Clinical Genetics: Overview of genomic technologies in clinical diagnostics Flashcards

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1
Q

Give a brief overview of what PCR (polymerase chain reaction) is and how it works

A
  • PCR is used to amplify a specific region of DNA, e.g. a gene or gene exon associated with a disease
  • 3 stages: Denaturation, annealing and extension
  • 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, e.g. sanger sequencing
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2
Q

What is fragment analysis?

A
  • PCR followed by capillary electrophoresis which sizes the PCR product accurately
  • Instead of being represented by bands as with gel electrophoresis the PCR products are represented by peaks
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3
Q

What can fragment analysis be used for?

A
  • Can be used to detect repeat expansions or other small size changes in allele size (up to a few hundred bp)
  • This is important because repeat expansions can cause diseases such as Huntingdon’s disease
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4
Q

What is Huntingdon’s disease?

A
  • It is a severe neurodegenerative disorder caused by CAG repeat expansion in the Huntingtin (HTT) gene
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5
Q

How does the CAG expansion in the Huntingtin gene causes Huntingdon’s disease?

A
  • Normal HTT gene has < 27 CAG copies; Intermediate 27-35 copies; Pathogenic > 35 copies
  • Expanded protein produced from HTT gene with CAG repeat expansion is toxic and accumulates in neurons causing cell death
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6
Q

How is Huntingdon’s disease diagnosed?

A
  • Diagnosed with fragment analysis
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7
Q

Give a brief overview of sanger sequencing

A
  • Cycle Sequencing technique; based on the same principles as PCR
  • Used to re-construct a nucleotide sequence
  • Each of the 4 dideoxyribose nucleoside triphosphates (ddNTPs) in reaction mixture has a different dye so we can determine the nucleotide sequence.
  • Can sequence up to 800bp of sequence per reaction so good for sequencing single exons of genes
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8
Q

What are some of the disadvantages of sanger sequencing?

A
  • Slow
  • Low-throughput
  • Costly to perform for large numbers of samples
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9
Q

What can sanger sequencing be used for?

A
  • Can be used to identify single nucleotide polymorphisms (SNPs), or mutations
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10
Q

Give a brief overview of Fluoresence in situ hybridisation (FISH)

A
  • Used to microscopically detect large chromosomal abnormalities such as:
    • Extra chromosomes
    • Large deleted segments
    • Translocations
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11
Q

How does fluoresence in situ hybridisation work?

A
  • Design Fluorescent probe to a chromosomal region of interest
  • Denature probe and target DNA
  • Mix probe and target DNA together (hybridisation)
  • Probe binds to the target DNA on the chromosome of interest
  • Target fluoresces or lights up
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12
Q

Give a brief overview of Array CGH (comparative genomic hybridisation)

A
  • Used for detection of sub-microscopic chromosomal abnormalities
  • Patient DNA labelled Green
  • Control DNA labelled Red
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13
Q

How does Array CGH work?

A
  • Patient DNA and control DNA are extracted from samples
  • Patient DNA labelled with Cy3, green and control DNA labelled with Cy5, red
  • They are then mixed together and hybridised to the microarray
  • Patient and control DNA compete to hybridise to the microarray
  • Each spot on the array is then scanned to identify the colour of fluoresence it produces
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14
Q

What does each colour of fluoresence on the microarray for array CGH represent?

A
  • No colour fluoresence = Equal hybridisation of patient and control DNA
  • Red = More control DNA hybridised than patient DNA so there’s a loss of DNA in patient in that position in genome
  • Green = More control DNA hybridised than control DNA so there’s a gain of DNA in patient in that position in genome
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15
Q

What is MLPA?

A
  • Multiplex ligation-dependent probe amplification (MLPA)
  • It’s a variation of PCR that permits amplification of multiple targets
  • MLPA is used to detect abnormal copy numbers at specific chromosomal locations
    • Can also detect sub-microscopic (small) gene deletions/partial gene deletions
  • Usually used to see if there’s any abnormalities in a gene known to be involved in a specific condition
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16
Q

How does MLPA work?

