Pharmacogenomics Exam 1

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TPMT Drugs

- Azathioprine - 6-Mercaptopurine

Genome Variability in Humans

- 99.8% of the genome is identical in humans, with only about 2% of the whole genome codes for genes. The remaining of this DNA is considered "junk" DNA. This DNA is not clinically meaningful or has any repercussions for biology. Studies are coming out that suggest the 98% of the genome is important, but they do not deal with proteins and pharmacogenetics.

CYP2B6 Drugs

- Bupropion - Efavirenz - Nevirapine - Cyclophosphamide - Ifosfamide

CYP1A2 Drugs

- Coffee - Theophylline - Estrogen - Clozapine - Olazapine - 2-aminoacetylfluorene (2AAF)

CYP2C19 Drugs

- Diazepam - Omeprazole - Pantoprazole - Lansoprazole - Clopidogrel - Voriconazole

Examples of Biomarkers

- Drug metabolites - DNA - RNA - Protein - Imaging - Laboratory Values (LDL-C, Glucose, etc.)

Phase II Metabolism Reactions

- Glucuronidation (UGT) - Sulfation (SULT) - Glycine (ACoAS) - Acetylation (NAT) - Methylation (COMT, TPMT) - Conjugation (GST)

CYP2D6 Drugs

- Imipramine, Trimipramine, Doxepin - Perphenazine, Thioridazine, Olanzapine - Tamoxifen - Codeine, Tramadol - Carvedilol, Metoprolol, Propranolol, Timolol - Propafenone

UGT1A1 Drugs

- Irinotecan - Tranilast - Bilirubin

NAT2 Drugs

- Isonaizid - Sulfasalazine - Hydralazine - Procainamide

CYP2A6 Drugs

- Nicotine - Cotinine - Coumarin - Methylnamylnitrosamine (MNAN) - Nicotine-derived Nitrosamine Ketone (NNK)

Phases of the Gartner Hype Cycle

- Phase 1 is the Technology Trigger, where we discover that we can do something. X=0 and Y=0 - Phase 2 is the Peak of Inflated Expectations, where we brainstorm all the different utilizations of the idea and these ideas are usually over the top and probably not possible. The line slopes up sharply to a peak visibility. - Phase 3 is the Trough of Disillusionment, where we realize we cant do all the things we thought we could do with the technology, and the line slopes down sharply to a trough, but doesn't hit y=0. - Phase 4 is the Slope of Enlightenment, where we start to discover all the realistic things we can do with the technology, and the line slopes up more linearly for a short time. - Phase 5 is the Plateau of Productivity, where we reach the maximum we can do with the technology, and we begin to become efficient with the technology and only make slight improvements. The line doesnt go down, but very, very, very slightly goes up over time.

Limitations of Precision Medicine

- Reimbursement pathway of testing is not established - Ethical issues with genetic testing and data sharing - Integration of pharmacogenomics, personalized medicine and the payer and regulatory environment is still ongoing - Clinicians are generally not educated concerning the availability of tests, their associated drugs and the outcomes. - The response to the medication may be the result of multiple genes.

Potential Benefits of Precision Medicine

- Shift the emphasis in medicine from reaction to prevention - Predict the susceptibility to disease, improve disease detection and preempt disease progression - Customize disease-prevention strategies - Prescribe more effective drugs and avoid prescribing drugs with predictable side effects - Increase patient adherence to treatment by targeting the right patient with the right drug - Improve quality of life - Reduce the time, cost and failure rate of pharmaceutical clinical trials - Revive drugs that have failed in clinical trials or were withdrawn from the market - Control healthcare costs by avoiding unnecessary costs when drugs are proven ineffective for the patient.

Important Phase II Enzymes for Pharmacogenomics

- UGT1A1 - UGT1AB - UGT2B7 - NAT2 - GSTM - TPMT - COMT

CYP2C9 Drugs

- Warfarin - Phenytoin - Glimepiride - Tolbutamide - Glyburide

Factors Causing Genetic Drift

1. Bottleneck Effect - A drastic reduction in a population from some outside event (volcanoes, earthquakes, landslides, pandemics) that causes a dramatically reduced genetic variation in the population that remains. - This new smaller population may not be able to adapt to new selection pressures. 2. Founder Effect - Occurs when a new colony is started by a few members of the original population. There is a reduced genetic variation in this new colony and can lead to speciation - The Founder Effect is what caused a lot of our genetic diversity throughout the world.

CYP2C19 Drug Consequences

1. Diazepam: - Increased sedation in PMs due to prolonged half-life of Diazepam 2. Omeprazole, Pantoprazole and Lansoprazole - Increased cure rates in PMs due to increased half-life of parent drug. - Decreased cure rates in EMs and UMs due to decreased half-life of parent drug. 3. Clopidogrel - Decreased response in PMs due to lower transformation to the active metabolite

Describing Genetic Structure

1. Genotype frequencies 2. Allele frequencies

CYP2D6 Drug Consequences

1. Imipramine, Trimipramine, Doxepin - Diminished response in UMs and increased CNS ADEs in PMs 2. Perphenazine, Thioridazine, Olanzapine - Diminished response in UMs and increased EPS in PMs 3. Tamoxifen - Poor efficacy in PMs due to low transformation into the active metabolite 4. Codeine - Increased risk of ADEs in UMs due to toxic concentrations of the active metabolite, morphine. 5. Tramadol - Increased risk of respiratory depression or nausea in UMs due to an increase in active metabolite concentrations. - Less analgesia in PMs 6. Carvedilol, Metoprolol, Propranolol, Timolol - Increased ADEs in PMs due to increased drug concentrations

Germline Editing Ethical Issues

1. Inaccurate or incomplete editing (the CRISPR method done in the twins may have been sloppy, so other genes may have been affected). 2. Difficultly predicting harmful effects of germline editing. 3. Obligation to consider potential implications for the individual and future generations 4. Once the changes are introduced into the population, they are difficult to remove (can only be done at initial life- so a single cell). 5. Genetic enhancements to subsets of the population could exacerbate inequities or be used coercively 6. Ethical considerations in purposefully altering human evolution.

CYP2D6 Star Allele Types

1. Increased Function- xN (the Copy Number Variation) 2. Normal Function- s1, s2, s33, s35 3. Decreased Function- s9, s10, s17, s29, s41, s59 4. No Function- s3, s4, s5-8, s14, s36, s38, s40, s42, s44, s56, s62 In regards to the CNV, someone can have multiple copies of the normal functioning alleles, but someone can have duplicates of a decreased function gene or non-functional genes.

Real-World Applications of Precision Medicine

1. Non-invasive Prenatal Testing 2. CAR T-Cell Therapy 3. SCID Cure 4. Cell-Free DNA 5. Induced Pluripotent Stem Cells

Techniques for Cell Membrane Transport

1. Passive Diffusion 2. Facilitated Diffusion - Typically small (monomer) molecules, polar (water soluble), charged ions (Ca, Cl, Na, K), Glucose, and membrane transporters. 3. Active Transport - Vesicular Transport (Exocytosis, Endocytosis, Budding) 4. Pore (Convective) Transport 5. Ion Pair Formation

Social Issues for Pharmacogenomics

1. Race and Ethnicity 2. Testing in Vulnerable Populations - Prisoners, Children, etc... 3. Incidental Findings - Familial relationships and risk for disease 4. Availability of Testing - Cost of testing and reimbursement - Will only the rich be able to rest their genome? 5. Knowledge/Education of the Public - Reasonable expectiations - Direct to Consumer testing 6. Knowledge/Education of Health Professionals - Judicious interpretation of tests - Incidental findings - Implication of clinical standards and quality-control measures.

Legal Issues for Pharmacogenomics

1. Regulatory Approval 2. Informed Consent 3. Disclosure and Reporting Requirements 4. Data and Sample Ownership 5. Intellectual Property

Where Pharmacogenomics Really Matters

1. Single Pathway to Bioactivation 2. Narrow Therapeutic Window Drugs 3. Toxic Off-Target Adverse Effects

BS Genome Testing

1. TeloYears- A company that will test your DNA and measure the length of your telomeres. They will report essentially how old you are relative to your telomeres. A lot of speculation with how accurate their numbers are, and patients can interpret the data in a lot of good/bad ways. 2. Ancestry Musical DNA- Will create a playlist of music based on your genome. A lot of people felt weird using their DNA for a music streaming program. 3. Genetic Test for Infidelity- Tests AVPRA1 gene.

Assumptions of HWE

1. The organism is a diploid (2 copies of each chromosome) 2. The organism sexually reproduces. 3. The generations are non-overlapping 4. The organism mates at random 5. There is a large population size 6. There is negligible migration 7. There is negligible mutation 8. There is no selection on genes

Reasons for Discordant Results in CYP2D6 Trials for Tamoxifen

1. They did not genotype many of the rarer, poor metabolizer alleles 2. They did not account for concurrent use of other drugs metabolized by CYP2D6 in many cases. 3. Different doses of Tamoxifen were used in several trials. 4. They did not assay endoxifen levels 5. The trials had limited power (poor metabolizers are rare) 6. Unknown variants in other genes whose products are involved in tamoxifen metabolism.

