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[MBD] NoNa > Overview of Genomic technologies in Clinical diagnosis > Flashcards

Overview of Genomic technologies in Clinical diagnosis Flashcards

1
Q

What are some different 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

Describe PCR

A
  • PCR is used to amplify a specific region of DNA
  • Design specific primers which are oligonucleotide sequences =complimentary to amplicon
  • Primers flank the region you want to amplify.
  • Each cycle doubles the amount of DNA copies of your target sequence
  • Purpose: Amplify enough DNA molecules = have sufficient material for downstream applications
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3
Q

What are the three steps of PCR -

A

Denaturation -High temperature permits separation of DNA strands

Annealing- Cooler temperature allows primer to attach to ends of target sequence

Extension-Temperature allowing thermostable DNA polymerase to add nucleotides at the 3’ end of primers

1 PCR cycle doubles amount of DNA

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4
Q

What is Capillary electrophoresis (fragment analysis)

A

PCR based assay
Following PCR we carry out capillary electrophoresis
(This is a technique which separates DNA molecules based on electrophoretic mobility )
-We size the PCR product
-This can be used to detect small size changes (up to a few hundred bp)/repeat expansions

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5
Q

What is a repeat expansion disorder ?

A

Genetic diseases that are caused by expansions in DNA repeats.

Example- Huntington’s disease (severe neurodegenerative disorder )

Cause -CAG repeat expansion in the HTT gene

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

Expanded protein is toxic and accumulates in neurons causing cell death

Can be diagnosed using capillary electrophoresis (fragment analysis)

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6
Q

Describe Sanger sequencing

A
  • Sequence the PCR product, modified PCR - dideoxy chain termination
  • 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 genes
  • Slow, low-throughput and costly to perform for large numbers of samples
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7
Q

What can sanger sequencing be used for?

A

We can identify single nucleotide polymorphisms (SNPs), mutations, monogenic diseases

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8
Q

Describe FISH (Fluorescent in situ hybridisation)

A

It is used to detect (Microscopic) large chromosomal abnormalities

Cells from patients spread during metaphase + use FISH

Detects:
•Extra chromosomes
•Large deleted segments
•Translocations(chunks of chromosomes moving from one place to another)

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9
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 on DNA
  5. Target fluoresces/lights up so we can see our target
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10
Q

What type of observations can we make using FISH

Outline two ways FISH can be used

A

Special Karyotyping

-pairs of chromosomes are manipulated to have distinctive colours.
makes it easier to detect chromosomal abnormalities
(Trisomy 21)- 3 copies of 21 -Down syndrome

Target specific

  • Probe for specific chromosomes
  • 2 copies of chromosome 18, 3 copies of chromosome 21
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11
Q

What is Array CGH (comparative genomic hybridisation )

A

Used to detect sub-microscopic chromosomal abnormalities (too small to see with FISH)

Uses microarray:
Patients DNA labelled Green
Control DNA labelled Red

Mix together red + green samples + hybridise to microarray + detect green/red signal

Can see patient sample whether it has net DNA loss/gain
Increased green signal = gain
no net red/green = equal hybridisation = patient and control DNA sample is the same = no net loss/gain

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12
Q

Describe MLPA

A

Multiplex ligation-dependent probe amplification

  • Variation of PCR that amplifies multiple targets
  • Probe= two oligonucleotides -recognise adjacent target sits on DNA
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13
Q

What can MLPA be used to detect?

A

Use:
-To detect abnormal copy numbers at specific chromosomal locations

-Can detect sub-microscopic gene deletions/partial gene deletions (not genome-wide like array-CGH)

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14
Q

How does MLPA work?

A

Three step process

  1. Hybridisation
    - Two probes are hybridised to denatured template DNA (1 forward primer+1 reverse primer)
  2. Ligation
    - The 2 forward and reverse probe oligonucleotides join together into a complete probe

3.Amplification
PCR amplification of the target to generate an amplified library

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15
Q

How can we analyse the amplified product library following MLPA?

