Role of Genetic Polymorphisms i R t T i A tin Responses to ... · Role of Genetic Polymorphisms i R t T i A tin ... among the experimental animals that are being used ... facts about

Role of Genetic PolymorphismsRole of Genetic Polymorphismsi R t T i A ti R t T i A tin Responses to Toxic Agentsin Responses to Toxic Agents

• Definitions• “Forward genetics” and toxicologyg gy• “Reverse genetics” and toxicology• Genetic markers• SNPs and their use in toxicology• Ethical, Legal and Social Issues (ELSI), g ( )

“Toxicology is concerned with the interaction between xenobioticsd bi l i l l l di tl i di tl d d i th DNA dand biological molecules directly or indirectly coded in the DNA, and

can be regarded as a branch of GENETICS.”Michael F.W. Festing (2001)Michael F.W. Festing (2001)

Gregor Mendel (1822 – 1884)

TERMINOLOGYTERMINOLOGY

Gene: A sequence of DNA bases that encodes a proteinAllele: A sequence of DNA basesAllele: A sequence of DNA basesLocus: Physical location of an allele on a chromosomeLinkage: Proximity of two alleles on a chromosomeLinkage: Proximity of two alleles on a chromosomeMarker: An allele of known position on a chromosomeDi t N b f b i b t t ll lDistance: Number of base-pairs between two allelescentiMorgan: Probabilistic distance of two allelesPh A d b bl h ( i )Phenotype: An outward, observable character (trait)Genotype: The internally coded, inheritable informationPenetrance: No. with phenotype / No. with allele

Modified from M.F. Ramoni, Harvard Medical School

The 80s Revolution and the Human Genome Projectthe Human Genome Project

Genetic Polymorphisms: naturally occurring DNA markers that id tif i f th d i di id lidentify regions of the genome and vary among individuals

The intuition that polymorphisms could be used as markers sparkled the revolutionthe revolution

On February 12, 2001 the Human GenomeProject announced the completion of a firstProject announced the completion of a firstdraft of the human genome and declared:

“A SNP map promises to revolutionize both p pmapping diseases and tracing human history”

SNP are Single Nucleotide Polymorphisms – subtlevariations of the human genome across individuals


DISTANCES ON A GENETIC MAPDISTANCES ON A GENETIC MAP

• Physical distances between alleles are base-pairsB t th bi ti f i t t t• But the recombination frequency is not constant

• A useful measure of distance is based on the probability of recombination: the Morganprobability 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)physical distance of 1 million base pairs (1Mb)


MORE TERMINOLOGYMORE TERMINOLOGY

Physical Maps: maps in base-pairsHuman physical map: 3000Mb (Mega-bases)Human physical map: 3000Mb (Mega-bases)

Genetic Maps: maps in centiMorganGenetic Maps: maps in centiMorganHuman Male Map Length: 2851cMHuman Female Map Length: 4296cMHuman Female Map Length: 4296cM

Correspondence between maps:Correspondence between maps:Male cM ~ 1.05 Mb; Female cM ~ 0.88Mb


Single Gene (Mendelian) diseases:

Simple and Complex TraitsSimple and Complex TraitsSingle Gene (Mendelian) diseases:

Autosomal dominant (Huntington)Autosomal recessive (Cystic Fibrosis)( y )X-linked dominant (Rett)X-linked recessive (Lesch-Nyhan)

T d 400 i l di h b id ifi dToday, over 400 single-gene diseases have been identified

Problem: traits don’t always follow single-gene modelsy g gComplex Trait: phenotype/genotype interactionMultiple cause: multiple genes in several loci determine p p ga phenotype in conjunction with non-genetic factors (accidents of development, social factors, environment, infections other factors)infections, other factors)Multiple effect: gene causes more than one phenotype


Toxicology Toxicology ≈≈ GeneticsGeneticsThere is substantial polymorphism in genes that determine the

response to xenobiotics both in humans and animals

This has important implications for toxicology and pharmacology:

• adverse reactions to drugs cause thousands of deaths each year and g ymany of those are associated with susceptible phenotypes

• are we protecting the most sensitive in human population when occupational/environmental limits of exposure are established?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 LD50 in rats)?