CYP2C19 Phenotypes with Diplotype

1. Ultrarapid Metabolizer - One or more gain of function alleles - s17/s17 or s1/s17 2. Extensive/Normal Metabolizer - Type copies of the normal functional allele - s1/s1 3. Intermediate Metabolizer - One reduced and one non-functional allele - s1/s2 or s1/s3 4. Poor Metabolizer - No functional alleles - s2/s2, s2/s3 or s3/s3

CYP2C9 Drug Consequences

1. Warfarin: - Increased bleeding in PMs due to reduced metabolism of S-Warfarin 2. Phenytoin: - Increased risk of neurological symptoms in PMs due to increased levels 3. Glimepiride, Tolbutamide, Glyburide - Increased risk of hypoglycemia in PMs

23andMe and the Shutdown

23andMe previously provided info regarding mutations affecting disease risk, including many cancers and Parkinson's. These risk reports were based on literature that was not entirely proven, or very obscure and not 100% creditable studies. These reports also offered specific tips and guidance on how to reduce risk. There was no genetic counseling offered by 23andMe. In 2013, the FDA ordered 23andMe to stop providing analyses of people's risk factors for diseases until the test accuracy the diseases could be validated. Essentially 23andMe was shut down. In 2015, 23andMe re-entered the market, but they now only provide information on those 36 tests approved by the FDA (where previously they provided data on 254 diseases). The new 23andMe focuses on carrier status, ancestry and genetic traits like hair and eye color, widow's peak and earwax type.

Race/Ethnicity Bias in Genetic Studies

80% of our genetic study tests have been on white/Caucasian people. Studies in whites are not normally valid across to other race/ethnic groups because there are genetic differences. Without research, clinical testing is not useful for non-whites.

Haplotypes

A Haplotype is a set of linked SNPs on the same chromosome. SNPs are inherited in such a way where often times they are linked. SNPs are often inherited together, so if you inherit one SNP, odds are you will inherit another SNP at another site. A Haplotype can be simply considered as a binary string since each SNP is binary. Haplotypes are essentially chunks of DNA where we have multiple SNPs represented. They can characterize more variation when looked at compared to just using one SNP. Since they are inherited, Haplotypes are essentially chucks of genotype. So, like genotypes, we inherit a copy of a haplotype from mom and one copy from dad.

Impact of Metabolizer Status on Drug Response

A Poor Metabolizer of a drug will have a lot longer of a half-life for a certain drug compared to someone who is a normal metabolizer or an Ultra Metabolizer. If a Poor Metabolizer of a drug is on chronic therapy with one of these drugs, their steady state concentration can be extremely higher than these other two groups, and it can take a lot longer to achieve steady state. There is more time for the drug to accumulate in the body, allowing for a higher concentration.

SNP Considerations

A Single Nucleotide Polymorphism is a genetic variation when a single nucleotide (A, T, C, or G) is altered and kept through heredity. - A SNP is a single DNA base variation found in >1% of a population. - A mutation is a single DNA base variation found in <1% of a population. SNPs are the most frequent form among various genetic variations, and 90% of human genetic variations come from SNPs. SNPs occur about every 300-600 base pairs and about 40 million have been identified. All SNPs started out as a mutation and just grew in frequency in the population. All SNPs are mutations, but not all mutations are SNPs! SNPs have become the preferred marker for association studies due to their high abundance.

Biomarkers

A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacologic responses to a therapeutic intervention. Biomarkers are essentially lab values or any characteristic of the body that is objectively measured to determine: - Presence/Absence of disease - Stage of the disease - Prognosis and risk - Prediction and/or readout of drug response - Adverse events

CAR T-Cell Therapy

A procedure where blood is taken from a patient who has diseased T-Cells or the T-Cells need to be armed with additional targets for certain cancers. The T-Cells are removed from the patient and genetically modified to add targets, reduce their effectiveness or fix something defective. The patient's immune system (bone marrow) is then ablated (destroyed) and the genetically modified T-Cell is re-added to the patient and the T-Cell essentially repopulates the immune system of the patient with the new data.

Codeine and Morphine Metabolism via CYP2D6

A small amount of codeine is metabolized into morphine via CYP2D6, and the status of a patient's CYP2D6 activity can have major implications on the affects of codeine. If someone is an ultra-rapid metabolizer, they will have a lot higher than expected morphine concentrations, which can increase the risk for ADEs and toxicity. Poor metabolizers will have a lot lower than expected morphine concentrations, increasing the risk of treatment failure. This is extremely important because there has been DEATHS because of this genetic variation! Example is from nursing mothers taking codeine and the infants die from an overdose of morphine because of the mother's ultra-rapid metabolism. Morphine gets into the breastmilk very easily.

CYP, Drug Metabolism and Variation

A small set of the CYP enzymes control the Phase I metabolism of many drugs and herbals and have genetic variation. CYP2D6 is involved in about 25% of Rx drugs and about 6% of Caucasians have an inactive form (a "null" variant) of this CYP! Two copies of this gene will cause zero CYP2D6, which can have major implications for drugs that go through CYP2D6 for metabolism. CYP3A4 is involved in about 50% of Rx drug metabolism, but not many pharmacogenomic relevance exists for it. CYP2D6, C9 and C19 are all polymorphic and have pharamcogenomic implications. Additionally, many drugs go through multiple different CYPs in different ways, increasing the importance of these CYPs and these drugs. Some drugs are inducers of CYPs, some drugs are inhibitors of CYPs and some are just substrates of CYPs. The CYPs are KEY FACTORS in predicting many drug interactions. Their roles in metabolism of newer drugs on the market are typically examined early in development. For many older drugs, the CYP role is much less known.

Synonymous Variation

A type of gene variation where the SNP is in the coding region, but no amino acid change happens. There is a change in the base pair nucleotide, but the amino acid does not change. There are examples of this type of variation changing the functionality of proteins, due to a change in the structure of the mRNA. The mRNA structure can be changed so it degrades faster, or sticks around longer, which affects the level the correlating protein in the body.

Pharmacogenomics and ADME

ADME is what the person does to the drug, and pharmacogenomics essentially affects any step in this process. Alterations and genetic differences in elimination rates of drugs and metabolites are important causes of PK adverse events. Absorption: Route of administration, food effects, bioavailability, protein binding. Distribution: Plasma, tissues, CSF, bile, etc. Metabolism (The major way pharmacogenomics affects the pharmacokinetics of a drug): Hepatic metabolism, influence of CYP450s, age, organ impairment, other drugs. Excretion (another major way pharmacogenomics affects the PK of a drug): Renal vs Hepato-biliary removal, organ impairment, differing clearance rates for different metabolites.

Co-Dominance

AKA Incomplete Dominance This is essentially expressed in the phenotype of the individual. Co-Dominance is when a heterozygote has an intermediate phenotype (so if RR is red, rr is white, Rr will be pink). The ABO blood groups are prime examples of Co-Dominance. Most pharmacogenomic variants will follow co-dominant inheritance patterns.

Missense Variation

AKA Nonsynonymous Variation This is a type of gene variation where the SNP is in the coding region and causes a change in the amino acid translated. This SNP is in an exon and the translation of the protein will be changed. The functionality consequence of the protein or the change in the structure of the protein is different based on the change of that protein.

Membrane Transporter Families

ATP-Binding Cassette Transporters Solute Carrier Transporters

ABC Transporters

ATP-Binding Cassette Transporters These are transporters that act through active efflux transport. Members include ABCC1, C2 and C3. Multidrug resistance-associated proteins (MRPs) are members of the ABC superfamily and are commonly known to affect drug disposition. MRP1 (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3). P-Glycoprotein (ABCB1) is another member of this family and is also associated with MRP1. There are no CPIC guidelines associated with PGP, but it still is very important for drug disposition. PGP is very important in chemotherapeutic drugs, and a number of mutations can affect PGP. However, due to the abundance of these mutations and the ability of other PGPs to be present, it is not as clinically relevant.

OAT1 and OAT3

Adefovir, Furosemide, Methotrexate Penicillin (and other Beta-Lactam Antibiotics)

The Telomere

After each DNA replication, a piece at the end of each chromosome is lost. The end of DNA sequences where this happens is called the Telomere. Telomeres protect the ends of the chromosomes, making sure valuable chromosomal DNA is not lost. Telomeres are thought to be "clocks" that regulate how many times an individual cell can divide. Every time a cell replicates their DNA, the telomere shortens. Healthy human cells are mortal because they can only divide a finite amount of times, depending on the length of the telomere. Smoking, obesity, lack of exercise and consumption of an unhealthy diet can increase the pace of telomere shortening, leading to illness and premature death. Some cancer cells can reproduce indefinitely (are essentially immortal) because they produce telomerase, which allows the telomere to lengthen indefinitely.

