What do we know about genetics?
- Chromosome abnormalities cause congenital defects
- Mutations for monogenic diseases
- Entire human genome
- Genetic variation identifies for complex diseases
What don’t we know about genetics?
- What all DNA codes for
- Causes of rare monogenic diseases
- Genetics variants affecting drug response
- Most of DNA variants affecting risk of complex diseases
- How genetics and environment interact to cause complex diseases
How is the neonatal heel prick test carried out?
- PCR used
- Primers used complementary to region of interest – one before and one after section
- Screened for 39 mutations of cystic fibrosis
- Use multiple PCRs in one tube (multiplexing)
- DNA ladder of known length used to compare
Sanger sequencing
- DNA denatured, the primer is added to the template (labelled), DNA polymerase, small number of dideoxynucleosides triphosphates added (OH removed)– only one type either A C G T (terminator), and four types of dNTPs.
- Repeated for the different types of ddNTPs
- Different lengths of fragments are made as normal dNTPs are also present
- At the end beaker number 1 will contain different fragment lengths
- Electrophoresis – separated according to mass
- Detection
What is next generation sequencing?
It can be used to find causes of monogenic diseases cheaply, quickly and find novel gene mutations in monogenic diseases. Whole exome sequencing – (just protein coding genes) helped to determine the genes that cause Miller syndrome (DHODH gene)
- Library preparation – DNA fragmentation
- Clonal amplification - PCR
- Cyclic array sequencing – pyrosequencing, sequencing by ligation, sequencing by synthesis
What is pyrosequencing?
Pyrosequencing is a method of DNA sequencing based on the sequencing by synthesis principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequencing relies on light detection based on a chain reaction when pyrophosphate is released.
What is sequencing by ligation?
Sequencing by ligation is a DNA sequencing method that uses the DNA ligase to identify the nucleotide present at a given position in a DNA sequence. It uses the mismatch sensitivity of a DNA ligase to determine the sequence of the target DNA molecule. It uses oligonucleotides.
Sanger sequencing vs NGS
approach:
- Sanger uses one reaction to sequence all exons of a gene
- NGS uses one reaction to synthesise multiple genes
Use:
- Sanger is used to identify unknown mutations by sequencing the whole gene
- NGS is used to analyse unidentified mutations in multiple genes
Pros:
- Sanger has high precision
- NGS can simultaneously do multiple genes and is cost effective
Cons:
- Sanger is expensive, takes time, one gene at a time and it poorly automated
- NGS requires high coverage for accuracy and an abundance of sequencing data
Pharmacogenomics
Studying the genetic basis for the difference between individuals in response to drugs - allows ‘right drug, right dose, right patient’.
Drugs are metabolised in the liver by enzymes - come from genes. Drugs become more expensive as they target a smaller population as profit less for companies
EXAMPLE : Getting the drug right - Type 1 Diabetes and MODY
Getting the dose right - anti leukaemic drug 6-mercaptopurine. TPMT (thiopuirine methyltransferase) gene mutation increases risk of bone marrow
toxicity. Affected mutation people given lower doses
How will genetic information be useful in healthcare?
What are some practical/ethical issues?
- Will help dosage of certain drugs
- Disease risk prediction
- All genetic information can be stored as a genetic profile
Practical Issues:
- Huge amount of information - expensive and time-consuming
- Difficult to decide which information is clinically relevant
Ethical Issues:
- Right ‘not to know’ - incidental findings
- Protection of data
- Equality of access to genetic information
What are direct-to-consumer genetic tests?
Companies can sequence your genome and look for certain genetic markers e.g. BRCA1
Monogenic Diseases:
- Can provide carrier status information (e.g. Tay Sachs)
- Can predict risk of late-onset disease (e.g. BRCA1/2)
- Essential to provide GENETIC COUNSELLING - some of the information may be very sensitive
Complex Diseases:
- Limited clinical utility
- May cause undue alarm
- May offer false reassurance
- Data privacy concerns
What are the applications of whole genome sequencing?
- Identify new gene mutations in monogenic disease
- Identify the differences between normal cells and cancer cells - allows the use of targeted treatments instead of using generalised poisons.
CRISPR
- Cas9 a prokaryotic enzyme used in bacterial defence
- Can be used for gene editing
- Sequence needed for binding of Cas 9 is NGG
- Immunotherapy in cancers (leukaemia) - reengineer T cells so they target cells that express certain proteins made by cancer cells
- Can target wrong genes so could be dangerous
What are the limitations of PGD?
- IVF - physically and emotionally demanding - expensive
- Only suitable for diseases where the genetic/chromosomal abnormality is known
- Can only select for traits that are present/absent in the embryos obtained - can’t design the baby
What is allowed in PGD in the UK?
- Severe early onset genetic disease e.g. Tay Sachs
- Severe late onset genetic disease e.g. Huntingdon’s Disease
- Disease with incomplete penetrance (symptoms are NOT always present in an individual with a disease-causing mutation) e.g. BRCA1/2 mutations
- To choose tissue-matched baby than can provide umbilical cord blood to treat sick sibling
- NOT ALLOWED to choose the sex of the baby for non-medical reasons
What are the ethical issues surrounding PGD?
- Involves discarding unused embryos
- Disability rights arguments
- Slippery slope - designer babies
- Eugenics - improving a population by controlling breeding to increase the occurrence of desirable characteristics
What type of genetic tests are used in PGD?
- Fluorescence In Situ Hybridisation (FISH) - detects chromosomal conditions e.g. Down Syndrome
- FISH is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity. It is used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
- PCR and DNA Sequencing - detects mutations in single genes
What is sequencing by synthesis?
After each base is incorporated a snapshot is taken and the wavelength of light corresponding to the base is recorded