• genotyping of individuals from a sample of blood DNA is becoming increasingly easy so it is possible to genotype people for loci that are thought to control susceptibility to certain drugs/xenobioticsthought to control susceptibility to certain drugs/xenobiotics

Adapted, in part, from M.F.W. Festing, Tox. Lett. 120:293-300 (2001)

…loci that are thought to control susceptibility to certain drugs/xenobiotics: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 xenobiotics

Problem: relatively little research is done on the genetics of tibilit d t i l i t i l t bsusceptibility and toxicologists in general seem to be

unaware of the extent of genetic variation in responseamong the experimental animals that are being used

Problem: modes of action of an overwhelming majority of established toxic substances are still largely unknown (not even worth mentioning scores ofunknown (not even worth mentioning scores of compounds that are being newly developed)


Genotype-Phenotype Interactions in Complex Biological Systems

Age

Environment

Adapted from: Huang, 2002

Genetics in ToxicologyGenetics in Toxicology

Phenotype (e.g., toxic symptoms, cancer)

net

ics”

Studying mechanisms

Genes that control susceptibility/resistancerwar

d G

e Studying mechanisms

of action

Genes that control susceptibility/resistance

“For

enet

ics”

Genotype (gene knockout, polymorphism, etc.)

Studying mechanisms

ever

se G

e

Ph t

Studying mechanisms

of action

“Re Phenotype


“Forward Genetics” and Toxicology“Forward Genetics” and ToxicologyDifferent animal strains nearly always respond differently to theDifferent animal strains nearly always respond differently to thesame agent/dose unless the toxic insult is so dramatic that all theanimals die very quickly

Examples of strain differences (rats) in response to xenobiotics:3,2’-dimethyl-4-aminobiphenyl prostate tumors

48% F344, 41% ACI, 13% LEW, 7% CD, 0% Wistar

N-methyl-N-nitro-N-nitrosoguanidine(MNNG) stomach adenocarcinomas

67% WKY, 60% S-D, 53% LEW, 23% Wistar, 6% F344, , , ,

There is no such thing as an “animal strain that is particularly g p ysusceptible/resistant to carcinogenesis” !


Current Approach:

i l di l iAnimal studies Human population

CD-1Pharmaceutical Industry

Single genome-basedrisk prediction

y

B6C3F1

Genetically Diverse National Toxicology Program

yHuman Population

“Forward Genetics” and Toxicology“Forward Genetics” and Toxicology

Designing an IDEAL “forward genetics” animal study forg g g yinvestigating genetic variability in response to a toxic agent:

S th k f t b t tibilit i diff t t i f d t• 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 to elicit a suitable response

E d i t h ld b tit ti ( b f t )• Endpoints should be quantitative (e.g., number of tumors)


Parental strains and derivation of five major types of mouse genetic resources yp g

Each of the sequenced strains is shown in a different color depending on the origin. The four wild-derived strains, denoted by asterisks, are CAST/EiJ (M. m. cataneus) in red, PWD/PhJ (M. m. muculus) in blue MOLF/EiJ (M m molossinus) in muculus) in blue, MOLF/EiJ (M. m. molossinus) in purple, and WSB/EiJ (M. m. domesticus) in green. The remaining 12 classical laboratory strains are shown in green reflecting the predominant contribution of the M. m. domesticus subspecies to these strains. The shade of green denotes the different origin of the classical strains, with the darker shades denoting strains of Swiss origin (FVB/NJ and NOD/LtJ), the yellow-green denoting a strain of Asian origin (KK/HlJ), and intermediate shade denoting Castle or C57-related strains (129S1/SvImJ A/J AKR/J BALB/cBy C3H/HeJ (129S1/SvImJ, A/J, AKR/J, BALB/cBy, C3H/HeJ, DBA/2J, BTBR T+tf/J, and NZW/LacJ).