Allele, Exon, Forward/Reverse Strands

Allele: - A combination of one or more base pairs of DNA on a particular chromosome. It is one of the many versions of a gene. Exon: - Portion of a gene that encodes for a protein. Usually there are multiple exons in one gene that all together create a protein. Forward/Reverse Strands: - The two total strands of a double-stranded DNA. These are complimentary to each other and may encode important information.

Precision Medicine

An emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment and lifestyle for each person. While this is a new concept (in terms of genetics), we really have always practiced Precision Medicine because we always try to individualize treatments to the patient using their lifestyle and their environment.

Hardy-Weinburg Equilibrium

An equation that determines the expected ratio of genotype groups and the frequency of alleles within a population. p^2 + 2pq + q^2 = 1, where p is the major allele frequency of A and q is the minor allele frequency of a. 1. If only random mating occurs, then the allele frequences will remain unchanged over time. 2. After one generation of random-mating, genotype frequencies are given using the equation above.

Narrow Therapeutic Index Drugs

Another area where pharmacogenomics really matters. Testing for variants here can prevent overdosing on medications that have tight windows of effectiveness vs ADEs. Warfarin, Azathioprine and Tacrolimus are prime examples. A small effect on genetic variation may have a dramatic effect on the medication.

Off-Target Serious Adverse Effects

Another area where pharmacogenomics really matters. This is typically immune-mediated adverse effects where if the person has some sort of rogue immune variant, it will cause serious adverse reactions such as SJS, TEN, long QT or myopathy. Carbamazepine, Allopurinol, Phenytoin, Simvastatin.

Migration

Another reason for genetic structure changes. Migration is when individuals move into a population. Termed "gene flow," the individuals introduce new alleles to the population. This is essentially the reason for ancestry! Founder populations move to different areas and mix their alleles with the endemic founder population, leading to new populations.

Genetic Drift

Another way genetic structure can change. This is typically considered like a "sampling error." Genetic drift is genetic change by chance alone, and is a very small portion of populations. If a population is randomly sampled and a genetic drift change is seen, it is essentially a misrepresentation of the population. Genetic Drift is more prevalent in small populations and causes the most disruption. Genetic Drift is essentially a random change in allele frequency, and is only happens in two situations: - Bottleneck Effect - Founder Effect

Natural Selection

Another way genetic structures can change. Natural selection is when certain genotypes produce more offspring and other genotypes fail. Certain genotypes confer better "fitness" when it comes to survival or reproduction, and they outgrow the other "less fit" genotypes. Natural selection also causes a species to adapt to new conditions or fail.

Non-Random Mating

Another way that causes genetic structure changes. Mating causes a combination of alleles into genotypes and non-random mating causes these allele combinations to also be non-random.

OATP1B1 Substrates

Atorvastatin, Bile Acids, Bilirubin Caspofungin, Enalapril, Fexofenadine Irinotecan, Methotrexate, Rifampin Rosuvastatin, Simvastatin

PGP Representative Substrates

Atorvastatin, Cyclosporine, Digoxin Fexofenadine, Fluphenazine, Irinotecan Ketoconazole, Loperamide, Lopinavir Rifampin, Sirolimus, Tacrolimus, Testosterone

Golden State Killer and DNA Testing

Between 1974 and 1986, the Golden State Killer committed more than 50 rapes and 12 murders. They didnt have much info about the killings, but they did have some DNA evidence. All other evidence was circumstantial. Law enforcement officials uploaded the killer's DNA sequences to a free online database (GEDmatch) where people can share their DNA data from analysis companies like 23andMe and Ancestry. They searched for relatives who have submitted their DNA and found a lot of 2nd and 3rd cousins. Other information like genealogical records, approximate age, and crime locations allowed law enforcement to locate the killer. In April 2018, police arrested a 72-year old Joseph James DeAngelo based on this search of the genome. It is estimated that 60% of caucasians can be found using this method.

BSEP Representative Substrates

Bile Acids Pravastatin

CYP Enzyme with Star Allele Nomenclature

CYP 2 D 6 s4E CYP (Gene/Enzyme name), 2 (Family), D (Subfamily), 6 (Polypeptide), s4E (Star Allele name, 4 is the allele and E is the allele subtype).

CYP1A2 Pharmacogenomics

CYP1A2 is responsible for the metabolism of coffee, estrogen, clozapine, theophylline, olazapine and certain pro-carcinogens such as 2-aminoacetylfluorene (2AAF). CYP1A2 is predominantly expressed in the liver and accounts for about 10% of total hepatic P450 content. CYP1A2s1F has been shown to increase the inducibility of CYP1A2 expression by caffeine, smoking and omeprazole treatment than that of CYP1A2s1A. This enhanced induction affects the increased metabolism of CYP1A2 substrate drugs with altered therapeutic outcomes or an increased risk of certain cancers.

Major CYP450 Players in Drug Metabolism

CYP2A6, B6, C9, C19, D6, E1 AND CYP3A4 are responsible for metabolizing the most clinically important drugs. The major pharmacogenomic players are 2C9, 2C19 and 2D6. CYP3A4 actually does not have much variability and pharmacogenomic activity. It has none of the major mutations like missense or frame-shift, making it not very important in pharmacogenomics. CYP2D6 presents mainly in the liver and is a major player in drug metabolism, from antidepressants to antihypertensives to chemotherapy. It has MANY mutations.

Examples of P450 and Drug Combinations

CYP2C9 - Warfarin - Phenytoin - Glimepiride, Tolbutamide, Glyburide CYP2C19 - Diazepam - Omeprazole, Pantoprazole, Lansoprazole - Clopidogrel - Voriconazole CYP2D6 - Imipramine, Trimipramine, Doxepin - Perphenazine, Thioridazine, Olanzapine - Tamoxifen - Codeine, Tramadol - Carvedilol, Metoprolol, Propranolol, Timolol - Propafenone CYP3A4/5 - Tacrolimus - Cyclosporine CYP1A2 - Coffee, Theophylline - Estrogen - Clozapine, Olazapine - Certain pro-carcinogens such as 2-aminoacetylfluorene (2AAF) CYP2A6 - Nicotine, Cotinine - Coumarin - Methylnamylnitrosamine (MNAN) - Nicotine-derived Nitrosamine Ketone (NNK) CYP2B6 - Bupropion - Efavirenz, Nevirapine - Cyclophosphamide, Ifosfamide

Tamoxifen and CYP2D6 Metabolism

CYP2D6 metabolizes Tamoxifen into its active metabolite Endoxifen. Endoxifen is the compound responsible for Tamoxifen's MOA, which is a breast cancer medication. 6-10% of the European population is deficient in this enzyme! Again, ultra-fast metabolizers may have an extra benefit because they have more CYP2D6, so more Tamoxifen gets metabolized to Endoxifen. Poor metabolizers will not see an effect (or a much more reduced effect) with Tamoxifen because they do not metabolize as much to Endoxifen. There is no guideline for Tamoxifen metabolism, however. This is because the evidence is controversial and contradictory. Several small trials have suggested decreased efficacy of Tamoxifen in poor (intermediate) metabolizers in adjuvant therapy in the treatment of metastatic disease. All these trials were retrospective and the largest trial was only a statistically significant association to a univariate analysis. Other trials have not confirmed these results.

CYP3A4/5 Drugs and DPYD Variant

CYP3A4/5 - Tacrolimus - Cyclosporine DPYD - Capecitabine and Fluorouracil

The Cytochrome P450 Enzyme

CYP450s are the major Phase I metabolism in the liver, and they are responsible for oxidation of the compound to make it possible for conjugation via Phase II metabolism. The majority of drugs that we have are metabolized via the CYP3A family (3A4, 3A5, 3A7), but other major players include CYP2D6, CYP2C9 and CYP2C19 (ordered in decreasing prevalence). There are 57 different active genes involved in CYP enzymes and 17 different families of the enzymes. CYP1, 2 and 3 are the ones primarily involved in drug metabolism. The CYP enzymes are membrane-bound enzymatic proteins that are mostly involved with oxidation, peroxidation and reductive metabolism and are involved in >90% of drug transformation

Components of Population Genetics

Changes in allele frequencies and/or genotype frequencies through time due to: - Mutation - Migration - Natural Selection - Genetic Drift - Non-random mating As the human population keeps growing the mutations can either increase or decrease in terms of their frequency.

World's First Genetically Altered Humans

Chinese scientist Dr. He Jiankui (trained in the US), used CRISPR to modify the embryos of two twin girls, Lulu and Nana. The scientist was on unpaid leave from the university in China where he worked. He modified the CCR5 gene in the embryos, which is a chemokine receptor that is accessible by HIV to infect WBCs. The girls may be resistant to potential infections of HIV. Both copies were shut off in Lulu, but only one copy was shut off in Nana. The father of the twins had HIV. It is said that the consent was misleading (as they may have been told they were testing an HIV vaccine). He also defied the unofficial moratorium on editing human embryos intended for pregnancy. The university and the scientific community largely denounced his work.

