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Molecular Basis Of Disease > Overview Of Genomic Technologies In Clinical Diagnostics > Flashcards

Overview Of Genomic Technologies In Clinical Diagnostics Flashcards

1
Q

What is PCR used for and how does it work (briefly)?

A
  • PCR is used to amplify a specific region of DNA (via denaturation, annealing and extension)
  • Primers (short stranded DNA complementary to region) flank the region you want to amplify.
  • Each cycle doubles the amount of DNA copies of your target sequence
  • Amplify enough DNA molecules so that we have sufficient material for downstream applications
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2
Q

What is fragment analysis and what is it used to detect?

A
  • PCR based assay
  • PCR followed by capillary electrophoresis
  • Here we are sizing the PCR product -> working out the relative length (BP)
  • Can be used to detect repeat expansions or other small size changes (up to a few hundred bp)
  • Repeat expansion = a triplet that causes a disorder where the higher the number of triplets, the higher the severity of disease gene
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3
Q

State an example of a repeat expansion disorder and how its diagnosed?

A
  • Huntington’s disease - severe neurodegenerative disorder
  • Caused by CAG repeat expansion in the Huntingtin (HTT) gene
  • Normal < 27 copies; Intermediate 27-35 copies; Pathogenic > 35 copies
  • Pathogenic: Expanded protein is toxic and accumulates in neurons causing cell death
  • Diagnosed with fragment analysis
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4
Q

What is sanger sequencing and how does it work?

A
  • Cycle Sequencing; based on the same principles as PCR
  • Process:
  • Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence.
  • Read the dyes to obtain DNA sequence
  • Can identify single nucleotide polymorphisms or mutations
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5
Q

Why is it not used for large numbers of samples?

A
  • Good for sequencing single exons of genes
  • Slow, low-throughput and costly to perform for large numbers of samples
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6
Q

What is FISH? What does it use?

A
  • FISH = Fluorescent in situ hybridisation
  • Uses: Cultured cells, metaphase spread
  • All the genetic material is condensing into chromosomes. These chromosomes then become visible.
  • During this stage, the nucleus disappears and the chromosomes appear in the cytoplasm of the cell.
  • Microscopic (5-10Mb)
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7
Q

What is FISH used to detect?

A
  • To detect large chromosomal abnormalities
  • Extra chromosomes
  • Large deleted segments
  • Translocations
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8
Q

State the process for how FISH is done?

A
  1. Design Fluorescent probe to chromosomal region of interest
  2. Denature probe and target DNA
  3. Mix probe and target DNA (hybridisation)
  4. Probe binds to target
  5. Target fluoresces or lights up
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9
Q

State examples of FISH processes and 2 disorders it can detect?

A
  • Processes: Special karoytoping (label different chromosomes with different lights) and target specific FISH
  • Disorders: Trisomoy 21 and Down syndrome
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10
Q

What is Array CGH and what is it used to detect?

A
  • Array comparative genomic hybridisation
  • Used to detect sub-microscopic chromosomal abnormalities (which FISH can’t do)
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11
Q

Describe how Array CGH works and the subsequent result scenarios?

A
  1. Patient DNA (green dye) and control DNA (red dye)
  2. Applied to microarray and hybridised
  3. Microarray measures fluorescent signals and generates plot
  4. Green indicates DNA gain, red shows DNA loss (both chromosomal abnroamlities.
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12
Q

What is MLPA?

A

Multiplex ligation-dependent probe amplification (MLPA) is a variation of PCR that permits amplification of multiple targets.

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13
Q

What is MLPA used to detect?

A
  • We use MLPA to detect abnormal copy numbers (Repeats of sections of genome) at specific chromosomal locations
  • MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions
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14
Q

What does each probe consist of in MLPA?

A

Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA

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15
Q

Describe the probes used for MLPA

A
  • Two probes are used
  • One probe oligonucleotide contains the sequence recognized by the forward primer
  • The other contains the sequence recognized by the reverse primer.
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16
Q

Describe the process of MLPA?

A
  1. Hybridisation occurs of both probe oligonucleotides to their respective targets
  2. Ligations occurs both probes into complete probe
  3. PC amplification occurs of complete probe to form amplified library
  4. Perform fragment analysis (capillary electrophoresis) of MLPA product
    a. Allows to see size/dosage of products forms
17
Q

How are results of MLPA worked out?

A
  • The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome
18
Q

What are MLPA results used for?

A
  • An important use of MLPA is to determine relative ploidy (how many chromosome copies?) at specific locations.
  • For example, probes may be designed to target various regions of chromosome of a human cell
19
Q

What is next gen sequencing used for and why is it used over sequential testing?

