Gene Sequencing / Testing RFLP analysis SNPs (single nucleotide polymorphisms) More than 1.4 million SNPs were identified in the initial sequencing of the human genome, with over 60,000 of them in the coding region of genes (Evans and McLeod 2003). (but even silent mutations can affect phenotype)
The accuracy of DTC genome-scan tests has been questioned, but Venter et al. found that the genotypes, or particular DNA bases observed, of an individuals markers from 23andMe and Navigenics agreed more than 99.7% of the time. Same for most other tests.
Do predicted disease risks have any clinical validity? Genotype-phenotype correlation? Certain disease risks can be better predicted than others. What the tests do disagree on, however, is the disease risk…
Pharmacogenetics Study of genetic variation that gives rise to different responses to drugs It is estimated that genetics can account for 20 to 95 percent of variability in drug disposition and effects. Nongenetic factors include: age, organ function, concomitant therapy, drug interactions, and the nature of the disease.
Pharmacogenomics – Under the PM Umbrella Better medication choices 100,000 Americans die annually and 2,000,000+ are hospitalized due to adverse reactions to medications Predict individual reactions to dugs Safer dosing options More exact dosing, optimum result/side effect balance Improvements in drug development Exclude genetic variations from certain clinical trials, speeding up drug design time
Polygenic Determinants of Drug Response Nine possible combinations of drug-metabolism and drug-receptor genotypes and the corresponding drug- response phenotypes. Each yields a different therapeutic index (efficacy:toxicity ratios) ranging from 13 (65 percent:5 percent) to (10 percent:80 percent). Differences in drug sensitivity and renal clearance
Proteomic Profiling Relevant in the identification and predisposition to disease Joshs presentation
Metabolomic Analysis First from urine analysis Networks of metabolite feedback pathways regulate gene and protein expression. Metabolites also can mediate signaling between organisms. Biomarkers of disease (diagnostics) The metabolome is therefore most predictive of phenotype (Fiehn 2002; Weckwerth 2003). However, …an understanding of the resulting data is limited owing to a fundamental lack of biochemical and physiological knowledge about network organization…
Metabolite target analysis Metabolic profiling MetabolomicsMetabolite fingerprinting all metabolites, present in a cell or sample. Comprehensive analysis of entire metabolome under a given set of conditions. the intention is not to identify each observed compound but to compare patterns or fingerprints of metabolites that change in response to disease or toxin exposure. Use statistical tools to look for major differences. focus on one specific metabolite group of metabolites, i.e those associated with a specific pathway. Extraction method (capillary electrophoresis, gas or liquid chromatography) specially designed for compounds in class to eliminate unwanted/ irrelevant metabolites. Metabolomic Analysis
SOME BRIEF CASE STUDIES Applications of Personalized Medicine
Personalized medicine today yesterday Cytochrome P450 genotyping test Enzyme group cytochrome P450 (CYP450 Many types of medications(including antidepressents, anticoagulants, proton pump inhibitors, etc) Determine dosing and effects of these drugs. Thiopurine methyltransferase test Thiopurine Thiopurine methyltransferase (TPMT) UGT1A1 TA repeat genotype test Irinotecan (Camptosar) UGT1A1 enzyme Dihydropyrimidine dehydrogenase test 5-flourouracil (5-FU) Dihydropyrimidine dehydrogenase enzyme Responsible for breaking down 5-FU
Uses in Muscular Dystrophy: Becker and Duchenne MD – same family of disease; Duchennes more severe than Beckers because generally the reading frame is preserved in BMD while it is not in DMD. DMD – death around age 20; BMD – life expectancy may be reduced, but some have a normal life span. Severity partially depends on mutation. Dystrophin is the largest known gene in the human body, located on the X chromosome. 79 exons ~15% caused by premature stop codons Phenotype-genotype correlation studies
Gentamicin treatment in DMD/BMD Aminoglycoside antibiotic synthesized by Micromonospora Works by binding the 30S subunit (inhibition site) of the bacterial ribosome, interrupting protein synthesis (stop codon readthrough) Gentamicin treatment of Duchenne and Becker muscular dystrophy due to nonsense mutations. (Wagner et al 2001) Some success in mdx mouse model – suppressed truncation of protein and improved phenotype. Cons: highly nephrotoxic; can have psychiatric side effects.
Ataluren (PTC-124) and PRO051 Mutation specific Both aim to restore reading frame: Ataluren does this through ribosomal stop codon readthrough PRO051 does this through exon skipping (block splicing machinery) Duchenne Becker phenotype
Nonsense mutations result in a premature stop codon (UAG, UAA, or UGA) and cause a truncated protein. Works best on UGA stop codon. Concept applicable to other diseases that also result from nonsense mutations, such as cystic fibrosis and nonsense- mutation hemophilia A and B (nmHA/B). Ataluren mechanism
As mentioned before, only ~15% have nonsense mutations. Others have deletions, inversions, insertions, point mutations that do not result in a stop codon… Many specific sequences will be required for effectively treating the majority of patients with DMD. Will each specific sequence need to go through full testing required by the Food and Drug Administration (FDA)? Toxicity standards?
Analysis of Dystrophin Deletion Mutations Predicts Age of Cardiomyopathy Onset in Becker Muscular Dystrophy
Irinotecan Treatment for cancer that works by inhibiting topoisomerase 1, which prevents DNA from unwinding. Clinical studies have revealed significant associations between UGT1A1*28 and irinotecan toxicity. A recommended strategy for irinotecan-dose adjustments based on individual genetic factors has not yet been fully established.
Selzentry (Pfizer) CCR5-tropic HIV treatment (CD4 immune cells) Ineffective for CXCR4-tropic strains of HIV Trofile assay to determine patients strain of HIV Detect virus that does not act through CCR5 at levels as low as 0.3% of a viral population Clinical trial patients selected with Trofile assay
The Food and Drug Administration, in a harbinger of likely future actions, has just approved a drug- diagnostic combination for AIDS patients who are running out of treatment options … the hallmark of personalized medicine. Edward Abrahams Personalized Medicine Coalition, 2007