BCRP Representative Substrates

Ciprofloxacin, Erythromycin, Estradiol Etoposide, Irinotecan Metabolite (SN-38) Methotrexate, Nitrofurantoin, Rosuvastatin Sirolimus, Tacrolimus

OCT2 Substrates

Cisplatin, Oxaliplatin, Metformin Verenicline, Procainamide, Amiloride

Clopidogrel Bioactivation

Clopidogrel is bioactivated in a two-step process (a double-prodrug, essentially). What is important here is that CYP2C19 is involved in BOTH of these steps, and 2C19 is a major polymorphism. This variation will cause a major implication in metabolism of Clopidogrel to its active drug, and therefore the active effect. CYP2C19 has 4 star alleles, which are the major polymorphisms of the enzyme. s1 is the wild type, s2 is a Splice Site Polymorphism, s3 is a Nonsense Mutation and s17 is a Promoter Mutation. s2 and s3 decreases CYP2C19 activity, which makes it so Clopidogrel cannot be metabolized as well to the active metabolite, reducing the effect of the drug. s17 increases transcription of 2C19, which increases the prevalence of 2C19. In this case, the person will be an ultrametabolizer of clopidogrel because they have more 2C19 enzymes.

Clopidogrel and Genetic Variation

Clopidogrel is used for antiplatelet therapy, usually given after someone has a stent in place to prevent thrombosis at that stent. It is an irreversible inhibitor at the P2RY12 receptor on platelets, preventing the binding of ADP, which stimulates clotting. It is commonly given with aspirin. Clopidogrel requires GI absorption and then hepatic biotransformation to its active form. The initial dose is 300mg, followed by 75mg daily. Clopidogrel has major pharmacogenomic variation and importance, especially because it is such an important medication and prevents a life-threatening clot. About 85% of clopidogrel is inactivated before it is even absorbed. The other 15% is metabolized in the liver to active metabolites via CYP3A4, 3A5 and 2C19.

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats. This is a genome editing technique that: - Targets a specific section of DNA - Makes a precise cut/break at the target site - Can render a gene non-functional or replace one version of a gene with another

Reproductive Issues

Complex and potentially controversial procedures, use of genetic information in reproductive decision making and reproductive rights. There are tests we can do to maximize intelligence in offspring!

Data and Sample Ownership/Intellectual Property and Pharmacogenomics

Data and Sample Ownership - Availability of biobank samples without patient consent. - Before 2 years ago, discarded/unused blood samples from patients could be given to biobanks for research and study, without the patients consent. Intellectual Property - Commercialization of products, including property rights (copyright, patents and trade secrets) and accessibility of data and materials. - Is there a patent to the human gene?

Stigmatization

Entities may use genomic testing of the patient to stigmatize based on the patient's genetic differences. The impact of slurring/branding due to an individual's genetic differences.

The Omics

Epigenomics: The study of all the epigenetic changes in the genome. Transcriptomics: all the messenger and RNA and Proteomics: All the proteins Interactomics: All the interactions between all molecules Metabolomics (AKA Metabonomics): All the metabolites, drugs and otherwise. Microbiomics: Presence and action of endogenous bacteria/viruses

Omics

Essentially a "catch all" term that encompass all the various genome terms. Omics is the analysis of various biological molecules that has developed into their own scientific disciplines: - Genomics - Epigenomics - Transcriptomics - Proteomics - Interactomics - Metabolomics - Microbiomics

Incidental Findings in Pharmacogenomics

For example, if CYP2C19 became associated with an increased Alzheimer's risk, that would essentially remove CYP2C19 from a lot of the genomic tests that are offered (because of the implications). A real example is Apolipoprotein E (ApoE), which is a major cholesterol carrier that supports lipid transport and injury repair in the brain. ApoE polymorphic alleles (ApoE4) are actually the main genetic determinants of Alzheimer's Disease risk. Some previous studies report less effect of statins in carriers of the E4 for loweing cholesterol levels. But now there is also this incidental finding between E4 carrier status and Alzheimer's. Association between ApoE SNPs and lipid response during statin therapy has not been confirmed.

GSTM, COMT Drugs

GSTM - Busulfan COMT - Levodopa

Gene, Genome, Genotype

Gene: - The fundamental physical and functional unit of heredity. A gene is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes for a specific functional product (i.e., a protein or an RNA molecule). Genome - The entire set of genetic instructions found in a cell (in humans), including 23 pairs of chromosomes, found in the nucleus, as well as a small mitrochondrial chromosome. Genotype - The combination of two alleles at a particular location in the genome. Each of these two alleles comes from a different chromosome.

Genetic Discrimination

Genetic testing can be used by schools, work, insurers, courts, and others to determine long-term health, intelligence, and other aspects of the patient. We need to ensure fairness in the use of genetic information by insurers, employers, courts, schools, adoption agencies, the military and all other entities.

GWAS

Genome-Wide Association Studies GWAS is used extensively in disease genetics and pharmacogenomics. GWAS is systematic testing of millions of SNPs across the genome for a phenotype. This is a technique where we are trying to find a polymorphism that affects a response. To perform a GWAS, we need a control genome that does not have the response, and the test genome that does get the response. We genome all the possible SNPs in each genome and compare them to each other to see what SNPs associate with response vs non-response. This is different than SNP testing because we are looking at the ENTIRE genome to see if there are any associations with the response and the person's entire genome, vs a single SNP or what is already known. A GWAS was used to determine the association between statin-induced myopathy and SLCO1B1 (OATP1B1 transporter). 85 people were tested who had simvastatin myopathy and compared to 90 control genomes, and it was found that one SNP was extremely tightly correlated between all the myopathy patients.

Genomics vs Pharmacogenetics

Genomics is essentially disease genetics, disease prognostics and disease diagnostics. We are evaluating a person's genes for the likelihood or presence of a certain disease (usually cancer). There are a lot of implications on clinical options but also what that means for the patient's life. That genetic testing will essentially reveal details about relatives and family members. Did those other people consent to this information? Did they talk to a genetic counselor? How does this information affect them? Do we inform them as well? Pharmacogenetics has a lot less ethical considerations, as it only deals with how we deal with medications. It analyzes genes and SNP profiles for metabolism/action and determines an optimal medicine response. It does not reveal if someone and those related to that someone has a higher risk for cancer, only that their CYP enzymes are slow/fast. We have a lot of options if someone's CYP mutation renders a medication ineffective compared to determining a disease risk.

Haplotype, Mutation, Phenotype

Haplotype: - A set of DNA variations, or polymorphisms, that tend to be inherited together. The Haplotype takes into account the alleles on the same strand, not across strands of DNA. Genotypes go across strands. This is why when we genotype people, we do not know what strand the allele is on, so we do not know what the haplotype is. Mutation: - A change in the DNA sequence. Germ-line mutations (SNPs) occur in the eggs and sperm and can be passed to offspring whereas somatic mutations (most of them) occur in the body cells and are not passed on. Phenotype: - The observable traits of an individual person. A phenotype could be a disease, laboratory value, a drug response variable, etc.

DPYD Variant

Has a CPIC guideline with capecitabine and fluorouracil

Human Migration over Time

Human migration has been predicted based on mitochondrial DNA. Mitochondrial DNA is distinct from genomic DNA and is mostly inherited directly by the mother (there are now exceptions to this dogma). Mitochondrial DNA is very tough, and is resistant to a lot of oxidative stress and change. So Mitochondrial DNA can be used to trace back lineages. Most of the diversity we observe in the human population is from the ancestral (African) population. Africa was where the first humans emerged and is the source of human diversity. Because the African population has been around the longest, they have the most diversity when it comes to mutations and SNPs. The African population also has the lowest amount of linkage disequilibrium because they have been around the longest.

Genotypes vs Haplotypes

Humans are diploid, so haplotypes become limited when we genotype someone. Genotypes are the genetic make-up of an organism, and actually shows us the base pairs on each chromosome, making it difficult to see the haplotypes (as haplotypes take into account multiple genes on the same chromosome). Remember Halotypes are characterization of multiple genotypes. If we have genotype data from two SNPs, we don't know what chromosome the base pairs are located on and in order to do that, we have to generate a haplotype, which is called Phasing. Genotypes only tell us the alleles at each SNP locus, but we do not know the connection of the alleles at different SNP loci. There could be several possible haplotypes for the same genotype, and we would need to actually sequence the DNA to determine the true haplotype.

Informed Consent/Disclosure and Pharmacogenomics

Informed Consent - Patient awareness and understanding of what results mean and how the results will be used. Disclosure and Reporting Requirements - Whether new genetic results or new knowledge of risk will be reported. - Genetic literature is always evolving and new studies will always come out and may have new implications based on the genomic testing.

UGT1A1 and Irinotecan

Irinotecan is inactivated and excreted from the body after glucuronidation by UGT1A1. Homozygosity for the 7-repeat allele, or UGT1A1s28 is associated with SEVERE toxicity. The patient experiences diarrhea and low WBC counts, mainly.

CYP Enzyme Variation for ALL CYP-Metabolized Drugs

It is not appropriate to extrapolate a patient's CYP variation to all drugs that are metabolized by the enzyme. The variants have substrate-specific effects and each drug has to be studied individually to determine how the CYP450 polymorphisms affect that drugs metabolism. CYP variation is often used to direct clinical care when the evidence is limited or conflicting, which is not appropriate.

