Molecular Genetics Flashcards
(23 cards)
What is the difference between a germline and a somatic mutation?
Germline mutation
- mutation in the gametes
- Heritable mutation in germ cells
- Constitutional mutation in offspring: present in all cells of body
- Occurs soon after fertilisation or passed on from parent with
constitutional mutation
- Test DNA extracted from blood
- All inherited mutations are passsed in germline mutations, can pass
from an unaffected parents
Somatic mutation
- mutation in any other cell line other than the gametes
- Non-heritable mutations
- Only affects certain cells/ cell types in body
- Mutation occurs early in development or later in life
- Test affected tissue eg tumour tissue
Mosaic mutation
- presence of two or more populations of cells with different genotypes in one individual developed from a single fertilised egg
How will the child be affected in the following mutations?
Germline:
A - father has mutation in all cells (germline and somatic), genotype is a
fully dominant constitutional mutation
B - father with mosaic mutation in germline and somatic cells
C - father has a germline mosaic mutation
De Novo germline:
D - father has mutation in a single sperm cell and transmits it to the child
E - mutation occurs in zygote within the first few mutations
A - this will be transmitted to the child, who will be similarly affected.
B - Child will be heterozygous in every cell and will probably have worse symptoms than the parent.
C - the father will not show any symptoms, but will pass the mutation to child who will be fully affected.
D - the child will have the genotype but it will not be seen in the parent, they will be heterozygous in every cell but it cannot pass it on to further generations.
E - somatic mutation has occurred early in the child, so it will occur heterozygously in every cell.
F - mutation has occurred later on in development so will only affect specific cell types and lead to a milder phenotype.
Define diagnostic testing
Confirm or exclude a genetic disease in a symptomatic patient
Define carrier testing
Identify asymptomatic adult who is a carrier of an AR or X-linked recessive disorder
Family history of disease or member of an ethnic group at increased risk of genetic disease
Mutations associated with an ethnicity
Define predictive and presymptomatic testing
Asymptomatic individuals with a family history of a genetic disorder and a potential risk of developing the disorder
Identify patients with increased risk of developing disease
Screen for diseases that manifest later in life; breast cancer
Define prenatal testing
Identify foetus with a genetic condition before birth
Define newborn screening
Test of newborn to identify conditions that require immediate initiation of treatment to prevent death or disability
Define pharmacogenetic testing
Targeted tests for variants associated with drug dosage choice or adverse reactions
When is Sanger sequencing used?
Small genes
Complex genes
Direct mutation tests
Mutation confirmations - already know the mutation, so testing family members. Or know the gene cause of the disease already
When is NGS used?
MiSeq: - large single genes
- small gene panels
NextSeq: - large gene panels
- exomes
What is pyrosequencing and when is it used?
A real time PCR enzymatic reaction, ensuring pyrophosphate intake for DNA formation
Real time detection of sequencing events
Rapid and accurate quantification of variants in short DNA sequences
Useful for mosaic detection
What are the different types of fragment analysis?
MLPA/ Dosage: detection of large deletions and duplications
ARMS: detect single base changes/ small deletion
Methylation-specific PCR: differentially amplify methylated regions of interest
Fluorescent PCR & Triplet-primed PCR: identify expanded triplet repeat alleles
Commercial kits: detect a set of specific mutations
Sequenom: detection of somatic mutations in tumours
Restriction digest: detect mutations which disrupt restriction enzyme site
Linkage analysis: indirect genetic testing in absence of known mutation (tack haplotypes through.a pedigree to see if someone is low or high risk - used for unknown mutations)
What is the process of reporting and interpreting genetic data?
Data analysed: commercial software, in-house methods
2 independent checks of data: Genotyper and validator
Report generated: results interpreted within context of the referral
Further testing/ referral to genetics recommended, as appropriate
Limitations of test disclosed, eg Sanger sequence cant pick up large deletions or insertions
Authorised by competent clinical scientist
What is next generation sequencing?
