Pharmacogenomics Flashcards
What is a haplotype?
An inheritable, proximal block of SNPs on the same chromosomes
Example of SNP variation
TAS2R38 gene variation and PTC taste
- Mendelian Inheritance pattern
- 3 Phenotypes; Super-taster, taster, non-taster
- As an inheritance pattern, has been inherited after species divergence
TD50
Dose at which toxicity occurs in 50% of cases
ED50
Dose at which the drug is effective in 50% cases
G6PD Deficiency: what it is
- Glucose 6 Phosphate Dehydrogenase- enzyme that metabolises glycosides
- G6PD deficiency is an X-linked recessive disorder with 140 single-base changes
- 5 classes of enzymatic variation
- 400 million affected worldwide, most common Sub-Saharan africa
How is G6PD deficiency diagnosed
Quantitative spectrophotometric analysis
Flow cytometric assay (assesses RBC G6PD activity)
Classes of enzymatic variation in G6PD deficiency
Classes 1-5
Class I- severe, deficient haemolytic anaemia
Class IV- normal activity
Class V- increased activity
G6PD deficiency in Malaria
G6PD creates NADPH which removes oxidants, such as those generated by antimalarials
- Primaquine (antimalarial treatment) produces excess oxidants, which can’t be removed by NADPH supply
- Destroys RBC membranes and causes haemolytic anaemia
- Sx: Acute haemolysis, jaundice, tiredness, SOB, dark urine
- CI in pregnancy
Other drugs CI in G6PD deficiency
Sulfasalazine
Nitrofurantoin
Isoniazid- what for, features etc.
Nicotinic acid derivative used in TB treatment
- Blocks mycolic acid synthesis (component of the mycobacterial cell wall)
- Metabolised to its active form by N-Acetyltransferase 2
NAT-2 Variation
3 SNPs in NAT2 gene; two are synonymous
- Species and geographical variation
- Rapid, intermediate and slow acetylators
Symptoms of slow acetylators with Isoniazid
Peripheral neuropathy, hepatotoxicity, neuritis
Meta-analysis of acetylator studies:
- NAT2 variation accounts for 88% variability of isoniazid metabolism
- Rapid acetylators have higher rates of microbiological failure, particularly for combination therapy
- ADRs also more common
- Genotype/phenotype disconcordancy in 5% patients
Further PK issues with TB treatment
Isoniazid and Rifampicin both first-line
Variation in Rifampicin activity may also be associated with SNPs of SLCO1B1
How was CYP2D6 variation established
Variation in Sparteine activity with increased effects in 7% mothers (induces uterine contractions)
- Sparteine is metabolised by CYP450 into Sparteine-N1-Oxide, then into 2 inactive metabolites
CYP2D6 variation
CYP2D6 metabolises 25% of clinically-used drugs, importantly Theophylline, COCP, Warfarin, Carbamazepine, and Phenytoin
- Phenotypes: Ultrarapid (UM), Extensive (EM), Intermediate (IM), Poor (PM)
- EM most common in caucasians
Effects of codeine and CYP2D6 status
O-methylation of codeine to morphine is essential for its analgesic activity, but only 10% metabolism
-ADRs are more common in UMs e.g. respiratory depression, with standard PO 25mg TDS in one patient
Clinical study of variation of Codeine and CYP2D6
EMs vs PMs (single PO 75mg codeine dose)
- Increased pain thresholds to nociceptive laser stimuli
- Reduced urine codeine concentration
Clinical study of variation of Tramadol and CYP2D6
AUC (blood-drug concentration) is lower for both enantiomers of Tramadol in PMs vs EMs
- Pain Pressure
- Nociceptive reflex
- Cold pressor reflexes
Have different responses in PMs and EMs
-PMs only had significant response to pressure-pain tolerance and nociceptive reflex
SEs: nausea, dizziness, tiredness
Opioid toxicity in neonates/mothers
-Neonates breastfeeding from UM mother died (concentration in breastmilk was too high; neonates have impaired morphine mwtabolising/eliminating capacity)
- Opioid exposed newborns may have greater methylation of the CYP2D6 gene and related genes- reduced expression, lower metabolism
- In a methadone maintained model of opioid dependent mothers
Epidemiology of Bipolar Disorder
Significant cause of global morbidity (QALYs)
Increased mortality vs. non-bipolar patients
-Suicide increased (but lower than unipolar depression)
-Medical deaths increased
Bipolar I
Manic and depressive episodes
Bipolar II
Hypomaniac and depressive episodes
Cyclothymic disorder
2 years of hypomaniac periods
Bipolar NOS- other medical conditions causing a mood disorder
Symptomatic features of Bipolar
Mania
Hypomania
Depression
Subsyndromal Depression
Features of Mania
Extreme happiness w/euphoria, grandiosity, impulsivity, libido, social intrusiveness
- Psychotic Sx (delusions, hallucinations, formal though disorders)
