Overview of Genomic Technologies in Clinical Diagnostics Flashcards

1
Q

List some genomic technologies used today

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

How does PCR work

A
  • Fundamental for many DNA applications 3 step process
  • 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 for downstream
    applications
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3
Q

How can we use PCR for fragment analysis?

A
  • PCR 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)
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4
Q

Explain how we can diagnose Repeat expansion diseases

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
  • Expanded protein is toxic and accumulates
    in neurons causing cell death
  • Diagnosed with fragment analysis
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5
Q

How does Sanger sequencing work

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.
  • Up to 800bp of sequence per reaction; Good for sequencing single exons of the gene
  • Slow, low-throughput and costly to perform for
    large numbers of samples
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6
Q

How can we use Sanger sequencing to detect mutations

A
  • We can identify single nucleotide
    polymorphisms (SNPs), or
    mutations
  • Detection of a mutation in a family by use of Sanger
    Sequencing
  • R1042G mutation in gene C3
    segregates with affected
    individuals
  • Mutation causes disease cutaneous vasculitis
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7
Q

What is Fluorescent in situ hybridisation

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

How does FISH work

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

How does Array CGH work

A
  • Similar to microarray technology
  • Array comparative genomic
    hybridisation
  • For detection of sub-microscopic
    chromosomal abnormalities
  • Patient DNA labelled Green
  • Control DNA labelled Red
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10
Q

How do we analyse Array CGH

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 parent
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11
Q

What is MPLA

A
  • Multiplex ligation-dependent
    probe amplification (MLPA) is a variation of PCR that permits the amplification of multiple targets
  • Each probe consists of two
    oligonucleotides which
    recognize adjacent target sites on the DNA
  • We use MLPA to detect
    abnormal copy numbers at specific chromosomal locations
  • MLPA can detect sub-microscopic (small) gene deletions/partial
    gene deletions
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12
Q

How can we use MLPA to analyse for mutations?

A
  • Perform fragment analysis (capillary electrophoresis) 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 the 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
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13
Q

How does MPLA work

A
  • One probe oligonucleotide contains the sequence recognized by the forward primer, and 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 prob
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14
Q

What is Next Generation Sequencing

A

Wider range of tests in a shorter time for less money

Current strategy: Disease panels:

  • Enriching to sequence of 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
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15
Q

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
  • 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
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16
Q

How do we do Exome sequencing?

A
  • Target enrichment
  • Capture target regions of interest with baits
  • Potential to capture several Mb genomic regions (typically 30-60 Mb)
17
Q

should we do whole genome sequencing

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
18
Q

What are the challenges in exome and genome sequencing?

A

Result interpretation is the greatest challenge:

  • 20,000 genetic variants identified per coding genes ‘exome’
  • 3 million variants in a whole human genome
    Ethical considerations:
  • Modified patient consent process
  • Data analysis pathways – inspect relevant genes first
  • Strategy for reporting ‘incidental’ findings

Infrastructure and training (particularly IT and clinical scientists)

19
Q

How is data from whole genome sequencing analysed

A

Interpretation of clinical genomes currently has a substantial manual component Whole genome sequencing is NOT trivial

20
Q

What is the 100,000 genomes project?

A
  • 100,000 genomes project
  • Bring direct benefit of whole genome
    sequencing and genetics to patients
  • Enable new scientific discovery and medical
    insights
  • Personalised medicine
21
Q

What are the classification of mutations made by genomic England

A

Variants within virtual panel divided into three tiers:

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
22
Q

What is the NHS Diagnostic Laboratory

A
  • Accredited laboratory: ISO standard 15189 for Medical Laboratories
  • Scientific, technical and administrative staff
  • Provide clinical and laboratory diagnosis for genetic disorders
  • Liaise with clinicians, nurses and other health professionals
  • Provide genetic advice for sample referrals and results
23
Q

What is Clinical Validity

A

How well the test predicts the phenotype

24
Q

what is Clinical Utility

A

How the test adds to the management of the patient

25
Q

What genetic testings do they carry out

A

Diagnostic:

  • Diagnosis
  • Management and Treatment
  • Interpretation of pathogenicity

Diagnostic testing is available for all Consultant referrals:

  • Clinical Geneticists most common referrers

Predictive:

  • Life choices, management

Informed consent:

  • Implications for other family members

Carrier (recessive):

  • Life choices, management
26
Q

What are the diagnostic test outcomes

A

Pathogenic mutation
Normal variation:

  • Polymorphism
    Novel variant:
  • Investigations to establish clinical significance…
27
Q

How do we establish a mutation as pathogenic

A

Mode of inheritance

Genetic 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
28
Q

How do we interpret results from this genetic testing

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?