Pheno-conversions

It is possible for someone to be taking another drug or doing another action that inhibits (or even induces) the target CYP enzyme. This can make the person look like a poor, intermediate, extensive or an ultra-rapid metabolizer. Ex. Antidepressant use is common in breast cancer patients, as depression is common in cancer and antidepressants can help treat the hot flashes that are associated with Tamoxifen use. SSRIs (Fluoxetine and Paroxetine) inhibit CYP2D6, which can change how the person metabolizes their Tamoxifen to Endoxifen. Level of inhibition varies between different antidepressants, with Paroxetine causing the most inhibition and Venlafaxine causing no inhibition. A study was done in 2010 that was a population-based cohort study of women receiving Tamoxifen adjuvantly for breast cancer treatment, and mortality from cancer was increased in the group using Paroxetine concurrently with tamoxifen!

Linkage Disequilibrium

Linkage Disequilibrium is what creates haplotype blocks. If there is equilibrium of genetic information, then there is a balance of genetic information. SNP 1 is independent of SNP 2. However, this is not true for certain SNPs. If SNP1 results in SNP2 being inherited, then they are considered to be in high Linkage Disequilibrium. LD measures the degree to which alleles at two sites are linked and to be in complete LD means that the two mutations are completely co-inherited (in complete linkage). We can measure LD using either R squared or D prime and range from 0-1. 1 indicates perfect LD whereas 0 indicates no LD. TagSNPs typically have high LD. To determine LD we look at TagSNPs and the correlations between multiple SNPs. If we have correlated SNPs, or in complete LD, we only need to look at one SNP to predict the other SNP.

Reimbursement of Pharmacogenomic Testing

Many PGx tests are reimbursed through insurance. Typically the patient must have an indication for the drug (unless going through 23andMe). If going through the physician, the tests are more expensive, but they may be covered because of the clinical benefit the test may provide. Insurance will not pay for a panel, only for the individual drug-gene pair as we need an indication for the test. Cost-effectiveness of panel-based genotyping is higher than genotyping each drug-gene pair individually. So testing individual drug-gene pairs increases the cost of genotyping. DNA testing, however, is good throughout lifetime (for the most part). Clinically, PGx testing is done with the understanding that the patient will pay for the test if the cost is not reimbursed through the insurance.

MATE1

Metformin Cimetidine Procainamide

OCT1 Substrates

Metformin Tropisetron Oxaliplatin

Examples of Epigenetics

Methylation - Methyl groups bind to DNA and limit the expression of various genes Histones - Protein spools of DNA winds around, and the tightness of this winding affects the level of gene expression and can modify the chromatin MicroRNAs - These can regulate gene expression in the body. MicroRNAs are really tiny strips of RNA that essentially silence the expression of genes.

Meiosis, Crossing Over and LD

More Crossing-Over means less LD because every time something crosses over, there is a chance the crossing over breaks between the two SNPs. There is less LD between variants if they are far apart (higher chance of a cross-over event) and less LD if they have more generations (more cross-over). The African population (ancestral) has the least amount of LD because they are the oldest of the humans, so they have the most generations.

Human Gene Patenting Case Study

Myriad Genetics discovered and developed a test for mutations in the BRCA1 and 2 genes, which greatly increased breast and ovarian cancer susceptibility. Myriad was awarded patents on the BRCA1 and 2 genes in the 1990s, and due to the patents, Myriad was the only place in the US to go and get these diagnostic tests. They set the cost at $3,340, which is very high compared to the cost to analyze an ENTIRE genome (about $1000). In 2013, the US Supreme Court ruled that isolated human genes cannot be patented. DNA molecules engineered by man are eligible for patents, but NOT naturally existing genes or gene mutations. This decision likely gives greater access to genetic testing and encourages greater scientific research. Scientists can now look at the BRCA 1 and 2 genes a lot easier than before as it is no longer a patented gene.

NIPT

Non-invasive Prenatal Testing - This is a new test that can indicate genetic abnormalities of the fetus in utero. Originally this was done via an amniocentesis, which was invasive and involved sticking a needle into the placenta and sucking out amniotic fluid. This procedure carried a 0.6% chance of a miscarriage. - Now it has been discovered that fetal DNA is present in the mothers bloodstream, so mom's blood can be taken and this fetal DNA can be separated an analyzed for genetic abnormalities. NIPT can be performed beginning around the 10th week of pregnancy.

Pharmacogenomics Place in Therapy

Pharmacogenomics is essentially just one piece of the puzzle, one that if we combine the data with other biologic and omic data, we can improve drug safety and efficacy. There are times where testing for pharmacogenomics is great for the patient, but there are also times where testing is not even worth it.

Circulating Cell-Free DNA

Not fully developed application, but this is a technique that can determine if a patient is rejecting a transplanted organ and can determine if there is a release of dying tumor cells or active metastases. This stems from the natural process of Cell-Free DNA, which is DNA that has been released from the cell after the cell lyses. We can measure the cell-free DNA and sequence it to differentiate between patient and donor DNA in the blood. This can help determine if someone is rejecting their transplant (as the cells in the transplanted organ are dying and releasing their DNA, which is different than the patient), or determine if there is cancerous tumors. This may also have applications in identification of infectious bacteria or viral particles.

SLC Transporters Meaningful Transporters

OATP1B1 (SLCO1B1)- Liver OCT1 (SLC22A1)- Intestine and Liver OCT2 (SLC22A2)- Intestine and Liver MATE1 (SLC47A1)- Kidney and Liver OAT1 and OAT3 (SLC22A6 and 22A3)- Kidney

Single Pathway To Bioactivation

One example of where pharmacogenomics really matters. This is where the medication is a pro-drug to the active drug and metabolism of the pro-drug via some enzyme leads to effectiveness. This metabolism is exclusively one enzyme, typically a CYP450 enzyme. Clopidogrel, Tamoxifen and Codeine are all prime examples of this single pathway to bioactivation. If someone is a poor metabolizer at the enzyme that breaks these into the active drug, then the patient will not get activity. If someone is an ultra metabolizer, then they will have too much activity and experience toxicity.

Characteristics of Intestinal Transporters

Oral drugs are primarily absorbed in the small intestine. Small intestine transporters significantly impact the extent and the variability of drug absorption, which impacts its pharmacokinetics. Drug absorption is dependent on the passive permeability of the administered drug, the dose and other factors. ABC and SLC Transporters are present in the apical membrane of the enterocyte and may facilitate intestinal drug absorption or reduce the bioavailability of a drug. They are also located in other locations, which can determine what effect the drug has.

ABC Transporter Meaningful Transporters

PGP (ABCB1)- Brain, Intestine, Kidney and Liver BCRP (ABCG2)- Brain, Intestine, Kidney, Liver and Placenta BSEP (ABCB11)- Liver

PGP Pharmacogenomic Variation

PGP, or ABCB1, is a huge gene, and has about 1200 SNPs that have been identified. The functionality of the variants depends on the gene expression levels (which impact the protein available), the substrate binding efficiency, and conformational changes that impact substrate binding (slient). PGP has major ethnic differences, but it does not seem to have any clinical effect.

Passive Permeability and Dose Factors for Drug Absorption

Passive Permeability - High passive permeability of a drug will limit the role of transporters in drug absorption. Drugs with low intrinisic passive permeability are more subject to manipulation by transporters. Dose - High doses of a drug may saturate intestinal transport processes, thus minimizing their impact.

Overpromise of Precision Medicine

Patients in the community typically have a tendency to oversell the promise of precision medicine. A lot believe there will be a distinct chemical molecule for each individual patient, which is not possible. We need to curb this initial enthusiasm and adjust everyone's expectations back to reality. Pharmacogenomics and genetic testing is not a "cure-all" and only one more piece to the puzzle. Precision Medicine follows the Hype Cycle, and there is much disappointment in the public eye when we discover the limitations to the technology, but we eventually incorporate what we can do into practice and ultimately find benefit in the technology.

Penetrance

Penetrance of a disease-causing mutation is the proportion of individuals with the mutation who exhibit clinical symptoms. For example, if a mutation in the gene responsible for a particular autosomal dominant disorder has 95% Penetrance, then 95% of those with the mutation will develop the disease and 5% will not. Mendelian Disease, such as Cystic Fibrosis or Sickle Cell Disease have 100% Penetrance. If someone has this gene/mutation, they will have the disease. Hypertension, on the other hand, is a low Penetrance disease, as developing it is due to a variety of factors and a variety of genes.

Promoter Mutations

When a SNP is inserted in a promoter region. The major consequence here is a change in the gene expression and ultimately the protein that is translated.

Ethnic Differences in IM and PM of CYP2D6

Poor Metabolizer alleles are more common in the European population whereas IM alleles are much more common in the East Asian and African populations. East Asian IMs show similar in vitro CYP2D6 activity compared with PMs in the European Populations. There may be gene-gene or gene-environment interactions at play.