The figure also shows schematically the derivation process for five types of resources, recombinant inbred lines (BXD); chromosome substitution strains (B.P), Collaborative Cross (CC), heterogeneous stocks (Northport HS) and laboratory strain stocks (Northport HS), and laboratory strain diversity panel (LSDP)

Mamm Genome. 2007 July; 18(6): 473–481

Recombinant inbred strains (RIs)Recombinant inbred strains (RIs)

C57BL/6J (B)C57BL/6J (B) DBA/2J (D)DBA/2J (D)femalefemale malemaleC57BL/6J (B)C57BL/6J (B) DBA/2J (D)DBA/2J (D)

fullyinbredfully

inbredBXDBXD

F1F1isogenicisogenic

chromosome pairchromosome pair

F1F1

hetero-geneoushetero-

geneousF2F2

geneousgeneous

20 generations

brother-sister

matings

20 generations

brother-sister

matings

BXD RIStrain set

BXD RIStrain set

Recombined chromosomes are needed for

mapping

Recombined chromosomes are needed for

mappingInbredInbred

+ … ++ … +BXD1BXD1 BXD2BXD2

matingsmatings

BXD80BXD80

mappingmappingb edIsogenicsiblings

b edIsogenicsiblings

Image Credit: genenetwork.org

Genetic Prediction of Chemical-Induced Organ Injury

A Multi-Strain Model

A multi-strain factorial design which captures the genetic diversity

NZW/LacJ

BuB/BnJTranslate Risk

C57BL/6J

Cast/EiJGenetically Diverse

AKR/Jy

Human Population

Adapted from Petkov et al. Genome Res. 2004; 14: 1806-1811

Profiling Liver Toxicity to APAP in a Genetically Diverse Population

Dose response to liver injury: ALT (24 h)

Dose response to liver injury (4 h) vs survival (24 h)Multi-strain profiling of APAP-induced liver injury:

% liver necrosis (24h), reduced GSH (4h), ALT (24h), ALT (4h)

• Once a susceptible/resistant strains have been identified, loci can be mapped• In mice, Recombinant Inbread strains (susceptible x resistant) can be

generatedgenerated• A set of RI strains can be tested for the susceptibility to agent “X”• Once the phenotype have been established, mice can be genotyped to

determine which loci segregated with susceptibility/resistance

From Zhou et al. (2005)

Problems: large number of animals (100-300, or more)resolution of the genetic mapping is only about ± 20 cM (mouse genome is ~50K genes and 1900 cM 1cM ≈ 0.5 Mb) so the identified locus can contain ~500 genes


“Collaborative Cross” The Resource for Forward Genetics Research

Images from Threadgill DW

“Reverse Genetics” and Toxicology“Reverse Genetics” and Toxicology

A knockout or over-expressor animal strain, or animals with a known polymorphism(s) in important genetic regions

Dose with a chemical(s)

Evaluate the phenotype

Looks MUCH easier than “Forward Genetics” experiment! Let’s do it!p

Problems: if mutant to non-mutant comparison is being made, the geneticbackgrounds MUST be identical !gif the strains have been crossed, care is needed to ensure that the observed differences are not due to a gene closely linked to the gene of interestgenes do not act alone! Several alleles may be important, their effects can be additive or epistatic


Genetic MarkersEven though we share most DNA, there are variations (polymorphisms)

Polymorphic: two or more forms of the same gene or genetic markerPolymorphic: two or more forms of the same gene, or genetic marker exist with each form being too common in a population to be merely attributable to a new mutation

Classes of polymorphic genetic markers:Classes of polymorphic genetic markers:Single Nucleotide Polymorphisms (SNP): single base differences in populationSingle Nucleotide Polymorphisms (SNP): single base differences in population Microsatellites: short tandem repeat (e.g. GATA, 2 – 6 bp long)Minisatellites: simple sequence repeats (10 – 40 bp long)V i bl N b f T d R t th b f tVariable Number of Tandem Repeats: the number of repeats may varyRestriction Fragment Length Polymorphisms: presence/absence of a siteDeletions, Duplications, Insertions: alterations on a chromosome levelComplex haplotypes: combinations of the above

Genetic Markers

Coding:

Single Nucleotide PolymorphismsS g e uc eot de o y o p s s

Restriction Fragment Length Polymorphisms

Deletions Duplications InsertionsDeletions, Duplications, Insertions

Non-coding:Microsatellites

MinisatellitesMinisatellites

Variable Number of Tandem Repeats

Restriction Fragment Length Polymorphisms

Single Nucleotide Polymorphisms

Deletions, Duplications, Insertions

Genetic Markers

• Polymorphisms (allelic variations) are essential to:– Study inheritance patterns

M h t d h t th ti b– Map phenotypes and anchor genes to the genetic map by co-segregation analysis

– Determine change in function: resistant/sensitive populations

• Genetically determined variability among humans is due to a difference in 0.1% of the genomic sequence!g q

• Polymorphisms can be silent, or be exhibited at levels of:M h l– Morphology

– Protein– DNA

Chromosomal rearrangements: Deletions Duplications InsertionsDeletions, Duplications, Insertions

Deletions: a certain part is lost, for example abc ac

Insertions: a part is added, for example ac abcp

Duplications: can be tandem, for example abc abbc, or not for example

Insertion Deletion not, for example abc abcabc

Reversals: a part is turned around, head to tail abc cba

Transpositions: two parts change places, for example p , pabcd acbd

Minisatellites

• Original DNA fingerprinting technique• Relies on stretches of tandemly repeated sequences• Relies on stretches of tandemly repeated sequences

(usually 15 - 100bp)• Alleles show high variability in numbers of repeatsg y p

Genotyping using minisatellites:• Digest genomic DNA• Run out on gel

S th bl t d b ith di l b ll d t DNA• Southern blot and probe with radiolabelled repeat DNA• Individuals appear with a set of bands unique to them,

although each band is shared with one of their parentsalthough each band is shared with one of their parents

Microsatellites

• Number of repeats varies greatly between individualsNumber of repeats varies greatly between individuals• Make up to 10-15% of the mammalian genome• Believed to have no function• Have high mutation rates• Used in forensic analysis• Can be amplified by PCR – fragments that are generated

have different length due to different number of repeats

Microsatellites are highly polymorphic due to potential for “skipping” during DNA replication

Restriction Fragment Length Polymorphisms (RFLPs)

• Consider two alleles having slightly different sequences

GAATTC GCATTCGAATTC GCATTCCTTAAG CGTAAG EcoRI will cut the first but not the second

Variations of a single base between individuals:Single nucleotide polymorphisms (SNPs)

Variations of a single base between individuals:

A most common form of genetic variation in humansThought to be a major cause of genetic diversities amongThought to be a major cause of genetic diversities among different individuals in drug response, disease susceptibility... A SNP must occur in at least 1% of the populationp pOccur every 500-1000 bpAbout 50,000 – 100,000 SNPs in coding sequencesSNPs may occur in coding regions:

cSNP: SNP occurring in a coding regionrSNP: SNP occurring in a regulatory regionrSNP: SNP occurring in a regulatory regionsSNP: Coding SNP with no change on amino acid


Single nucleotide polymorphisms (SNPs)

• Two bases (one for chromosome) for each locusB f th A T C G l t SNP h• Because of the A-T C-G complement, a SNP can have only two variants: (AT) or (CG)

• A SNP is a variable with two states:• A SNP is a variable with two states:Major allele: Allele (AT) or (CG) more frequentMinor allele: Allele (AT) or (CG) less frequentMinor allele: Allele (AT) or (CG) less frequent

• An individual can be, for each polymorphic locus:Homozygous on major alleleHomozygous on major alleleHeterozygous on major/minor alleleHomozygous on minor alleleHomozygous on minor allele

The role of SNP analysis through all stages of drug development

Target Identification: disease association studies identify SNPs in candidate genes. The proteins encoded by such genes may represent novel drug targets

Target Validation: population analysis determines the level of variation within a candidate gene. The presence of several SNPs will generate a large number of potential variants and such candidates can be eliminated

Lead Identification: screens can be developed to identify lead compounds that interact with each variant of the drug target

Lead Validation: biological assays can be performed that incorporate different lead compounds and all variants of the target proteinlead compounds and all variants of the target protein