- Cognitive Sx (racing thoughts, distractibility, disorganisation, in-attentiveness)
Treatments for Bipolar
Mania
- Antipsychotics (Haloperidol, Risperidone, Olanzapine)
- Lithium +/ Sodium valproate if first line ineffective
Long Term management
-APs OR Lithium +/ Sodium Valproate
Lithium treatment in Bipolar
The most-prescribed drug
Requires monitoring
Exerts clinical effects by inhibition of Inositol Monophosphatase, Glycogen Synthase Kinase 3B, and Adenylyl Cyclase
Response to lithium may be a familial trait which clusters in families
- Twin studies have shown lithium prophylaxis is better in twins whose co-twin also has BPD
Purpose of Genome wide association studies
Identifying loci that increase disease risk and predict treatment response
GWAS of Chinese individuals and lithium response
Chen 2014
High specificity/sensitivity (linkage disequilibrium) for 2 SNPs in Glutamic Acid Decarboxylase Like 1 Enzyme (GADL1) on Chr3
- Inclusion criteria: clinical course, adherence, minimising the influence of other medications
- Patients with SNPs and rapid disease cycling had a better treatment response
-GADL1 similar to GAD65/67, underexpressed in bipolar patients
Follow up Chen 2016 study (bipolar)
Carriers of T allele have lower frequencies of recurrent affective episodes than non-T carriers during period adherence
-One GADL1 SNP and medication adherence contribute to treatment response
Additional GWAS study for lithium response
Squassina 2011:
GWAS of 200+ Sardinian patients
- GWAS and Quantitative trait analysis
- SNP in intron 1 of the ACCN1 gene
- Encodes a cation channel with high lithium permeability and Na+ affinity
Genetic variants and lithium response GWAS
4 SNPs on Chr21 on long non-coding RNA genes
- Strong linkage disequilibrium to lithium response
- lncRNAs are important regulators of gene expression in the CNS and may decrease during manic episodes
Evidence against GADL1 involvement in lithium variation
Birnbaum 2014
Microarray/ RNA sequencing identified minimal GADL1 expression throughout the brain
Autopsies identified minimal GADL1 protein expression
Western blot of mouse brain lysates shows that this reduces with age
Role of GADL1 in the Kidney
Is abundantly expressed and involved in PLP-dependent taurine synthesis
Taurine may cross the BBB to interact with glutamate NMDA receptors
-Relation to GADL1 in bipolar
Switching in Bipolar
The sudden transition from one mood polarity to the other
PM CYP2D6 patients likely to experience switching more frequently after introducing a new drug
Pharmacogenetic testing relevant e.g. Amplichip CYP450 test
Features of lung cancer
Non-Small Cell most common (75%)
50% cases inoperable so cytotoxic chemotherapy is first line treatment
- SEs of chemo: Nausea, vomiting, myelosuppression, alopecia, impotence, teratogenicity, carcinogenicity
-17% 5 year survival
EGFR pathway in NSCLC
15% of the 55% of NSCLC patients with mutations have an EGFR mutation
Increase protein expression/phosphorylation in EGFR-dependent pathways which increase Ras/Raf, PI3K/AKT pathway activation
- Induce proliferation and angiogenesis
EGFR mutations
Normally in the kinase domain
Increase sensitivity to TK inhibitors (Gefitinib, Erlotinib)
L858R mutation: increases Gefitinib sensitivity
T790M mutation acquired resistance to TK inhibitors
Benefits of Gefinitib treatment in L858R/ Exon 19 NSCLC patients
Improved clinical outcomes
In vitro: 50x sensitivity to Gefitinib treatment
HER2 and breast cancer
HER2 o/e in 20% breast adenocarcinomas
Mutation is associated with worse untreated prognosis
Phase 2 RCT of Trastuzumab with HER o/e breast cancer
Vogel 2002
Phase 3 RCT
114 women with o/e identified by IHC, FISH
Trastuzumab administered at 2 doses
Clinical benefit demonstrated showing no disease progression at 12 months vs. controls
P3 RCT: effectively enhanced first-line chemotherapy
EGFR in Gastric/GOJ cancers
Bang 2010 P3 RCT
Multi-centre P3 RCT to account for ethnic differences
Inclusion based upon IHC/FISH
Improved overall and progression-free survival when used adjunctively to chemotherapy
- No effect upon number/magnitude of ADRs
Effects of BCHE mutations on Succinylcholine/ Mivacurium activity
Gatke 2007
Mutations responsible for variation in BChE prolong drug activity
BCHEFS126- produces a truncated protein lacking an active site
BCHE328D- produces an inactive protein due to a radical AA change
Have extensively prolonged succinylcholine activity- prolonging respiratory depression and apnoeas
May also have sever reactions to Donepezil etc.
