Personalized medicine Flashcards
Reasons for genetic testing
- Likelihood of developing disease
- Choosing the most appropriate drug
for treatment
• Pharmacogenomics &
personalised medicine - Legal
- Ancestry
Pharmacogenomics (PGx) – treatment regimens
• The aim is to leverage an individual’s genomic data to the treatment of disease so that: 1. The right drug is prescribed 2. The correct dose and dosage schedule is determined 3. Avoid adverse side effects • Drugs are not equally effective in all individuals • Some people respond more favourably over others
Pharmacogenetics example
CYP2D6 gene
• CYP2D6 gene encodes debrisoquine hydroxylase, expressed primarily in the liver • Responsible for the metabolism of 25% of drugs (such as paracetamol & codeine) • There are 70 different CYP2D6 alleles
Pharmacogenomics - Targeted therapies
Use genomic data to develop targeted therapies to suit the unique genotype of the individual • ~25% of breast cancers showed HER-2 overexpression • Correlated with increased invasiveness • Herceptin® mAb blocks HER-2 signalling • Partially successful – problem heterogenicity of tumor genotypes**
Genotyping
• Detects SNPs over entire genome or in targeted
genomic regions
• Allows for the identification of SNPs that have been
associated with particular diseases*
• *determined from prior GWAS study (studies)
• Can screen for a lot of markers at once (4 million or so)
Whole-genome sequencing
• Provides context to the genetic variations observed
• Provides entire genome sequence to analyse
• Allows for the identification of ‘rare’ mutations not previously characterised
• Typically completed using Next-generation sequencing (NGS)
• Several different technologies exist, with new ones
emerging
NGS vs genotyping
NGS Complete information All variants may be identified (incl rare / new) Expensive (in comparison) Diagnosis
Genotyping Partial information Only identifies known variants Cheaper (in comparison) Slower (in comparison)** Rapid (in comparison) Determination of risk (disease prevention)
NGS outlook
• Costs per genome coming down
• Technologies developing greater throughput
• Leading to the NGS revolution
• Power of NGS in healthcare is determined by size of our datase
Towards WGS implementation in healthcare
Current focus on rare diseases
GWAS analysis
Manhattan plot Each ”dot” is a SNP, reported based on its p value for association Threshold for GWAS significance (5 x 10 -8)
Polygenic risk scores (PRS)
• A measure of an individual’s liability for a particular phenotype (disease)
• Based on GWAS data
• Confers a degree of ‘weight’ to the predictive power of NGS or genotyping data for an individual
• Individuals with a higher weighted PRS may be more likely to develop the trait
• Limited by the size / source of the dataset – mostly European (at
present)
Liability
Liability is a term used to collectively describe all the genetic and environmental factors that contribute to the development of a multifactorial disorder.
Clinical utility of PRS
Potential to impact clinical practice (and related fields) and the management of complex diseases to the same extent as WGS in the diagnosis of rare diseases
• Will be even more accurately derived when WGS cost reaches that of
genotyping (ie WGS replaces genotyping)
• Redirect resources from treatment to prevention in complex diseases
(from ”diagnose and treat” to ”predict and prevent”)
Direct to consumer (DTC) genetic testing
DTC refers to a genetic test you can complete at home
without a healthcare provider, doctor prescription.
• You collect a DNA sample and send it to the company.
• They analyze it and produce a report on your genetics
• There are many different types of tests available for:
o ancestry
o kinship
o lifestyle/health factors
o disease risk
DTC genetic testing vs traditional medical testing
test initation DTC: patient Traditional; healthcare worker Quality control: DTC:test quality largely unregulated traditional: regualted, quality system Data interpretation regulation Traditional: data interpreter are licensed
Ethical implications of DTC genetic testing
- Do you want to know?
• If the trait has no known cure / treatment, would you want to know? - Reliability of data for non-European populations
• Limitations of PRS - Individuals may be unprepared for the results
• What if the report returns an unexpected finding? Where can the consumer seek more information / treatment options
• Increased load on clinicians - Who should know the results?
• Are relatives / life partners entitled to know the results of the test, if it
has the potential to effect them? - Privacy concerns
• What safeguards are in place to prevent employers / insurance providers / etc from obtaining the results?
Epigenetics
”Heritable” changes in gene expression (phenotype) that do not involve changes in the DNA sequence (genotype)
What are the the major mechanisms of epigenetic regulation?