A
  • Current strategy: Disease panels
  • Disease panal: sequences only the known disease genes relevant to the phenotype
  • Panels expandable to include new genes as they are published
  • Potentially pathogenic variants confirmed by Sanger sequencing
  • Has replaced SS for almost all Sequencing tests in the lab
20
Q

In regards to whole genome sequencing, what will it not replace in 1. cystic fibrosis 2. capillary-based methods 3. large sized chromosla aberrations

A
  • NOT all tests will automatically move to whole genome sequencing
  • Panels/single gene tests may still be more suitable for some diseases, e.g. cystic fibrosis
  • Capillary-based methods: Repeat expansions, MLPA, family mutation confirmation Sanger sequencing
  • Array-CGH: large sized chromosomal aberrations
21
Q

What are the common issues surrounding exome and genome sequencing? (PART 1)

A
  1. Result interpretation is the greatest challenge
    - 20,000 genetic variants identified per coding genes ‘exome
    - 3 million variants in a whole human genome
    - Difficult to identify pathogenic mutations due to large number of mutations
  2. Ethical considerations
    - Modified patient consent process
    - Data analysis pathways - inspect relevant genes first
    - Strategy for reporting ‘incidental’ findings
    - Incidental findings = identification of pathogenic mutations of other disease not discovered for person - raises ethical issues of whether to tell person
22
Q

What are the common issues surrounding exome and genome sequencing? (PART 2)

A
  1. Infrastructure and training (particularly IT and clinical scientists)
    - For interpretation of genes
23
Q

What stage of whole genome sequencing is the most time-consuming and why?

A
  • 3 steps: data generation, data processing and automated interpretation and manual intrepration
  • Interpretation of clinical genomes currently has a substantial manual component - longest step
  • Whole genome sequencing is NOT trivial
24
Q

What is the 100,000 genomics project?

A
  • Bring direct benefit of whole genome sequencing and genetics to patients via company genomics england
  • Enable new scientific discovery and medical insights
  • Personalised medicine
25
Q

State the source of genes for the 100,000 genomics project

A
  • England - wide collection
  • GMCs (genomic medicine centres)
26
Q

Who/what is being sequenced?

A
  • Rare diseases - index cases (initial patient of disease outbreak) + families
  • Cancer - germline and tumour samples
27
Q

How are mutations classified by genomics England in the 100,000 genome project?

A
  • Classified as variants within a virtual panel into 3 tiers
  • Tier 1 variants: Known pathogenic, Protein truncating (shorten length?)
  • Tier 2 variants: Protein altering (missense), Intronic (splice site)
  • Tier 3 variants: Loss-of-function variants in genes not on the disease gene panel
  • Expert review is required for this
28
Q

Revise at flash card 20
What is exome sequencing?

A
  • There are ~21,000 genes in the human genome
  • Often we are only interested in the gene protein coding exons or ‘exome’ represents 1-2% of the genome
  • Some ~80% pathogenic mutations are protein coding
29
Q

Why is the exome sequencing used over the genome?

A
  • More efficient to only sequence the bits we are interested in, rather than the entire genome
  • Costs £1,000 for a genome, but only £200-£300 for an exome
30
Q

Revise at flash card 20
State the general technique for exome sequencing?

A
  • Technique: Target enrichment
  • Mix with RNA - hybridise - purify via magnetic beads -> capture beads -> wash beads and digest RNA -> Amplify and sequence
  • Capture target regions of interest with baits
  • Potential to capture several Mb genomic regions (typically 30-60 Mb).
31
Q

What is the NHS diagnostic laboratory and state its main aim?

A
  • Provide clinical and laboratory diagnosis for genetic disorders
  • Provide genetic advice for sample referrals and results
  • The main role of the lab is to help Consultants reach a genetic diagnosis for individuals and families to help guide treatment and clinical management (management plan)
32
Q

What factors are used to measure the effectiveness of NHS diagnostic lab tests and what network has a network of all the genetic disease?

A
  • Perform specific tests with proven:
  • Clinical Validity: How well the test predicts the phenotype
  • Clinical Utility: How the test adds to the management of the patient
  • UKGTN (UK genetic testing network)-approved tests:
  • In-depth and up-to-date knowledge of the genetic diseases covered
33
Q

What is the NHS diagnostic laboratory used for (5)?

A
  • Diagnostic: Diagnosis! Management and Treatment! Interpretation of pathogenicity
  • Predictive: Life choices, management
  • Carrier (recessive): Life choices, management
  • Diagnostic testing is available for all Consultant referrals
  • Clinical Geneticists most common referrers
  • Informed consent: Genetic counselling, Implications for other family members
34
Q

State the 3 possible outcomes of diagnostic tests?

A
  • Pathogenic mutation: The variant is responsible for causing disease
  • Normal variation: Polymorphism
  • Novel variant: Investigations to establish clinical significance, Effect of gene is unclear basically
35
Q

How do we establish whether a mutation is pathogenic?

A
  • Mode of inheritance
  • Genetic databases of published and unpublished data: See previous history of gene
  • Nonsense, frameshift, splice site (exon+/-2 bp) mutations
  • Missense intronic mutation!:
  • In-silico tools for missense and splicing mutations
  • In genetics, a missense mutation is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. It is a type of nonsynonymous substitution.
36
Q

Describe how diagnostic tests results are intrepreted?

A
  • Do not report known polymorphisms
  • Have a conservative approach to reporting novel mutations of uncertain pathogenicity: Uncertain significance’, ‘Likely to be pathogenic’
  • Request samples from family members - look for segregation of genes
  • How pairs of gene variants are separated into reproductive cells
  • If gene can’t be found - Continue testing other genes?

Molecular Basis Of Disease (30 decks)

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