Population Genetics

Population genetics is the study of the change of allele frequencies, genotype frequencies and phenotype frequencies. When we talk about population genetics, we are talking about microevolution (the evolution *within* the species).

Other SNP Terminology

RS: - This is the reference sequence number, and is a standardized number for the genomic variation. Unless the variant is totally new, it will have a corresponding RS number in the National Center for Biotechnology Information (NCBI). Nucleotide Sequence - A SNP can be named based on the nucleotide sequence. - For example, a guanine for cytosine substitution at nucleotide number 1165 is 1165C>G Amino Acid Sequence - A SNP can be named based on their amino acid sequence. - For example, an arginine for glycine substitution at amino acid number 389 is Arg389Gly or R389G

Ethical Issues for Pharmacogenomics

Really all these issues pertain to disease genetics over pharmacogenetics: 1. Genetic Discrimination 2. Stigmatization 3. Privacy and Confidentiality of Genetic Information 4. Conceptual and Philosophical Implications 5. Reproductive Issues 6. Balancing Harm and Benefit

SNP, Wild Type, Diplotype

SNP: - A Single Nucleotide Polymorphism, which is a variation in a single nucleotide of the genetic sequence. This is a common form of variation in the human genome. Wild Type: - The typical trait or genome that occurs most abundantly in nature. Diplotype: - The genotype of haplotypes. This is the combined haplotypes from both strands of DNA.

Dimorphism

SNPs are usually assumed to be dimorphic, which means the mutation/change of the base pair at that site is assumed to be a binary variable. The probability of a repeat mutation at the same SNP locus is quite small. However, there are some SNPs that have been found to be trimorphic (three allele options). Trimorphic SNPs are relatively rare, because there would have to be a second mutation at that same site, which is very rare. The nucleotide on a SNP locus is called a Major Allele if the total allele frequency is >50% or a Minor Allele if the total allele frequency is <50%.

Phase II Metabolism and Pharmacogenomics

Several classes of Phase II biotransformation enzymes catalyze conjugation reactions (glucoronidation, sulfation, acetylation, etc). Probably the most important Phase II enzymes are UGT, SULT, NAT, GST, TPMT, ACoAS, and COMT. These enzymes contribute to the majority of clearance pathways that exist for most drugs. The Phase II enzymes that have the most pharmacogenomic relavence are TPMT and NAT. They both have CPIC guidelines associated with them. TPMT is actually one of the oldest pharmacogenomic examples

SCID Cure

Severe Combined Immunodeficiency Same sort of concept as CAR T-Cell Therapy. We take out some of the bone marrow of the patient and genetically modify the progenitor cells so the ADA gene (which is responsible for SCID presentation) is activated. These progenitor cells are grown to make sure the ADA gene is present and active, then the patient's bone marrow is ablated and the progenitor cells are added back to the patient.

SNPs

Single Nucleotide Polymorphisms These are the *KEY* to human variability. 90% of genetic variation found in the human genome is a SNP. A SNP is a DNA sequence variation at a single nucleotide (a substitution to a different base pair) that may alter the function of the encoded protein and is kept through heredity. Changing a base pair can change the codon that section translates to. If that codon is changed, then the amino acid there can be different and the protein is altered. Based on the amino acid that is there the protein can be wildly different and have dramatic effects on protein function. Polymorphisms are common and contribute to common diseases and influence our response to medications. Each person has about 20-40 SNPs or Di novo mutations, which is the error rate of DNA replication. While they do contribute to diversity, the vast majority of them mean nothing clinically. To be considered a SNP, it needs to be at a frequency in the population of >1%. If it is <1% of the population, then it is considered a mutation.

Where Pharmacogenomics Matters

Single pathway to bioactivation is the MAJOR place where pharmacogenomics matters. This is where the drug is a prodrug and must be metabolized to the active form. If someone is a poor metabolizer, they will not get the drug effects. If someone is an ultra metabolizer, they will get too much of the drug effects and may experience ADEs. Phase I Metabolism is this step. Example drugs: - Encainide - Clopidogrel - Tamoxifen - Codeine

SLC Transporters

Solute Carrier Transporters (SLCs) This is another important class of drug carriers and does have some pharmacogenomic relevance. Members include the Organic Anion Transporter Protein (OAT or OATP) and the Organic Cation Transporter Protein (OCT, or OCTP). SLCs as membrane transporters may function as: - Passive transporters - Ion-coupled transporters - Exchangers with organic anions and cations. SLC1B1 and simvastatin have pharmacogenomic relevance.

CYP2D6 Genetic Variation

Star Allele polymorphisms in CYP2D6 were the first CYP variations described, and there are >70 variants described in this gene! These variants, however, either cause no change in function, a small decrease in function or a significant decrease in function. Extensive Metabolizers, Intermediate Metabolizers and Poor Metabolizers, respectively. Extensive Metabolizer, again, is the standard allele against which all others are compared (it is considered the wild type). People can also have fast CYP2D6 metabolism, which is not so much star allele polymorphism, but from duplications of the gene. People who are ultra-rapid metabolizers at CYP2D6 have multiple copies (up to 16 copies) of that gene, so they have an abundance of the enzyme. This is termed Copy Number Variation (CNV).

Star Allele Nomenclature

Star alleles can refer to a specific SNP or a combination of SNPs. Generally s1 represents the reference sequence of the gene or the "wild type." A patient without any polymorphisms will be s1/s1. However, the wild type is not always the most common sequence, but it is the reference sequence. These alleles are generally numbered in order of discovery, so there is no consistency across other genes or star alleles.

Star Allele

Star alleles typically represent a haplotype, so each copy of the genome may be s1. The diplotype, the genotype of the haplotype is going to be s1/s1. s1 is typically normal, reference sequence. There are no mutations. For the VAST MAJORITY of star alleles, s2 and s3 variants decrease the function of the CYP enzyme they are associated with. In regards to haplotypes, someone who is an s1, s2, s3, etc will have a normal genome for that gene all the way up to that one polymorphism. If someone has two polymorphisms in a gene, that is technically assigned a different star allele number (so if someone is a s8 then that might mean they have a s2 and an s3 polymorphism). When we test for particular variants in genotyping, we only look at that one particular location in the gene, so we cannot be 100% sure they are that variant unless we genotype the entire gene to find out if they have other polymorphisms in that gene.

Genome Editing Applications

Technically illegal in current society, but there are a lot of useful applications: 1. Basic understanding of human biology, the role of specific genes and processes 2. To create and study models of human genetic disease in vitro 3. To treat disease (somatic cells) 4. Germline changes to avoid/prevent disease 5. Germline alterations to give "genetic enhancement."

CPIC Guidelines and Example Drugs

The Clinical Pharmacogenomic Implementation Consortium (CPIC) group writes and establishes practice guidelines for clinically significant pharmacogenomic medications. CYP2D6: TCAs, SSRIs, Antipsychotics, Opioids, Tamoxifen, Propafenone CYP2C9: Phenytoin, Warfarin, Antihyperglycemics CYP2C19: TCAs, SSRIs, Clopidogrel, Voriconazole CYP3A4/5: Tacrolimus

GINA Law

The Genetic Information Nondiscrimination Act of 2008, which was passed in response to a lot of these potential ethical and moral concerns about genetic testing. Passed in response to concerns about genetic discrimination by employers and insurers. Insurers may not use genetic information to make enrollment or coverage decisions for patients. Employers may not use genetic information in making decisions regarding admission or employment. This act does not include protections from genetic discrimination in life, disability or long-term care insurance. These are gaps that should be taken care of, because these entities could raise their coverage rates or not cover you based on genetic profile.

Gartner Hype Cycle

The Precision Medicine movement follows the Gartner Hype Cycle, which is a general 5 phase function of Time vs. Visibility (essentially focus on the topic/knowledge on the topic). - Phase 1 is the Technology Trigger - Phase 2 is the Peak of Inflated Expectations - Phase 3 is the Trough of Disillusionment - Phase 4 is the Slope of Enlightenment - Phase 5 is the Plateau of Productivity

Genetic Exceptionalism

The belief that genetic information is special and deserving of greater considerations of consent to and privacy of sequencing and analysis than any other form of medical data (EMR, Lab Tests, diagnoses, etc). A reason for this belief is that genetic testing has implications to family members, and the genetic test is essentially constant and will not change in life. Genetic information is for a large part much more innocuous than other forms of personal medical data. There are certainly implications to the genetic data, and patients should know these implications, but as long as they consent to that, genetic exceptionalism is not that big of a factor.

Proteomes

The complete set of all proteins produced under a given set of conditions. The Proteomes term can be applied to: - The complete set of proteins for a given organism - A specific subset of proteins present in a particular cell type or under specific growth conditions Proteomes vary because they reflect genes that are actively expressed at any given time. Proteomics is still relatively limited as problems can remain with purification and stability of proteins.