Lead Optimization: knowledge of polymorphisms affecting the target can be used to develop drugs that work more efficiently over a broader group of patients or to identify drugs that work more efficiently in specific genotypesin specific genotypes

Preclinical Testing: animal models can be developed incorporating all known variants of the target to provide more accurate predictions of drug efficacy in humans

Clinical Trials: trials can be carried out with groups of patients selected on the basis of genotype, to specifically test for adverse drug reactions at particular doses

SNP discovery and SNP genotyping

SNP di d t ti f l l hiSNP discovery: detection of novel polymorphisms• DNA sequencing• In silico: comparing the sequences of genomic clones or ESTs p g q g

deposited in public and proprietary databases• Single strand conformational polymorphisms

SNP genotyping: identification of specific alleles in a known polymorphism1.Allele discrimination: allele-specific PCR, allele-specific single-base primer extension (mini-sequencing), allele-specific ligation, allele-specific enzymatic cleavage, etc.sequencing), allele specific ligation, allele specific enzymatic cleavage, etc.

2. Presence of allele(s) of interest in a given DNA sample:Fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, etc.

See details in: Twyman RM & Primrose SB, Techniques patents for SNP genotyping. Pharmacogenomics 4:67-79 (2003)

Well-studied genetic variants in human disease

From Taylor et al. Trends Mol Med 7:507-512 (2001)

Most drug-metabolizing enzymes exhibit clinically relevant genetic polymorphisms. Essentially all of the majorhuman enzymes responsible for modification of functional groups [phase I reactions (left)] or conjugation withendogenous substituents [phase II reactions (right)] exhibit common polymorphisms at the genomic level;those enzyme polymorphisms that have already been associated with changes in drug effects are separatedfrom the corresponding pie charts. The percentage of phase I and phase II metabolism of drugs that eachenzyme contributes is estimated by the relative size of each section of the corresponding chart. ADH, alcoholdehydrogenase; ALDH, aldehyde dehydrogenase; CYP, cytochrome P450; DPD, dihydropyrimidinedehydrogenase; NQO1 NADPH:quinone oxidoreductase or DT diaphorase; COMT catechol O-dehydrogenase; NQO1, NADPH:quinone oxidoreductase or DT diaphorase; COMT, catechol Omethyltransferase; GST, glutathione S-transferase; HMT, histamine methyltransferase; NAT, N-acetyltransferase; STs, sulfotransferases; TPMT, thiopurine methyltransferase; UGTs, uridine 5'-triphosphateglucuronosyltransferases. From Evans WE and Relling MV Science 286:487 (1999).

Cytochrome P450 genotyping

From: Flockhart DA and Webb DJ. Lancet (1998)

Frueh et al 2008.pdf

FDA OKs Genetic Test Linked to WarfarinSep 17 2007WASHINGTON (AP) - A genetic test that can reveal what patients are especially sensitive to the blood-thinner warfarin won federal approval Monday. Such screenings could prevent thousands of complications each year, health officials estimate.federal approval Monday. Such screenings could prevent thousands of complications each year, health officials estimate.

The approval of the test comes a month after warfarin, sold under the brand name Coumadin and in generic forms, became the first widely used drug to include genetic testing information on its label. The information can help doctors determine how best to prescribe the drug.

An estimated one-third of patients process the drug differently than do most others, exposing them to a higher risk of bleeding Research suggests that most of that sensitivity is due to variations in two genes The new test made by Nanospherebleeding. Research suggests that most of that sensitivity is due to variations in two genes. The new test, made by Nanosphere Inc. of Northbrook, Ill., can detect some of those variants.

One of the genes produces an enzyme that helps the body metabolize warfarin and other medicines; the second produces the blood-clotting protein that warfarin blocks.