• Histone modifications & chromatin remodelling • DNA methylation & CpG islands -Genomic imprinting • Non-coding RNAs -miRNAs, siRNAs, lncRNAs
Histone modifications
Chromatin structure is dynamic N-terminal regions of histone molecules can be chemically altered
Types of histone modifications
Histone modifications to N-terminal tails
Most modifications made to H3 or H4
Histone tails can be modified at multiple sites
• Acetylation
• Methylation
• Phosphorylation
Modifications are reversible
Naming histone modifications
• Modifications made to amino acids within histone tail
• Typically lysine (K), serine (S), arginine (R)
& tyrosine (Y)
• Named following the convention:
[Histone][Amino acid][Position of AA in tail][Type of mod][Number of mods]
E.g. trimethylation of arginine 11 in histone 3 N-terminal region
H3R11me3
Complexity of histone modifications
Different modifications to the same AA
can have different effects
• Histone modifications have strong effects on mRNA
transcription/expression
• Different modifications at the same residue (ie H3K9me1-3) can have opposite effects
Histone modifications in active vs silent genes
- Histone modifications are COMPLEX & typically act in combination
- Active genes have combinations of modifications in genetic components
- Silent genes typically have uniform modifications
Histone modifications in human diseases
Usually due to mutations in the histone modifying enzymes (histone acetyl transferases HAT, protein methyltransferases PMT) • Rare, severe conditions
DNA methylation
Different to histone modification
• Addition of a methyl (-CH3 ) group to position C5 of a cytosine residue
• Added by DNA methyl transferases (DNMTs)
Almost always methylates cytosine
residues that are adjacent to guanine
base (CpG dinucleotides)
• Often called CpG methylation
CpG islands
• CpG sites cluster in specific regions of the DNA sequence • Called CpG islands • Typically found in promoter and enhancer sequences • Methylation prevents the binding of transcription factors (& other components of the transcription machinery) – silencing gene transcription
DNA methylation patterns are..
DNA methylation patterns are tissue & cell specific
• Allows for tissue / cell specific gene silencing
• Prevents the aberrant expression of unwanted cell-specific gene product
Genomic imprinting
• Certain genes show the expression of ONLY
the maternal or paternal allele
• Genes show a parent-of-origin expression pattern
• DNA in the gametes are methylated with
parental methylation patterns
• NOTE – parental Me patterns might be different
to each other (paternal allele silent; maternal
allele active)
• Upon fertilisation MOST methylation marks
are erased
• Allows for germline cells to adopt tissue-specific
methylation patterns to allow for tissue
development
• Some genes escape the demethylation and
remethylation rounds
• Retain parental methylation marks
• These genes remain silenced, so expression
dependant on parental origin of allele
DNA methylation in cancer
Hypermethylated tumor suppressor gene turn it off
Hypomethylated oncogene turn it on
Non-coding RNAs (ncRNAs)
Gentically encoded RNA molecules that DO NOT encode a protein • Divided into 2 main classes based on length • Short (<30 ribonucleotides) • Further divided into: • MicroRNA (miRNA) • Short-interfering RNA (siRNA) • Long (<200 ribonucleotides) called long noncoding RNA (lncRNA)
MicroRNA (miRNA)
• Form of post-transcriptional regulation • Found in animals and plants • Tyically encoded from endogenous genes • Characterised by single strand, stemloop structure • Partial match with target genes (3’- UTR) • Allows for multiple targets
MicroRNA mechanism
miRNA encoded by gene in genome & transcribed
• Leads to single-stranded RNA molecule, that folds back on itself (stem-loop structure)
• Called Primary miRNA (Pri-miRNA)
• Pri-miRNA processed by Drosha (cleaves 5’ & 3’ tail)
• Exported from nucelus
• Now in the cytoplasm, DICER cleaves loop structure
• Leads to double-stranded RNA
• miRNA is protected by RNAinduced silencing complex (RISC) & Argonaute (Ago)
• Passenger strand discarded
• miRISC (miRNA + RISC) binds to target mRNA(s)
• Typically within 3’UTR
• Note degenerate nature
siRNA
• Encoded from exogenous (i.e. viral) as well as endogenous sequences • Double-stranded RNA – no stem-loop • Perfect match to target sequence • No degeneracy
siRNA mechanism
Double-stranded RNA processed
by DICER
• siRNA protected by RISC and Ago
• Passenger strand discarded
• Activated RISC binds to complementary sequence in
target mRNA
• This is a perfect match, so offers specificity in regulation - mRNA cleavage
RNAi-mediated silencing (Gene knockdown)
• Can be exploited to study gene function • Via exogenous delivery of genespecific dsRNAs (siRNA) in vitro / in vivo • Targeted mRNA degradation (cleavage) results in knockdown of expression
Long non-coding RNA (lncRNA)
- Up to 200 ribonucleotides long
- Similar to mRNAs
- Have single-stranded, 5’ methyl cap, 3’ poly A tail, spliced
- BUT no ORF
- Can have different subcellular locations
- ~30% of lncRNAs are nuclear based
- ~15% of lncRNAs are cytoplasmic based
- 55% found both in nucleus and cytoplasm
Mechanism of lncRNA
Guide • Help to recruit other proteins (such as chromatin modifying proteins) to local site Scaffold / Adapter • Help to scaffold multiple proteins together to form a complex, which subsequently interacts with DNA Decoy • Act as a decoy to sequester other miRNAs or transcription factors, preventing them from binding to DNA