Dominant and Recessive Traits

The dominance or recessiveness of a trait describes the phenotype of the individual based on the genotype. This is often confused with the major and minor allele concept. Dominant traits are when only 1 copy of an allele results in the trait being expressed (genotype is either AA or Aa). Recessive traits require 2 copies of an allele to be expressed (genotype must be aa).

Pharmacoethnicity

The ethnic diversity in drug response or toxicity.

Activity Score

The extent of someone's metabolism in regards to CYP2D6 is based on their activity score. The activity score takes into account the type of star allele they have and the amount of copies of the gene they have. The score is a quantitative value based on the sum of the allele activity scores. Allele 1 Score + Allele 2 Score = Total Score Normal function allele is 1, Decreased function is 0.5 and No Function is 0.

Importance of the HWE

The gene pool of a non-evolving population remains constant over multiple generations (allele frequency does not change). Shuffling of alleles by meiosis and random fertilization have no effect on the overall gene pool. Natural populations are NOT expected to actually be in HWE Deviations from HWE usually results in evolution. Mendelian genetics implies that genetic variability can persist indefinitely, unless other evolutionary forces act to remove it.

Central Dogma of Pharmacogenomics

The genetics of the person has direct effects on the overall pharmacogenomics. The pharmacotherapy affects the overall pharmacogenomics. All three (genetics, pharmacogenomics and pharmacotherapy) have direct effects on the overall Disease/Drug Response. The Disease/Drug Response ties in the drugs pharmacokinetics (Transporters, Plasma Protein Binding, Phase 1 and Phase 2 Metabolism, Elimination) and the pharmacodynamics (Receptors, Ion Channels, Enzymes, and Immune Molecules). Example: If you give someone enough lisinopril, chances are their blood pressure will go down. However, the patient's genetics may decide how much of an effect the lisinopril will have on their blood pressure.

Historic Milestone in Genomics

The human genome project was completed in 2003, which allowed for us to create a map of genes. We now have a reference point to see where genes lie on the chromosomes. When we sequence someone for their genome, we can reference this "map" and we can see where the mutations or variations the patient has based on this map. The first human genome (finished in 2003) took 10 years, utilized 20 sequencing centers and cost $2.7 billion. Today, only 1 sequencing center is needed and they can sequence a human genome in about 3 days for about $1,400!

SNP Location and Gene Function

The location of the SNP determines how impactful it is on a person's pharmacogenomics. For a SNP to have an effect on a person's translated protein, the SNP needs to be in an exon of the gene that is being transcribed. An exon is the genetic code of the functional parts of a protein. In between the exons are the introns, which are nonfunctional parts of the protein that when transcribed into mRNA eventually fall out. The final mRNA is just the transcribed exons. The exons are the only portions of DNA that are translated into a protein. If a SNP is located in a Promoter region, there will be no effect on the gene at all, as the promoter region just tells the polymerase when to start transcribing. If the SNP change is significant, it may increase or decrease the transcription of the corresponding protein, but it won't change the amino acid sequence of the protein. This same consequence could happen if the SNP is in a 5'-UTR (Untranslated Region). An example of this is VKORC1, Vitamin K Epoxide Reductase C1. This enzyme is the main target for warfarin. The SNP in this gene cause an increase in mRNA transcription, which increases the amount of protein that is transcripted so a person with this SNP will need more warfarin to inhibit the enzyme.

Functional Consequences of Genetic Variation

The major consequence of variation can result in a loss or gain of function. The consequence of the variation can be described in 6 different ways: 1. Synonymous 2. Missense/Nonsynonymous 3. Nonsense 4. Splice Site 5. Promoter 6. Frame Shift

Mutation

The major reason for genetic structure changes. A spontaneous change in the DNA. This change creates new alleles and is the ultimate source off all genetic variation. Typically a mutation happens due to a mistake during DNA replication. When DNA is copied, mistakes can happen. When the mutations happen they are typically low frequency, but the frequency can increase as the DNA is replicated again and again due to randomness or if it confers an advantage. Eventually they become polymorphisms.

Genetic Variation Influences on Drug Metabolism

The metabolism of many drugs involves both an initial conversion to a more polar form through an addition of an OH, NH2 or SH via Phase I Metabolism and/or a conjugation with another substance such as glucuronic acid, sulfuric acid, acetic acid, or an amino acid via Phase II Metabolism. Many drugs are hepatically metabolized with an initial oxidation via Phase I governed by one or more members of the CYP450 system. These enzymes, designated as CYP-numeral-letter-numeral, control much drug metabolism and have considerable genetic variation. The pharmacogenomics of Phase II Metabolism is an area of more recent intense focus. There are not a lot of pharmacogenomic stories for Phase II yet.

Tag SNPs

The problem of inferring the haplotypes from a set of genotypes is called haplotype inference. Predicting haplotypes from genotypes is called Phasing. Tap SNPs are a small subset of SNPs that is sufficient for performing association studies without losing the power of using all SNPs. SNPs can be linked, so we can generate information about a SNP from another SNP if they are linked (Tag SNPs). We can genotype SNP 1 and we dont have to do anything about SNP 2 because they are linked (Linkage Disequilibrium). This is important because it saves money. We can genotype a relatively small number of SNPs and learn a lot about all the other SNPs an individual can have. Linkage Disequilibrium is what creates haplotypes in a species.

Genomics

The study of all the variations in the genome. Genomics encompasses: - Pharmacogenomics: choosing the correct personalized medicine. - Nutrigenomics: choosing the best diet. - Toxicogenomics: predicting toxicities

Pharmacogenetics

The study of influences of specific variants or variation in *specific genes* on drug disposition and response.

Pharmacogenomics

The study of influences of variation in the human genome on drug disposition and response. Pharmacogenomics includes how variation affects: - Pharmacokinetics - Pharmacodynamics - Treatment efficacy and adverse side effects This is broader than Pharmacogenetics because Pharmacogenomics deals with the whole human genome, not the single gene.

Epigenomics

The study/analysis of an epigenetic trait, which is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Epigenetics is how much of the DNA is actually expressed. This can be affected based on the outside modifications to the DNA that change its actual expression (phenotype), NOT the actual sequence (genotype). There is growing evidence that epigenetic expression profiles can be passed between generations.

Induced Pluripotent Stem Cells

The technique of taking cell and treating it with a lot of different compounds and stimuli and turn it into a pluripotent stem cell, which can be used to grow any type of cell in the body.

Conceptual and Philosophical Implications

There are a lot of implications regarding human responsibility, free will vs genetic determinism and the concepts of health and disease that can be drawn from genetic testing. Genetic modifying people for example.

Ancestry in Pharmacogenomics

There are dramatic genetic differences between ethnic groups: - Allele Frequencies: Many SNPs occur only in some populations or have vastly difference prevalences. - Allele Linkage - Environmental/Social Factors There are also different pharmacogenomic markers by race/ethnicity and the utility of markers may be unclear for certain group and/or they may only be valid in specific race groups. Example is Warfarin. Certain variants are only found in individuals of African decent!

Types of Variations in the Genome

There are many types of variations: - SNPs are the most common - Structural Variation (big chunks of DNA that are being varied): Insertions, Deletions, Duplications and Inversions - Copy Number Varients - Variable Number Tandem Repeats

Future Challenges for Transporter Pharmacogenomics

There are other barriers where transporter proteins are utilized, naming the BBB and the placenta. Suboptimal or extraoptimal drug delivery could be partially mediated by these transporters. Research challenges include the diverse population and inclusiveness of all kinds of people and novel drug development vs new indications for approved drugs. Clinical challenges include the multiple treatment regimens most diseases require, the variation in response to therapy and unpredictable toxicity. Genomics is only one small piece of the therapeutic picture. There are also, of course, ethical challenges associated with all pharmacogenomics research.

SLCO1B1 and Statins

There is now a CPIC guideline for using statins (specifically simvastatin) and the SCLO1B1 gene. If a patient had 1 C allele, they had a 4.5 Odds Ratio for myopathy and if a patient had 2 C alleles, they had an Odds Ratio of 16.9! About 60% of myopathy cases are explained by this allele. SLCO1B1 transports simvastatin into the hepatocyte. Once in the hepatocyte it is metabolized by CYP enzymes. If the SLCO1B1 gene doesnt work, simvastatin does not get into the hepatocyte to be metabolized and there is accumulation of the drug in the plasma (therefore the muscle) and an increased risk of myopathy.

CYP3A4/5 Pharmacogenomics

These are the major CYP3A enzymes in humans and are responsible for about 50% of drug metabolism. CYP3A5 s6 and s7 are prevalently found in blacks at a frequency of 17% and 6%, respectively, whereas they are NOT found in whites or Asians. Immunosuppressive agents that are substrates (tacrolimus and cyclosporine A) exhibit a wide range of interpatient variation in pharmacokinetics, with multiple adverse reactions in renal transplant recipients. Other than these, there are not many examples of CYP3A polymorphisms.

