Overview of Genomic Technologies in Clinical Diagnostics

Question 1

What is PCR used for?

Answer 1

• used to amplify a specific region of DNA such that there are enough DNA molecules so that we have sufficient material for downstream applications
• each cycle doubes the amount of dna copies of your target sequence

Question 2

How do you identify the area to be amplified in PCR?

Answer 2

• complementary primers flank the region you want to amplify

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Question 3

Explain the process of PCR

Answer 3

3 step process

1. denaturation - template dna is denatured - high temp. allows separation of dna strands
2. annealing - cooler temp. allows primers to attach to ends of target sequence
3. extension - temp allowing thermostable polymerase to add nucleotides at the 3’ end of primers

Question 4

How can PCR product be analysed using fragment analysis?

Answer 4

The PCR product undergoes capillary electrophoresis so that we can identify its size in bp.
It can be used to detect repeat expansions or other small size changed (up to a few hundred bp)

Question 5

What is a repeat expansion disease? Give an example

Answer 5

• when there is a repetitive base pair repeat but it becomes pathogenic.
• e.g. Huntington’s diseases - CAG repeat expansion - more than 35 copies is pathogenic as expanded protein is toxic and accumulates in neurons causing cell death.
• diagnosed with fragment analysis

Question 6

How does sanger sequencing work?

Answer 6

• cycle sequencing; based on same principles as PCR
• you get the PCR product and then sequence it
• each of the 4 dna nucleotides has a different dye so we can determine the nucleotide sequence.
• can use this for exons of genes

Question 7

What are the disadvantages of sanger sequencing?

Answer 7

• slow
• low throughput
• costly to perform for large numbers of samples
• can only do on sections up to 800bp per reaction

Question 8

What is sanger sequencing used for?

Answer 8

to identify SNPs or mutations

Question 9

What is FISH? And what does it involve?

Answer 9

Fluorescent in situ hybridisation - used to see microscopic (can be seen with a microscope) chromosomal abnormalities - quite large (5-10Mb)
Involves using cultured cells from pts that are spread during metaphase to tease out chromosomes and probe them with FISH

Question 10

What can be detected using FISH?

Answer 10

• large chromosomal abnormalities
• • extra chromosomes
• • large deleted segments
• • translocations

Question 11

Explain the process of FISH

Answer 11

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!

Question 12

How can FISH be used?

Answer 12

• in spectral karyotyping - to identify number and structure of chromosomes
• for target specific FISH - to identify number and location of chromosomes

Question 13

What is array CGH and what does it involve?

Answer 13

array comparative genomic hybridisation

for detection of sub-microscopic chromosomal abnormalities (too small to see with FISH)

Question 14

Explain the process of array CGH

Answer 14

• patient DNA labelled green
• control DNA labelled red
  1- pt and control dna are labelled with fluorescent dyes and applied to the microarray
  2- pt and control dna compete to hybridise to the microarray
  3- the microarray scanner measures the fluorescent signals
  4- computer software analyses the data and generates a plot.

Question 15

What do the results of an array CGH show?

Answer 15

if a pt sample has a dosage loss or gain (chromosomal abnormality) thru excess of red or green dye

• equal hybridisation - pt and control dna sample is the same - no net loss or gain
• dna dosage loss - increased red signal in pt dna
• dna dosage gain - increased green signal in pt dna

Question 16

What is MLPA and what does it involve?

Answer 16

• multiplex ligation-dependent probe amplification is a variation of pcr that permits amplification of multiple targets
• it involves probes which consist of oligonucleotides that recognise adjacent target site on dna

Question 17

What is MLPA used for?

Answer 17

Detecting abnormal copy numbers at specific chromosomal locations (rather than genome-wide like array CGH)
It can detect sub-microscopic (small) gene deletions/partial gene deletions or single exon deletions

Question 18

Explain the process of MLPA

Answer 18

• you need two oligonucleotide probes to target region - one for each primer - forward/reverse
• Only when both probe oligonucleotides are hybridized to their respective targets, can they be ligated into a complete probe
  3 step process
  1. hybridisaition - hybridising both two probes to denatured template dna
  2. ligation - ligate and join together the forward and reverse probes
  3. amplification - pcr amplification of probe to form amplified library or product of our target

Question 19

How can MLPA product be analysed using fragment analysis?

Answer 19

• The MLPA product undergoes capillary electrophoresis to determine the dosage and size of it.
• So we are using MLPA to determine relative ploidy (how many chromosome copies) at specific locations.
• The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome.

