29. Overview of Genomic Technologies in Clinical Diagnostics Flashcards Preview

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Flashcards in 29. Overview of Genomic Technologies in Clinical Diagnostics Deck (26)
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1
Q

List some genetic technologies

A
  • PCR
  • Fragment analysis
  • Sanger Sequencing
  • Fluorescence in situ hybridisation (FISH)
  • Array - comparative genomic hybridization (Array CGH)
  • Multiplex ligation-dependent probe amplification (MLPA)
  • Next-Generation sequencing
2
Q

Describe polymerase chain reaction (PCR)

A

Fundamental for any DNA application

PCR is used to amplify a specific region of DNA; primers 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 to sequence or for other applications

3
Q

How would you perform fragment analysis after PCR?

A

PCR based assay followed by capillary electrophoresis

Here we are sizing the PCR product

Can be used to detect repeat expansions or other small size changes (up to a few hundred bp)

4
Q

Give an example of a repeat expansion disease that was diagnosed using fragment analysis

A

Huntington’s disease – severe neurodegenerative disorder

Caused by CAG repeat expansion in the Huntingtin (HTT) gene

Normal < 27 copies; Intermediate 27-35; Pathogenic > 35

Expanded protein is toxic and accumulates in neurons causing cell death

Diagnosed with fragment analysis

5
Q

Briefly, describe sanger sequencing

A

Cycle Sequencing; based on the same principles as PCR

Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence.

Here we are reading the dyes to obtain the DNA sequence. We can identify single nucleotide polymorphisms (SNPs), or mutations in this way

Can sequence up to 800 bp of sequence per reaction
Accurate (99.99%)
One reaction = one sequence

It is slow and low-throughput.
Costly to perform

6
Q

What does FISH stand for, and what would you use it for?

A

Fluorescent in situ hybridisation

To detect large chromosomal abnormalities

Abnormalities include:

  • Extra chromosomes
  • Large deleted segments
  • Translocations
7
Q

How would you perform FISH?

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 !

8
Q

What is array CGH, and what is it used for?

A

An array comparative genomic hybridisation

Used for detection of sub-microscopic chromosomal abnormalities

Patient DNA labelled Green and control DNA labelled Red

Patient array comparative genomic hybridisation profile

Increased green signal over a chromosomal segment in the patient DNA

Indicates a gain in the patient sample not present in the parents

9
Q

Describe how to perform an array CGH

A

STEPS 1-3: The patient and control DNA are labelled with fluorescent dyes and applied to the microarray.
STEP 4: The patient and control DNA compete to attach, or hybridise to the microarray
STEP 5: The microarray scanner measures the fluorescent signals
STEP 6: The computer software analyses the date and generates a plot.

The plot shows a patient array comparative hybridisation profile

An increased green signal over a chromosomal segment in the patient DNA indicates a gain in the patient sample that is not present in the parents

10
Q

What is MLPA?

A

One probe oligonucleotide contains the sequence recognized by the forward primer, the other contains the sequence recognized by the reverse primer.

Only when both probe oligonucleotides are hybridized to their respective targets, can they be ligated into a complete probe

11
Q

What do we use MLPA for?

A

We use MLPA to detect abnormal copy numbers at specific chromosomal locations

MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions

Perform fragment analysis of MLPA product

An important use of MLPA is to determine relative ploidy (how many chromosome copies?) as specific locations

For example, probes may be designed to target various regions of chromosome of a human cell

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

12
Q

Describe the advancement in the technology of next generation sequencing

A

Technological advances since the end of the human genome project
Decrease in the cost of DNA sequencing

Development of new NGS methods began 13 years ago with 454 pyrosequencing

DNA sequencing throughput jumped 10 orders of magnitude

Solexa sequencing-by-synthesis (SBS) developed end of 2005

Sequencing market is now dominated by Illumina SBS sequencing

13
Q

What is the current strategy with next generation sequencing?

