Overview of Genomic Technologies in Clinical Diagnostics

Question 1

Examples of genomic technologies used

Answer 1

• PCR
• Fragment analysis
• Sanger sequencing
• FISH
• Array comparative genomic hybridisation - Array CGH
• Multiplex ligation dependent probe amplification - MLPA
• NGS

Question 2

What is PCR?

Answer 2

PCR is a techqniue used for many DNA applications. It amplifies a specific region of DNA by flanking primers to that region. During each cycle of PCR, the amount of DNA copies of the target sequence is doubled. The purpose of this is to amplify enough DNA molecules so that there is enough material for downstream applications.

Question 3

What are the uses of the fragments produced from PCR?

Answer 3

• PCR based assays
• PCR followed by capillary electrophoresis
• These tecniques allow for the sizing of the PCR product so it can be used to detect repeat expansions or other small changes.

Question 4

What is huntington’s disease?

Answer 4

Huntington’s disease is a repeat expansion disease that is a severe neurodegenerative disorder. It is caused by CAG repeat expansion in the Huntington (HTT) gene. The expanded protein is toxic and accumulation of the toxic in the neurons causes cell death. This is diagnosied with fragment analysis.

Question 5

What levels of the Huntington’s proteins are normal, intermediate or pathogenic?

Answer 5

• Normal levels are less than 27 copies of repeats
• Intermediate is 27-35 copies
• Pathogenic is over 35 copies

Question 6

What is sanger sequencing?

Answer 6

It is a cycle sequencing based on the same principles of PCR. Each of the 4 DNA nucleotides have a different dye and you are able to determine the nucleotide sequence. This is used to identify SNPs or mutations. This process is very accurate however, it is very time consuming and expensive.

Question 7

Give an example where sanger sequencing is used

Answer 7

• Carried out on a family and the R1042G mutation was found in the gene C3. The mutation is linked to cutaneous vasculitis.

Question 8

What is Fluorescence in situ hybridisation (FISH)?

Answer 8

This technique is used to detect large chromosomal abnormalities, extra chromosomes, large deleted segments and translocations. It is good to see trisomy 21 or down’s syndrome.

Question 9

What is the process of FISH?

Answer 9

1. Design fluorescent probe to chromosomal region of interest.
2. Denature the probe and target DNA, this is important for the new hydrogen bonds to form between the target chromosome region and the probe.
3. Mix the probe and target DNA so they hybridise together.
4. Once the probe binds to the target region, it will fluoresce and you will be able to see the target area.

Question 10

What is array CGH?

Answer 10

It is array comparative genomic hybridisation. This is used for the detection of sub-microscopic chromosomal abnormalities. For example, the patient DNA is labelled green and the control DNA is labelled red. This allows for the patient array comparative genomic hybridisation profile to be made.
This is because there is an increased green signal in the patient which shows that there is an additional chromosomal segment in the patient DNA which is not present in the parents.

Question 11

What is multiplex ligation dependent probe amplification (MLPA)?

Answer 11

This is a variation of PCR that permits the amplification of multiple targets. Each probe consists of 2 oligonucleotides which recognise adjacent target sites on the DNA.

Question 12

What are the two probes used for?

Answer 12

1. One probe oligonucleotide contains the sequence recognised by the forward primer.
2. The other contains the sequence recognise by the reverse primer.
   Only when both primers are hybridised to their respective targets can they be ligated into a complete probe.

Question 13

What is the MLPA used for?

Answer 13

• Used to detect abnormal copy numbers at specific chromosomal locations.
• Also detect sub-microscopic (small) gene deletions or partial gene deletions.
• Quick way of looking at chromosomal abnormalities because it looks at a section of the genome compared to other methods which look at the whole genome.

Question 14

What happens to the product produced by MLPA?

Answer 14

First a fragment analysis (capillary electrophoresis) can be performed.
This is then used to determine the relative ploidy which is the number of chromosomes.
This is why MLPA is a quantitative assay. The peaks on the diagram tell the amount of copies present.
The probes can be designed to target various regions of the chromosome. The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have 2 copies of the chromosome.

Question 15

What has NGS replaced?

Answer 15

Sanger sequencing

Question 16

Examples of machines used in NGS

Answer 16

• Solexa sequencing by synthesis and illumine SBS sequencing.