A
  • Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA
  • One oligonucleotide probe contains the sequence recognized by the forward primer,
  • The other oligonucleotide probe 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
  • DNA that’s bound to complete probe is then amplified
  • Perform fragment analysis (capillary electrophoresis) on the MLPA product
17
Q

Why is MLPA an example of a quantitative assay?

A
  • Because the amount of MLPA product produced is proportional to the amount of starting DNA
18
Q

How is the data from MLPA analysed?

A
  • MLPA product/s produce peaks which represent the no. of copies of a chromosome at that particular area of the genome
  • The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome
19
Q

Why has next generation sequencing replaced sanger sequencing for almost all sequencing tests?

A
  • Higher DNA sequencing throughput
  • Provides a wider range of tests in a shorter time for less money
20
Q

Why is whole exome sequencing used more often tha whole genome sequencing?

A
  • 80% pathogenic mutations are protein coding
  • Therefore it’s more efficient and cheaper to only sequence the exome if we want to locate a pathogenic mutation within the genome
21
Q

How does whole exome sequencing work?

A
  • Use a process called Target enrichment
  • Take DNA frgaments and hybridise them with RNA baits that are complementary to the exons of the DNA fragments
  • Once hybridisation occurs the bait-bound DNA fragments will be “captured” using streptavidin coated beads
  • These beads are magnetic so using a magnet will separate the fragments containing exon sequences from the fragments that don’t contain them
  • Beads are then washed away and the RNA baits are digested leaving only the DNA fragments containing exons
  • Then perform next generation sequencing as normal
22
Q

It’s Universally accepted that genome sequencing will become commonplace in diagnostic genetics but not all tests will move to WGS, what tests won’t be replaced by WGS?

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
23
Q

What is the main disadvantage of Whole genome sequencing currently?

A
  • Data generation and data procesing is an automated process in whole genome sequencing
  • However, whole genome sequencing still requires manual interpretation which is ver time consuming and challenging
24
Q

What are some other disadvantages of whole genome sequencing?

A
  • Ethical considerations
    • Modified patient consent process needed
    • May also find defetcs/mutations that put future family at risk of a specific disease so strategy for reporting ‘incidental’ findings needs to be produced
  • Infrastructure and training (particularly IT and clinical scientists) needs to be put in place
25
Q

What is the NHS Diagnostic Laboratory?

A
  • Accredited laboratory that:
    • Provide clinical and laboratory diagnosis for genetic disorders
    • Liaise with clinicians, nurses and other health professionals
    • Provide genetic advice for sample referrals and results
26
Q

What is the difference between clinical validity and clinical utility?

A
  • Clinical Validity: How well the test predicts the phenotype
  • Clinical Utility: How the test adds to the management of the patient
27
Q

What is the UKGTN?

A
  • UK genetic testing network
  • Body that approves all the genetic tests used in the UK
28
Q

What are the 3 outcomes of a genetic test?

A
  • Pathogenic mutation
  • Normal variation (Polymorphism)
  • Novel variant - Investigations needed to establish significance
29
Q

How can you establish if a mutation is pathogenic?

A
  • Look at mode of inheritance and segregation of that mutation by studying entire families
  • Look at Locus-specific databases of published and unpublished data
  • Functional predictions of effect of mutation on gene function - Nonsense, frameshift, splice site (exon+/-2 bp) mutations
  • Missense/intronic mutation harder to prove if pathogenic as lots are tolerated within genome
    • In-silico tools used for missense and splicing mutations
30
Q

Use the following information from a case study of MFN2 to explain how the sibilings inherited this particular disease

Mitofusin 2 (MFN2) causes Charcot-Marie-Tooth disease type 2 (CMT2)

In one family there are two siblings with very severe early-onset CMT2 but parents unaffected

MFN2 sequenced by next generation sequencing: Both siblings homozygous for c.647T>C p.(Phe216Ser)

Both parents should be heterozygous c.647T>C p. but father is homozygous

A
  • MLPA was used to measure dosage of all MFN2 exons in the family
  • Found that children and father carry deletion of MFN2 exons 7-8
  • This means children aren’t homozygous for mutation instead they had one allele with the mutation and the other allele contained a deletion
31
Q

How are the different variants found by Genomics England classified?

A