A

An end to sequential testing

Wider range of tests in a shorter time for less money

Current strategy: Disease panels
Enriching to sequence 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

14
Q

Describe 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

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

Target enrichment

Capture target regions of interest with baits

Potential to capture several Mb genomic regions (typically 30-60 Mb)

15
Q

Describe genome sequencing

A

Universally accepted that genome sequencing will become commonplace in diagnostic genetics

Not all tests will automatically move to whole exome sequencing / whole genome sequencing

  • Panels/single gene tests may still be more suitable for some diseases.
  • Capillary-based methods: Repeat expansions, MLPA, Family mutation Sanger sequencing
  • Array-CGH: large sized chromosomal aberrations
16
Q

What are some drawbacks of exome and genome sequencing?

A

Result interpretation is the greatest challenge

  • 20,000 variants per coding genes ‘exome’
  • Need for good variant databases of well phenotyped cases/control sequences

Ethical considerations

  • Modified patient consent process
  • Data analysis pathways – inspect relevant genes first
  • Strategy for reporting ‘incidental’ findings

Infrastructure and training (particularly IT)

17
Q

The NHS diagnositc lab do?

A

The main role of the lab is to help Consultants reach a genetic diagnosis for individuals and families to help guide treatment and management.

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

Translational Research

18
Q

What are some requirements of an NHS diagnostic lab?

A

Accredited laboratory: ISO standard 15189 for Medical Laboratories.

Scientific, technical and administrative staff.

Provide clinical and laboratory diagnosis for inherited disorders.

Liaise with clinicians, nurses and other health professionals

Provide genetic advice for sample referrals and results

Carry out translational research for patient benefit

Train clinical research fellows, clinical scientists, post- and undergraduate students

19
Q

List some of the different tests the NHS lab performs.

A

Diagnostic

  • Diagnosis
  • Management and Treatment
  • Inform clinical trials

Family mutation

  • Diagnosis
  • 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
20
Q

What are some guidelines for diagnostic testing?

A

All referrals via Regional Genetics Centres

Close liaison with nurse specialists, genetic counsellors, clinicians during testing

International guidelines for predictive testing

Follow up at clinics, nurse led clinics, nurse telephone clinics as required

21
Q

What are the possible outcomes from diagnostic test?

A

Pathogenic mutation

Normal variation
- Polymorphism

Novel variant
- Investigations to establish significance

Previously published/detected variant of uncertain significance

22
Q

How would you interpret results from diagnostic testing?

A
  1. Which domain of protein affected?
  2. Segregation of mutation with disease in family in question
    - De novo dominant
    - If recessive, are they definitely on separate alleles – test parents
    - Detected in other affected individuals? Age of onset?
    - Has incomplete penetrance been described for this gene?
  3. Amino-acid conservation between species
  4. Nucleotide conservation
  5. Transcript analysis
    - Is the gene expressed in blood / fibroblasts?
    - In-vitro splicing experiments
  6. Functional studies
    - E.g. Ion channel function in Xenopus oocytes
23
Q

List some benefits of the 100,000 genome projects

A
  • Bring direct benefit of genetics to patients
  • Enable new scientific discovery and medical insights
  • Create an ethical and transparent programme based on consent and patient engagement
  • To kick-start the development of a UK genomics industry
  • PERSONALISED MEDICINE
24
Q

What was the 100,000 genome project?

A
  • UK – wide collection
  • GMCs (genomic medicine centres)
  • Who/what is being sequenced?
  • Rare diseases - families
  • Cancer – germline and tumour samples
25
Q

How were the results of the 100,000 genome project analysed using the genomics england panel app?

A

Genomics England Panel App
Community driven genetic interpretation
Crowdfunding research
‘Experts’ develop lists of possible genes than can cause a disease phenotype
These panels are reviewed by the community

Diseases have specific sets of virtual gene panels as a first port of call to look for pathogenic mutations

Thus we can focus on specific regions of the patients genome we think are important

26
Q

What are the 3 classification of variant by genomics England?

A

Classification of variants by genomics England
Maximise diagnostic efficiency
Balance sensitivity and specificity

Variants within virtual panel divided into three tiers

  1. Tier 1 variants
    - Known pathogenic
    - Protein truncating
  2. Tier 2 variants
    - Protein altering (missense)
    - Intronic (splice site)
  3. Tier 3 variants
    - Loss-of-function variants in genes not on the disease gene panel

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