Jensen 1995- determining genetic variation in Succinylcholine activity
Used differential inhibitors of BCHE
- There were no age/sex differences under 10 years
- Activity decreased with age
ALOX5 status and Asthma
ALOX5 encodes 5-Lipooxygenase (5LPO)- involved in leukotreine/ Arachidonic acid synthesis
5LPO encoded by ALOX5 gene; has a variable number of tandem repeats (VNTRs) in promoter region
-Only fully expressed with 5VNTRs
Cells with more/less VNTRs less active in vitro
Patients with mutant ALOX5 have a poorer response to Zileuton (5-LPO inhibitor used in asthma) by FEV1 score
What does the clinical pharmacogenetics implementation consortium (CPIC) do?
Create gene/drug practice guidelines for implementation of certain drugs
Variation in TMPT activity and Thiopurine drugs
What are the alleles- and effects?
Thiopurines e.g. 6-MP are purine analogues, prevent DNA formation
Have a narrow TI, increased risk of myelosuppression
3 alleles of activity:
- TPMT1: 90%, high activity
- TPMT*3A: 10%, intermediate activity
- TPMT*3C: 0.3%, low activity- increased risk of myelosuppression (6MP levels remain higher for longer)
Why are the effects seen in 3A/3C allele patients
The protein encoded by 3A/3C is degraded rapidly by a ubiquitin-proteasome mediated process
Naturally higher in neonatal RBCs vs age-matched adults
ALL and TPMT activity
Children with ALL have better outcomes if they also have low TPMT activity
- Higher concentrations of TGN metabolites can form from lower doses
- Allows dose lowering in deficient patients, so chemo can be delivered at maximum doses
- Reduced rates of mercaptopurine-induced neutropenias
Clinical implementations of thiopurine dosing
Genotyping, phenotyping (radiochemical, chromatographic techniques)
- Concordance of ~86%
- Can be influenced by RBC transfusion
3A individuals need to be treated with 1/10 of standard dose
High activity may need elevated dose
Genotyping is worth it- the cost of 400 genotyping tests is the same as 1 treatment of a TPMT associated ADR
Warfarin administration
Inhibits Vitamin K Epoxide Reductase (VKOR) to reduce clotting cascade activation
Is administered as R-Warfarin but the more potent S-Warfarin is inactivated by CYP2C9
Variation in warfarin activity- CYP2C9 and VKORC1
CYP2C9- 9% of variability
- *1 allele is highest activity
- *2 allele reduced activity
- *3 allele is minimal/no activity
VKORC1- 25% variability
- G allele- high liver expression
- A allele- low liver expression
May also have complete loss of function mutations
Warfarin Dosing Refinement Collaboration
Genotype-specific warfarin dosing
-2014 meta analysis suggests this does not reduce dosing-related ADRs
Other clinical considerations in warfarin dosing
Maintain an INR of 2-3
May be affected by other AC use
Interactions with other CYP450 drugs may occur
Inhibitors of CYP450
Grapefruit/Cranberry juice, valproate, miconazole, Erythromycin, Ciprofloxacin
Inducers of CYP450
Carbamazepine, Phenytoin, Rifampicin, Alcohol, BBQ meat, St Johns Wort
Indications of Carbamazepine
Focused/ Generalised Tonic/Clonic Seizures
Trigeminal Neuralgia
Side effects of CBZ
Anaemias and blood disorders
Ataxia
Dizziness, blurred vision, Nausea
Mechanism of action of CBZ, and how metabolised
Blocks voltage-gated Na channels in neurons
Metabolised by CYP3A4
What effects might CBZ have on CYP3A4
Induces it- may affect transcription/+ expression of CYP3A4/5 via the Pregnane X receptor
Effects of CYP3A4 genotype of CBZ
Korean epileptics study
35 Korean epileptic patients
Investigate CYP3A5*3 expression effects
CYP3A4 activity may vary 20x in vivo
Positive correlation between dose-requirement and serum level of CBZ in expressors and non-expressors
Oral clearance of CBZ in non-expressors was significantly lower and therefore serum levels were significantly higher
Carbamazepine and SCN1A
SCN1A- encodes VG Sodium channels in neurons
SNP7 of the SCN1A gene highly associated with maximum dose
Less of an issue with CBZ than with phenytoin
Starting doses are normally less than the final req.d dose
400mg OD vs 800-1200mg OD