Question 20

What is next generation sequencing and what does it involve?

Answer 20

• it is a more modern version of sequencing dna
• wider range of tests in a shorter time for less money
• its current strategy involves: 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

Question 21

What is the purpose behind exome sequencing?

Answer 21

• often, we are only interested in the exome which is 1-2% of the genome and since about 80% of the pathogenic mutations are protein coding, it is more efficient to only sequence the exome and not the introme because we aren’t interested in this part and would be more costly.

Question 22

Explain how exome sequencing is performed

Answer 22

• using target enrichment
• this involves capturing target regions of interest with baits
• has the potential to capture several Mb genomic regions (usually 30-60 Mb)
  1. create dna library from pt sample
  2. incubate dna library with biotinylated rna ‘baits’
  3. perform hybridisation so exons are baited
  4. use a purification column based around streptavidin - coated magnetic beads (attract biotin). This captures exon fragments.
  5. wash away any unbound fragments so ur left with an enriched library that you can sequence

Question 23

Will all genomic tests be moved to whole genome sequencing?

Answer 23

No! because
- 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 - can still use fragment analysis
• Array-CGH: large sized chromosomal aberrations - no need for whole genome

Question 24

What are the disadvantages of whole exome and whole genome sequencing?

Answer 24

• result interpretation is a chellenge as there are so many genetic variants
• ethical considerations
• • pt consent needed
• • inspect necessary genes first only
• • strategy for reporting ‘incidental’ findings
• infrastructure and training (esp IT and clinical scientists to interpret data)

Question 25

What is ‘The 100,00 Genomes Project’s’ purpose?

Answer 25

• a project to bring direct benefit of whole genome sequencing and genetic to patients
• and enable new scientific discovery and medical insights
• an example of personalised medicine

Question 26

How does ‘The 100,00 Genomes Project’ work?

Answer 26

• samples are collected in GMCs (genomic medicine centres) across England
• it then sequences rare diseases - people who are affacted and family members
• and cancer - germline dna from blood and tumour samples (to see what kind of mutation it has to help treatment research)

Question 27

How does ‘The 100,00 Genomes Project’ analyse data?

Answer 27

• they classify mutations
• so for each disease they have virtual gene panels whereby they have a list of genes that are known to cause that disease or phenotype and then when they identify mutations in those genes, they can interpret them in terms of pathogenicity. So when variants are picked up in these genes on these disease-specific gene panels, they group them into a tier.

Question 28

Explain ‘The 100,00 Genomes Project’ tiering system

Answer 28

Tier 1 - known pathogenic and protein truncating
Tier 2 - protein altering (missense) and intronic (splice site)
Tier 3 - loss-of-function variants in genes not on the disease gene panel

Question 29

What is the significance of the NHS diagnostic lab?

Answer 29

• where most genetic testing takes place
• accredited lab with ISO standard
• provide clinical and lab diagnosis for genetic disorders
• liaise with clinicians, nurses and other health professionals
• provide genetic advice for sample referrals and results
• tests have proven clinical validity (how well the test predicts the phenotype) and utility (how the test adds to the management of the pt)

Question 30

What is the main role of the lab?

Answer 30

to help consultants reach a genetic diagnosis for individuals and families to help guide treatment and clinical management

Question 31

What testing does the diagnostic lab do?

Answer 31

1. diagnostic
   - diagnosis
   - management + treatment
   - interpretation of pathogenicity (determining if its pathogenic)
2. predictive
   - life choices, management
3. carrier (recessive)
   - life choices, management

Question 32

Explain the possible outcomes of a diagnostic test

Answer 32

1. pathogenic mutation
2. normal variation (like polymorphisms - no impact on clinical phenotype)
3. novel variant (clinical significance is unclear and more investigations are needed)

Question 33

How do you establish if a mutation is pathogenic?

Answer 33

• mode of inheritance
• genetic databases of published and unpublished data
• Nonsense, frameshift, splice site (exon+/-2 bp) mutations (model effect of mutations on gene + see result)
• Missense/intronic mutation
• • In-silico tools for missense and splicing mutations (predict + model if changes are damaging to protein)

Question 34

What do we do after interpreting results from a diagnostic test?

Answer 34

• do not report known polymorphisms
• request dna samples from family members
• continue testing other genes
• report novel mutations of uncertain pathogenicity in a conservative manner