Question 17

Why is NGS so advanced?

Answer 17

• It has allowed the ability to sequence a wider range of tests in a shorter period of time for less money.
• Current strategy of NGS is to only sequence known disease genes relevant to the phenotype of the patient.

Question 18

When are gene panels expanded?

Answer 18

They are expanded when new disease linked genes are found.

Question 19

What technique is used to confirm potential pathogenic variants?

Answer 19

Sanger sequencing

Question 20

Exome sequencing

Answer 20

• Focuses on the coding areas of the genome
• 80% of the pathogenic mutations occur in the protein coding areas
• Target enrichment, capture the target regions of interest with baits

Question 21

Whole genome sequencing

Answer 21

• It is now widely accepted that genome sequencing will become the commonplace for genetic testing. However, not all tests will automatically move to WGS.

Question 22

When are panel or single gene tests more suitable than WGS?

Answer 22

More suitable depending on the situation/disease for example cystic fibrosis

Question 23

Examples of capillary based methods

Answer 23

Repeat expansion, MLPA and family mutation confirmation sanger sequencing may be more suitable than WGS at times

Question 24

What is Array CGH more suitable for?

Answer 24

More suitable for large sized chromosomal aberrations.

Question 25

What is the hardest thing about using WGS?

Answer 25

• the interpretation of the information.
• there are ethical considerations that need to be taken into account including patient consent, data analysis pathways and strategy for reporting incidental findings.

Question 26

Where are NHS diagnostic labs located and what do they do?

Answer 26

• Present in most large hospitals in the UK
• Provide clinical and lab diagnostics for genetic disorders
• Provide genetic advice for sample referrals and results
• In labs, be able to perform specific tests with proven
• Labs also carry out other functions that aren’t diagnostics
• Diagnostic test outcomes
• How to establish if a mutation is pathogenic
• Interpreting results

Question 27

What is clinical validity?

Answer 27

This is how well the test predicts the phenotype of the patient

Question 28

What is clinical utility?

Answer 28

This is how well the test adds to the management of the patient

Question 29

Who is able to use the clinical and lab diagnostics?

Answer 29

It is for clinicians, nurses and other health care professionals

Question 30

What is the main role of the lab?

Answer 30

The main role of the lab is to help consultants reach a genetic diagnosis for individuals and families to help guide treatment and clinical managment.

Question 31

What are the other functions that the NHS diagnostic lab can carry out besides diagnosis?

Answer 31

• Predictive tests: if a patient walks in with a genetic disease, they can test their child to see if they have the genes for that disease as well.
• Carrier: a patient can be screened to see if they are carrier for a genetic disease such as cystic fibrosis or sickle cells disease
• Informed consent: they are able to provide genetic counselling.

Question 32

What are the outcomes of diagnostic tests?

Answer 32

• Pathogenic mutation
• Normal mutation such as polymorphism
• Novel variant - a variant is not known so further investigations need to be taken to establish the significance.

Question 33

How to establish if a mutation is pathogenic?

Answer 33

1. Mode of inheritance
2. Locus-specific databases of published and unpublished data: whether its common in the population or not.
3. Nonsense, frameshift, splice site mutations: functional predictions, what the mutations will lead to for example, missing or extra proteins.
4. Missense and intronic mutations: these are the hardest to analyse.

Question 34

How are results interpreting?

Answer 34

• Do not report known polymorphisms only ones that are known mutations.
• Conservative approach to reporting novel mutations of uncertain pathogenicity: “uncertain significance” and “likely to be pathogenic”.
• Request further samples from family members

Question 35

What is the 100,000 genomes project?

Answer 35

• Made to bring direct benefits of genetics to patients
• It enabled new scientific discovery and medical insights
• Created an ethical and transparent programme based on consent and patient engagement
• Allowed personalised medicine

Question 36

How are possible genes that cause a disease clinically interpretated?

Answer 36

• Experts develop lists of possible genes that 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.
• Therefore, we can focus on specific genes of the patient’s genomes we think are important.
• Variants within virtual panel are divided into 3 tiers.

Question 37

What are the three variant tiers?

Answer 37

Tier 1: known pathogenic; protein truncating (nonsensE)

Tier 2: protein altering (missense); intronic (splice site)

Tier 3: loss of function variants in genes not on the disease gene panel.