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Fundamentals of P > Overview of genomic technologies in clinical diagnostics > Flashcards

Overview of genomic technologies in clinical diagnostics Flashcards

1
Q

List 7 Genomic 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

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2
Q

Polymerase Chain Reaction (PCR) - use

A

PCR is used to amplify a specific region of DNA

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3
Q

Polymerase Chain Reaction (PCR) - action

A

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 for downstream applications

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4
Q

Fragment analysis - define

A

PCR based assay

PCR followed by capillary electrophoresis

Here we are sizing the PCR product

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5
Q

Fragment analysis - use

A

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

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6
Q

Huntington’s disease - define

A

Huntington’s disease – severe neurodegenerative disorder

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7
Q

Huntington’s disease - cause

A

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

Expanded protein is toxic and accumulates in neurons causing cell death

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8
Q

Huntington’s disease - ranges of normal to pathogenic

A

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

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9
Q

Huntington’s disease - diagnosed using

A

Diagnosed with fragment analysis

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10
Q

Sanger Sequencing - define

A

Cycle Sequencing; based on the same principles as PCR

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11
Q

Sanger Sequencing - describe action

A

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

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12
Q

Sanger Sequencing - +ves/-ves

A

Up to 800bp of sequence per reaction
- Good for sequencing single exons of genes

Slow, low-throughput and costly to perform for large numbers of samples

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13
Q

FISH - to detect

A

To detect large chromosomal abnormalities

Extra chromosomes

Large deleted segments

Translocations

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14
Q

FISH - define

A

Fluorescent in situ hybridisation

Uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity

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15
Q

FISH - describe method

A

Design Fluorescent probe to chromosomal region of interest

Denature probe and target DNA

Mix probe and target DNA (hybridisation)

Probe binds to target

Target fluoresces or lights up !

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16
Q

Array CGH - define

A

Array comparative genomic hybridisation

ultra-high resolution way of objectively and quantitatively detecting. whether a patient’s DNA has losses (deletions) or gains (duplications, triplications. etc) which are pathogenic

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17
Q

Array CGH - use

A

For detection of sub-microscopic chromosomal abnormalities

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18
Q

Array CGH - results

A

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

19
Q

MLPA - define

A

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

20
Q

MLPA - action

A

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

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

21
Q

MLPA - use

A

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

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

22
Q

MLPA - describe action of the product

A

Perform fragment analysis (capillary electrophoresis) of MLPA product

23
Q

MLPA - describe involvement in ploidy

A

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

24
Q

Explain the Current strategy: Disease panels

A

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

25
Q

Explain reason for using 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

26
Q

Exome Sequencing - briefly describe action

A

Target enrichment

Capture target regions of interest with baits - streptavidin coated

27
Q

Exome Sequencing - capacity for results

A

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

28
Q

Describe which tests will not automatically move to whole genome sequencing

A

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

29
Q

Explain the challenge of result interpretation in sequencing

A

Result interpretation is the greatest challenge
20,000 variants per coding genes ‘exome’
3 million variants in a whole human genome

30
Q

Explain the ethical considerations in sequencing

A

Ethical considerations:

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

31
Q

Describe a challenge of genome/exome sequencing

A

Infrastructure and training (particularly IT and clinical scientists)

32
Q

The NHS Diagnostic Laboratory - main role

A

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

33
Q

The NHS Diagnostic Laboratory - perform specific tests with proven what

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

34
Q

The NHS Diagnostic Laboratory - list 5 roles

A

Diagnostic (diagnosis, treatment, pathogenicity)
Predictive (life choices)
Carrier (recessive)
Informed consent (counselling/implications)
Diagnostic testing is available for all Consultant referrals

35
Q

Diagnostic Testing - list regulations

A

All referrals via Regional Genetics Centres

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

Strict international guidelines for predictive testing

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

36
Q

Diagnostic Test Outcomes - list

A

Pathogenic mutation

Normal variation
Polymorphism

Novel variant
Investigations to establish significance

37
Q

How to establish if a mutation is pathogenic?

A

Mode of inheritance

Locus-specific databases of published and unpublished data

Nonsense, frameshift, splice site (exon+/-2 bp) mutations

Missense/intronic mutation
- In-silico tools for missense and splicing mutations

38
Q

Interpreting Results - list regu;ations

A

Do not report known polymorphisms

Conservative approach to reporting novel mutations of uncertain pathogenicity

  • ‘Uncertain significance’
  • ‘Likely to be pathogenic’

Request samples from family members

Continue testing other genes

39
Q

100,000 genomes project - define

A

UK Government project that is sequencing whole genomes from National Health Service patients

40
Q

100,000 genomes project - +ves

A

100,000 genomes project:

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
Personalised medicine

41
Q

100,000 genomes project - Who/what is being sequenced?

A

Rare diseases – index cases + families

Cancer – germline and tumour samples

42
Q

Explain how the use of the Genomics England Panel App allows us to focus on specific genes in pt’s genome we think are important

A

Genomics England Panel App

‘Experts’ develop lists of possible genes than can cause a specific disease

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 genes of the patients genome we think are important

43
Q

Compare Tier 1-3 variants

A

Tier 1 variants
Known pathogenic
Protein truncating

Tier 2 variants 
Protein altering (missense)
Intronic (splice site)

Tier 3 variants
Loss-of-function variants in genes not on the disease gene panel

44
Q

Describe the formation of the Tier 1-3 variant classification system

A

Classification of mutations by genomics England

Variants within virtual panel divided into three tiers

Expert review is required

Fundamentals of P (62 decks)

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