Systems Biology

This is a field of biology that aims at a systems level understanding of biological processes, where there are a bunch of parts connected to one another and they all work together. It attempts to create a predictive model of cells, organs, biochemical processes and complete organisms. Systems Biology integrates all the omic techniques, all the lab values, all the objective values for a patient in order to choose the best medication or create the best treatment plan. Systems Biology identifies elements in a patient (their genes, molecules, cells, etc), ascertains their relationships (co-expression, interactions, etc), and integrates that information to obtain a view of the system as a whole. How does the level of certain proteins affect the expression of the genome? How does the genomic mutation affect the kinds of proteins produced? How do all these intermingle to create disease?

P-Glycoprotein

This is the best characterized efflux transporter, and there are many overlaps and redundancies in the types of substrates it transports. PGP transports a wide variety of substrates that are structurally unrelated. Similar to CYP3A4, there is not a lot of variants that cause clinically significant effects. PGP transports either neutral or positively changed hydrophobic compounds such as antiviral agents, immunosuppressive agents and chemotherapy agents.

Transcriptomes

This is the set of all messengerRNAs ("transcripts") produced from a genome. Genome is all the hereditary information encoded in the DNA and/or RNA. The transcriptomes term can be applied to: - Complete sets of transcripts for a given organism - Specific subset of transcripts present in a particular cell type or under specific growth conditions Transcriptomes vary because they reflect genes that are actively expressed at a given time.

Metabolomics

This is the systematic study of the unique chemical fingerprints that specific cellular processes leave behind. - Example: We can analyze the metabolite profile to see if a genetic alteration causes a change in a phenotype or we can analyze the metabolite profile for a toxicology analysis Metabolites have a wide range of molecular weights and *large variations* in concentration The metabolome is MUCH more dynamic than the proteome and genome, which makes the metabolome more time sensitive

Frame Shift Mutation

This is when a SNP causes a change in the exon that is being transcribed. The SNP can be an insertion or a deletion, which causes a shift in what codons are being transcribed, which can completely mess with the translated protein. Inserted SNPs will shift the codons right while deleted SNPs will shift the codons left.

Heterozygote Advantage

This is when a heterozygote genotype (Aa) confers more fitness than a homozygote. This is usually seen when maintenance of both alleles (1 dominant and 1 recessive) confers better fitness, so evolution maintains both alleles instead of removing the less successful alleles out of the population. Sickle Cell Anemia is a prime example of the heterozygote advantage. Sickle Cell is considered a Mendellian disease, "deterministic." Most genes confer probability of something happening, but a Mendellian Disease is different in that if someone has two sets of that gene, they will get the phenotype. Heterozygous Sickle Cell gene confers better protection from malaria, so the heterozygous combination of alleles is maintained in the population. Cystic Fibrosis is another example.

Nonsense Mutation

This is when an SNP changes a codon from a regular amino acid to a STOP codon. This causes the transcription to terminate early. The resulting protein is not fully created and has no function. It gets degraded and does not provide any function to the body. A mutation like this causes someone to not have any functional protein from that gene.

Gene Mutations

Two types of genetic mutations: Inherited or Acquired Inherited (Germline) Mutations: These are alterations in the DNA that are inherited from a parent and are found in the DNA of virtually ALL of your cells. These are usually SNPs or Gene Duplications and have relatively small effects on the DNA structure. Acquired (Somatic) Mutations: These are alterations in the DNA that develop through a person's life. This is more akin to cancer, where we have a simple cell in the body that develops a chromosomal change. A major change in the DNA occurs due to this mutation, which usually has major effects on the DNA structure. Fortunately, a Somatic Mutation will not be inherited by prodigy.

Transporter Proteins and Pharmacogenomics

Transporter proteins play a key role in Pharmacokinetics because they are essentially the gatekeepers that can allow or prohibit drug function. They are present at every step in pharmacokinetics (ADME) and can have large effects on a drug's overall PK. There are not many CPIC guidelines for transporters, however. This is due to the fact that if someone has a polymorphism that affects the status of a transporter, other transporters around that affected transporter essentially pick up the slack. PGx of membrane transporters are concerned with the drug uptake and efflux into or through tissues. Membrane transporter proteins are involved in the absorption of drugs from the intestinal tract into the body, into non-intestinal tissues (liver, kidney, etc), or into specific target sites of action. Transporters work in concert with drug metabolizing enzymes to impact PK/PD disposition. Theoretically, transporter polymorphism has important effects on in vivo drug pharmacokinetics.

Genetic Linkage

Two mutations that are co-inherited (they are linked together). Usually these genes are close to each other on the chromosome and both mutations are either major allele A or minor allele a all the time. This is not a hard and fast rule... SNPs close together on the same chromosome do not have to be linked, they just have a higher chance to be linked.

Splice Site Mutations

When a SNP is inserted between exons and introns, which can change what exons are incorporated into the protein. Whole exons can be eliminated from the protein.

Phase II Metabolism Pharmacogenomics Drugs

UGT1A1 - Irinotecan: Neutropenia, Diarrhea - Tranilast: Hyperbilirubinemia - Bilirubin: Gilbert's Syndrome UGT1AB - Mycophenolate Mofetil: Diarrhea UGT2B7 - Diclofenac: Hepatotoxicity NAT2 - Isonaizid: Neurotoxicity - Sulfasalazine: Hemolytic Anemia - Hydralazine: Enhanced drug effect - Procainamide: Enhanced drug effect GSTM - Busulfan: Veno-occlusive Disease *TPMT* - Azathioprine: Myelosuppression - 6-Mercaptopurine: Myelosuppression COMT - Levodopa: Enhanced drug effect

UGT1AB, UGT2B7 Drugs

UGT1AB - Mycophenolate Mofetil UGT2B7 - Diclofenac

UGT, SULT, NAT, GST, TPMT, ACoAS, COMT

UGT: UDP-Glucuronosyltransferase SULT: Sulfotransferase NAT: N-Acetyltransferases GST: Glutathione S-Transferases TPMT: Thiopurine S-Methyltransferase Acyl-COAS: Acyl-CoA Synthetase COMT: Catachol-O-Methyltransferase

Harm vs Benefit

Uncertainties associated with genetic testing for susceptibilities and complex conditions (heart disease, breast cancer, etc) linked to multiple genes and gene-environment interactions.

Personalized Medicine

Use of information about an individual, including their family history, diseases, environmental factors and genetic information to personalize or individualize care. Personalized Medicine and Precision Medicine are essentially the same thing and encompass everything. Personalized Medicine takes into account a patient's pharmacogenomics and pharmacogenetics. Personalized Medicine is used to help determine a patient's disease risk prediction, define their disease phenotype and to guide treatment decisions.

Drug Efflux and PGP

Usually highly effective antineoplastic compounds (such as vincristine, vinblastine, daunorubicin or doxorubicin) fail to produce an adequate clinical response. Intrinsic or acquired multidrug resistance (MDR) may be due to the appearance of special membrane efflux proteins. Drug efflux is a significant cause of drug resistance. PGP is an example of many MDR-associated membrane proteins. PGP can transport drugs out of some cells, resulting in low drug levels in targeted cells. ABCB1 haplotypes have been proposed to predict the kinetics of drugs such as digoxin, cyclosporine and fexofenadine.

CYP2A6 Pharmacogenomics

We know that drug levels can be affected by the polymorphisms in this enzyme, but not to the point that clinical testing is super informative. Research is not to the point where we can be confident in making clinical decisions. CYP2A6 metabolizes nicotine, cotinine, coumarin, methylnamylnitrosamine (MNAN), and nicotine-derived nitrosamine ketone (NNK). High expresser individuals have been suggested to experience increased susceptibility to nicotine addition. Apparent ethnic variations in the CYP2A6 polymorphisms were found with about 1% of PMs in whites, but approximately 20% of PMs are in Asians.

CYP2B6 Pharmacogenomics

We know that drug levels can be affected by the polymorphisms in this enzyme, but not to the point that clinical testing is super informative. Research is not to the point where we can be confident in making clinical decisions. CYP2B6 is involved in the metabolism of bupropion, efavirenz, nevirapine, cyclophosphamide and ifosfamide. The most common CYP2B6 variant is the s6, which varies about 15-60% across different ethnic populations. Among the CYP2B6 polymorphisms, s6, s16 and s18 consistently exhibit lower activity for efavirenz in vitro and in vivo. A number of HIV patients receiving efavirenz experience CNS ADEs, which are suggested to be from the varying concentration of efavirenz in the plasma.

Privacy and Confidentiality

Where will this genetic information be stored? Who is allowed access to it? Patients can be identified fairly easily using their genome, and it does not take much of their genome to ID them. Currently genetic information is not considered identifiable information, according to the IRB. It may could be, if we get a hold of someone's genome.

Expected Genotype Frequencies using HWE

p2 + 2pq + q2 = 1, p2 is the %AA in the population, q2 is the %aa in the population and 2pq is the % Aa in the population. The possible range for an allele frequency or genotype frequency lies between 0 and 1. A frequency of 0 means complete absence of that allele or genotype from the population (no individual in the population carries that allele or genotype). A frequency of 1 means complete fixation of the allele or genotype. Fixation means that every individual in the population is homozygous for the allele (ie they have the same genotype at that locus).


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