Genomic DNA is fragmented into a library of small segments.
The fragments are accurately sequenced in parallel.
Individual sequence reads are reassembled by aligning to a known reference genome using bioinformatics.
The whole sequence is derived from a consensus of aligned reads across the genomic region.
Get as many reads alligned as possible to give greater depth of coverage and high quality data.
When you have lots of genes to do over a greater region it leads to lots of artefacts and so worsening
Involves huge data sets, so incidental findings are inevitable
What are examples of large, targeted gene panels analysed on NextSeq 500?
Large, targeted gene panels analysed on NextSeq 500:
- Retinal dystrophy: 176 genes - Ophthalmic Disorders: 203 genes (sub-panels) - Cardiac: 71 genes (sub-panels) - Metabolic: 225 genes (sub-panels)
What are gene panels?
Panels specifically designed based on the clinical indication of patient (reduce the likelihood of accidental findings and give ability to expand search if nothing is found) and use SureSelect enrichments
How is data analysis performed?
Automated exclusion of known validated polymorphisms
(anything over 1% in general population - must be varied population and must have good depth of coverage)
Assumed pathogenic changes: nonsense, frameshift, consensus splice site mutation
(Quite easy to classify. Assumed that these are definitely pathoggenic, but the more genes we are testingas part of a large panel the more careful we must be as there is are certain mutation spectrums associated with many genes. For example, a gene with a nonsense mutation isn’t associated with a disease but a misense will cause a dominant gain of function)
Remainder of variants: investigate available evidence
How are variants investigated for pathogenicity?
Use databases to determine whether the utation is commonly seen in the population - if it is then t is unlikely to be pathogenic.
Similarly if it is conserved across species it is ore likely to be in a functionally important domain and so more likely to be pathogenic.
Biggest wealth of data is in house, some panels have been tested for many years so we know what to expect - may contradict research?
What is the definition of an incidental finding?
Clear/likely pathogenic changes with no clinical relevance to the reason for referral.
- Clear dominant mutations in genes that cannot account for the phenotype/family history
- Recessive/carrier status - find heterozygous changes in recessive genes (re we missing a second change or are they a carrier)
- Changes predictive of other problems (eg retinal dystrophy mutations also cause bone density problems later in life)
How are variants of unknown significance further investigated?
Parental studies:
- check if recessive changes are in cis (same allele) or in trans
(different alleles)
- confirm homozygosity
- has change arisen de novo
- consider germline mosaicism and non-paternity for negative results
Segregation studies:
- is the variant present in other affected family members and absent in
unaffected relatives.
- complicated by non-penetrance, adult-onset diseases
Reassess variant pathogenicity at a later date:
- review new literature/ database results/ in-house data
What are the benefits of NGS?
Enables testing for heterogeneous disorders
May change the clinical diagnosis
Confirms mode of inheritance: calculate recurrence risk
Earlier medical intervention/ improved patient care
Efficient test, cost decreasing
Single pipeline for testing
Detects mosaic mutations at low levels
Wealth of data (particularly for exons): can inform pathogenicity of variants
Multidisciplinary approach
What are the limitations of NGS?
Incidental findings
Unreliable detection of large deletions/ duplications (now have CMB incorporated but only for large targeted panels, but not for genomes and exomes)
Deep intronic changes; how to analyse?
Data storage in NHS
Challenges for clinicians/counsellors
Capacity vs. coverage
Difficult variant interpretation
Carrier findings: implications for partner
What is the 10 000 genomes project?
Genomics England:
Company owned and majority funded by DoH
Sequence 100,000 whole genomes by 2017- extended to the end of 2018
Focus on Rare Disease & Cancer
Cancer: 50,000 genomes ~25,000 NHS patients - tumour genome and corresponding normal genome
Rare disease: 50,000 genomes ~15,000 NHS patients & 35,000
healthy relatives unhealthy patient compared to two healthy parents,
Aim to improve/ transform
‘Genomic Medicine’
