distance of 1 centiMorgan (cM) between two alleles means that they have 1% chance of being separated by recombination• A genetic distance of 1 cM is roughly equal to a physical distance of 1 million base pairs (1Mb)Modified from M.F. Ramoni, Harvard Medical SchoolPhysical Maps: maps in base-pairsHuman physical map: 3000Mb (Mega-bases)Genetic Maps: maps in centiMorganHuman Male Map Length: 2851cMHuman Female Map Length: 4296cMCorrespondence between maps:Male cM ~ 1.05 Mb; Female cM ~ 0.88MbMORE TERMINOLOGYMORE TERMINOLOGYModified from M.F. Ramoni, Harvard Medical SchoolSingle Gene (Mendelian) diseases:Autosomal dominant (Huntington)Autosomal recessive (Cystic Fibrosis)X-linked dominant (Rett)X-linked recessive (Lesch-Nyhan)Today, over 400 single-gene diseases have been identifiedProblem: traits don’t always follow single-gene modelsComplex Trait: phenotype/genotype interactionMultiple cause:multiple genes in several loci determine a phenotype in conjunction with non-genetic factors (accidents of development, social factors, environment, infections, other factors)Multiple effect:gene causes more than one phenotypeSimple and Complex TraitsSimple and Complex TraitsModified from M.F. Ramoni, Harvard Medical SchoolToxicology Toxicology ≈≈GeneticsGeneticsThere is substantial polymorphism in genes that determine the response to xenobiotics both in humans and animalsThis has important implications for toxicology and pharmacology:• adverse reactions to drugs cause thousands of deaths each year and many of those are associated with susceptible phenotypes• are we protecting the most sensitive in human population when occupational/environmental limits of exposure are established?• how to account for strain differences in susceptibility in animal studies (1000-fold differences have been reported for TCDD LD50in rats)?• genotyping of individuals from a sample of blood DNA is becomingincreasingly easy so it is possible to genotype people for loci that are thought to control susceptibility to certain drugs/xenobioticsAdapted, in part, from M.F.W. Festing, Tox. Lett. 120:293-300 (2001)…loci that are thought to control susceptibility to certain drugs/xenobiotics:Before we can correctly interpret genotyping results we need to:• gain a much better understanding of the genetics of susceptibility • know the mode of action of xenobioticsProblem: relatively little research is done on the genetics of susceptibility and toxicologists in general seem to be unaware of the extent of genetic variation in responseamong the experimental animals that are being usedProblem: modes of action of an overwhelming majority of established toxic substances are still largely unknown (not even worth mentioning scores of compounds that are being newly developed)Adapted, in part, from M.F.W. Festing, Tox. Lett. 120:293-300 (2001)Adapted from: Huang, 2002Genotype-Phenotype Interactions in Complex Biological SystemsAgeEnvironmentGenetics in ToxicologyGenetics in ToxicologyPhenotype (e.g., toxic symptoms, cancer)Genes that control susceptibility/resistance“Forward Genetics”“Reverse Genetics”Genotype (gene knockout, polymorphism, etc.)PhenotypeAdapted, in part, from M.F.W. Festing, Tox. Lett. 120:293-300 (2001)Studying mechanismsof actionStudying mechanismsof action““Forward Genetics” and ToxicologyForward Genetics” and ToxicologyDifferent animal strains nearly always respond differently to the same agent/dose unless the toxic insult is so dramatic that all the animals die very quicklyExamples of strain differences (rats) in response to xenobiotics:3,2’-dimethyl-4-aminobiphenyl Æ prostate tumors48% F344, 41% ACI, 13% LEW, 7% CD, 0% WistarN-methyl-N-nitro-N-nitrosoguanidine(MNNG) Æ stomach adenocarcinomas67% WKY, 60% S-D, 53% LEW, 23% Wistar, 6% F344There is no such thing as an “animal strain that is particularlysusceptible/resistant to carcinogenesis” !Adapted, in part, from M.F.W. Festing, Tox. Lett. 120:293-300 (2001)Adapted, in part, from M.F.W. Festing, Tox. Lett. 120:293-300 (2001)““Forward Genetics” and ToxicologyForward Genetics” and ToxicologyDesigning an IDEAL “forward genetics” animal study for investigating genetic variability in response to a toxic agent:• Survey the known facts about susceptibility in different strains of rodents• Small numbers of animals (4-6 per strain) of several strains should be used to characterize the response to the toxic agent “X”• At least 5 strains should be studied• Dose levels should be selected
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