Goals of pharmacogenomics Use drugs better and use better drugs! People inherit/exhibit differences in drug: Absorption Metabolism and degradation of the drug Transport of drug to the target molecule Excretion of the degradation products 100,000 deaths and 2.2 million adverse reactions reported each year 1
2 Dosage and drug specific to individual Get it right the first time Create individual metabolic profile Screening presymptomatic testing carriers preimplantation, prenatal, newborn screening disease susceptibility
DNA polymorphisms People differ in nucleotide sequence Coding sequences Non-coding sequences (most differences) Differences can be used as DNA markers Evolution To identify individuals To diagnose disease To diagnose genetic predisposition to disease 3
SNPs = Single nucleotide polymorphisms (handout and CH 8 pg 270) DNA sequence variations that occur when a single nucleotide in the genome sequence is altered Occur ~1/300 bases in human genome Make up ~ 90% of all human genetic variation Effort underway to map all human SNPs (~3 million) What is the size of the human genome in base pairs?
Many SNPs have no effect on cell function Non-coding region? Coding region? Others could: cause disease influence disease progression predispose to disease influence response to a drug 5
Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B José Luis Soto 1, Carmen M Cabrera 1, Salvio Serrano 2 and Miguel Ángel López-Nevot 1 analyzed 39 primary and metastatic melanomas and 9 melanoma cell lines C to T base change (intron1 of TP53 gene) compared with control. read a sequencing gel 6
7 Fig 10.20 open angle glaucoma (most common form, leading form of blindness in US)
SNP Read the gel results 8
Tumor gene Find the SNP
Example: Alcohol dehydrogenase SNPs Some SNPS in ALDH gene associated with extreme sensitivity to small doses of alcohol tachycardia, vomiting 13 SNPs found in human population 10
DNA Microarrays pg. 239, SARS handout Microarrays =small, glass slide (usually) spotted with short sequences from ~20,000 different genes at fixed locations oligonucleotides ~20 bases single stranded 11
Skin cancer microarray animation 1. Obtain sample phelgm, blood, tissue, tumor 2,3. Isolate mrna Expressed genes only Quantitative 4. Reverse transcribe mrna to cdna cdna more stable than mrna Fluorescently labeled nucleotides used 12
5. Coat microarray chip with tagged cdnas Hybridization - Immobilized target oligos found by probe DNA (the cdnas) and nucleotides hybridize Transcriptic microarray (vs. genomic)
14 6. Scan for fluorescence
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Transcriptional response in the peripheral blood of patients infected with Salmonella enterica serovar Typhi. Thompson LJ, et al Microarrays of peripheral blood to investigate host response of 29 individuals who contracted typhoid fever in Mekong Delta, Vietnam. Samples taken over a 9 month period encompassing acute disease, convalescence, and recovery. Typhoid fever induced a distinct and highly reproducible microarray pattern in the blood that changed during treatment and convalescence, returning in the majority of cases to the "normal" profile in healthy uninfected controls. Unexpectedly, there was a microarray pattern present at day 9 after infection that remained unchanged one month after infection and in some persisted as long as nine months despite a complete clinical recovery in all patients. Patients who retain the convalescent microarray pattern may be genetically or temporarily incapable of developing an effective immune response and may be more susceptible to reinfection, relapse, or the establishment of a carrier state. 16
The future (and present) Transcriptome Collection of all RNA transcripts in the cell (m, t, rrna) Tissue specific gene expression Which genes are on/off 17
Metabolome All the small molecules involved in metabolic pathways 3000 common metabolites in body tissues and biofluids People react differently to nutritional input, drugs 18
Proteome All proteins in the cell Larger than the genome Alternative splicing Post translational modification Different proteins are found in different cells 19
Microbiome Human gut and how it changes with disease 10X more bacteria in/on human body than human cells 20
21 600 species in oral cavity (most uncultivated, unnamed!) Over 1000 species in human gut Influence development, breakdown and absorption of nutrients, immunity, physiology Study microbes as community
Gnotobiotic mouse models born, removed from the mother by C-section in sterile environment Can be humanized 22
Genetic predisposition example: Alzheimer's disease SNPs ApoE gene 3 alleles E2, E3, and E4 each with a unique SNP GENETIC PREDISPOSITION AND RISK The E genes are susceptibility gene 23