Human Cytochrome P450 2C9 with bound Warfarin

Nature 424, 464-468 (2003)

Image Source: www.pharmgkb.org Walko&McLeod2009.pdf

The FDA Abacavir Warning (July 24, 2008)Abacavir (marketed as Ziagen) and Abacavir-containing MedicationsFDA reviewed data from two studies that support a recommendation for pre‐therapy screening for the presence of the HLA‐B*5701 allele and the selection of alternative therapy in positive subjects.Genetic tests for HLA‐B*5701 are available and all patients should be screened for the HLA‐B*5701 allele before starting or restarting treatment with abacavir or abacavir‐containing medications. Development of clinically suspected abacavir HSR requires immediate and permanent discontinuation of abacavir therapy in all patients, including patients negative for HLA‐B*5701.

Aromatic amines

Hetero-cyclic

iamines

N-oxidation

O t l tiO-acetylation:

NAT1 and NAT2

ci. (

1999

)

Reactive metabolites (acetoxy-derivatives)

et a

l. T

oxc.

Sccancer

Rapid acetylator

From

: Hul

la

Intermediate acetylator

Slow acetylator

PON1 polymorphism:PON1 – human serum paraoxonase, enzyme important for lipid metabolisms that is also involved in metabolism of organophosphate compounds

PON1R192 PON1Q192

Rapid hydrolysis Rapid hydrolysis p y yof paraoxon

p y yof sarin, soman,

diazoxon

Two-dimensional enzyme analysis to h t i PON1 l hi i hcharacterize PON1 polymorphisms in human

population: analyze hydrolysis of PON1 substrates, diazoxone vs. paraoxone (panel c)O PON1Q192/PON1Q192

From: Hulla et al. Toxc. Sci. (1999)

■ PON1Q192/PON1R192 PON1R192/PON1R192

Survival in women with epithelial ovarian cancer

From: Strange et al. Toxc. Lett. (2000)From: Introduction to Biochemical Toxicology 3rd Edition (2001) p. 128

• Several GST gene families have been identified

• Null-phenotypes are detoxification-deficient and more likely to suffer f f /formation of carcinogen-DNA adducts and/or mutations

• In general, GSTM1- and GSTT1-null are considered high-risk

Genomenewsnetworks.org

The genomes of more than 180 organisms have been sequenced since 1995. The Quick Guide includes descriptions of these organisms and has links to sequencing centers and scientific abstracts.

Ultra High Throughput Sequencing – Towards the “$1,000 Genome”Illumina® “SOLEXA” Genome Analyzer Roche® 454 Genome Sequencer

Seqanswer.com

Illimina.com

DNA Sequencing Transcriptome analysis

Gene regulation and control

DNA Sequencing Transcriptome analysis

Gene regulation and control

Roche.com & Nature Biotechnology

Ultra High Throughput Sequencing – Enabling GWAS Studies

amateurbrainsurgery.com Genome‐wide plots of available GWAS results for all associations P = 0.0001. (BMC Medical Genetics 2009)

coompgen.unc.edu

www.niehs.nih.gov/crg/ ornl.gov

From: Strange et al. Toxc. Lett. (2000)

Toxicogenetics: what’s next?Goal: When we find all polymorphisms in genes important

for metabolism/detoxification of xenobiotics, we can link them to particular drug or chemical toxicity and identif s sceptible pop lationsidentify susceptible populations

Problem: Simple research questions generate erroneous results (e.g. CYP2D6 polymorphisms and lung

CYP2E1 l hi d l h li licancer, CYP2E1 polymorphisms and alcoholic liver disease)

Problem: Biological complexity of mechanisms, ethnic variation, li i l h i b h i i dclinical heterogeneity, etc… both positive and

negative results are true?

( )Linking complex trait diseases to genetic polymorphisms requires (Todd, 1999):• large sample sizes and small p-values• Initial study + several replications• Genetic associations should make biological sense

Ph i l i ll i f l d h ld f i l l f h• Physiologically meaningful data should support a functional role of the polymorphism in question

Ethical, Legal and Social Issues in toxicogenetics are as complex as the studies of polymorphisms themselvesp p y p

http://genomics.unc.edu/articles/elsi_article.htm

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Role of Genetic Polymorphisms i R t T i A tin Responses to ... · Role of Genetic Polymorphisms i R t T i A tin ... among the experimental animals that are being used ... facts about