A

Using capillary electrophoresis
(fragment analysis)

-Used to determine ploidy(how many chromosome copies of amplified target)-specific locations.

-Signal strength of the probes is compared to those obtained from reference DNA sample
(contains two copies of the chromosome )

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16
Q

Describe Next Gen sequencing

A

Disease panels = Where we only sequence known disease genes relevant to the phenotype

17
Q

Describe Exome Sequencing

A

One of the common techniques of Next Gen sequencing

Only interested in the protein coding exons
(1-2% of the genome )

  • Pathogenic mutations -80% are protein coding
  • More efficient to sequence only these (not whole genome) + cheaper
18
Q

How do we actually perform whole exome sequencing?

A

-Target enrichment
= captures target regions of interest with “baits”
= potentially captures several Mb of genomic regions

-We incubate our RNA bases

19
Q

What is target enrichment ?

A

cost-effective and efficient method for researchers to capture specific regions of interest after library preparation for NGS

20
Q

What takes place once we create our DNA library

Whole exome sequencing ?

A
  1. Incubate our DNA library with RNA “baits “
  2. perform hybridisation and the exons are “baited”
  3. Purification column using magnetic beads (Streptavidin) which recognise biotin on RNA baits
  4. Capture the exons
  5. Wash away any unbound fragments,introns
  6. = enriched library (can then be sequenced)
21
Q

What has replaced SANGER sequencing?

A

Next generation sequencing

22
Q

Why will not all testing be suitable for Whole genome sequencing ?

A
  1. Panels/single gene tests may still be more suitable for some diseases, e.g. cystic fibrosis
  2. Capillary-based methods: Repeat expansions, MLPA, family mutation confirmation Sanger sequencing
  3. Array-CGH: large sized chromosomal aberrations
23
Q

What are some challenges with Exome and Genome Sequencing ?

A

Result interpretation :

  • 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)

24
Q

Describe the 100,000 genomes project

A
  • Whole genome sequencing
  • Direct benefit of whole genome sequencing and genetics to patients
  • New scientific discoveries and medical insights
  • Personalised medicine
25
Q

How is the 100,000s genome project carried out

A
  • England wide collection of samples
  • Rare disease -Index cases +families -inheritance patterns
  • Cancer treatments (Germline (blood) and tumour samples )
26
Q

Describe the clinical interpretation of the genome project

A 
Classifications of the mutations :
They are 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
27
Q

What is clinical validity ?

A

How well the genetic test predicts the phenotype

28
Q

What is clinical utility?

A

How the genetic test adds to the management of the patient

29
Q

What type of tests do the NHS Diagnostic laboratory do ?

A
  • Diagnostic + research
  • Predictive
  • Carrier (recessive)
  • Diagnostic testing is available when referred by Consultant
  • Informed consent process - patient + their family
30
Q

What are the diagnostic test outcomes ?

A
  • Pathogenic mutation
  • Normal variation( polymorphisms ) = diagnosis not reached
  • Novel variant (Investigations to establish clinical significance, whether variant impacts the disease)
31
Q

How do we establish if a mutation is pathogenic ?

A

-Mode of inheritance
-Genetic databases of published and unpublished
-Nonsense , frameshifts, splice site - functional predictions of mutation + model
-Missense/intronic mutation
(In-Silico tools for missense and splicing mutations to predict if protein changes are damaging)

32
Q

How do we interpret results ?

A
  • We do not report known polymorphisms

- Request further DNA samples from family - mutation segregation within the family

33
Q

What is MFN2?

A

Mitofusin 2 mutation causes Charcot-Marie-Tooth disease (CMT2)

  • Degeneration of the long nerves in the legs and arms = muscle wasting + sensory defects
  • Onset-childhood
  • Autosomal dominant +Autosomal recessive

[MBD] NoNa (28 decks)

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