Overview Of Genomic Technologies In Clinical Diagnostics

Question 1

What is PCR used for and how does it work (briefly)?

Answer 1

• PCR is used to amplify a specific region of DNA (via denaturation, annealing and extension)
• Primers (short stranded DNA complementary to region) 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

Question 2

What is fragment analysis and what is it used to detect?

Answer 2

• PCR based assay
• PCR followed by capillary electrophoresis
• Here we are sizing the PCR product -> working out the relative length (BP)
• Can be used to detect repeat expansions or other small size changes (up to a few hundred bp)
• Repeat expansion = a triplet that causes a disorder where the higher the number of triplets, the higher the severity of disease gene

Question 3

State an example of a repeat expansion disorder and how its diagnosed?

Answer 3

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

Question 4

What is sanger sequencing and how does it work?

Answer 4

• Cycle Sequencing; based on the same principles as PCR
• Process:
• Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence.
• Read the dyes to obtain DNA sequence
• Can identify single nucleotide polymorphisms or mutations

Question 5

Why is it not used for large numbers of samples?

Answer 5

• Good for sequencing single exons of genes
• Slow, low-throughput and costly to perform for large numbers of samples

Question 6

What is FISH? What does it use?

Answer 6

• FISH = Fluorescent in situ hybridisation
• Uses: Cultured cells, metaphase spread
• All the genetic material is condensing into chromosomes. These chromosomes then become visible.
• During this stage, the nucleus disappears and the chromosomes appear in the cytoplasm of the cell.
• Microscopic (5-10Mb)

Question 7

What is FISH used to detect?

Answer 7

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

Question 8

State the process for how FISH is done?

Answer 8

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 9

State examples of FISH processes and 2 disorders it can detect?

Answer 9

• Processes: Special karoytoping (label different chromosomes with different lights) and target specific FISH
• Disorders: Trisomoy 21 and Down syndrome

Question 10

What is Array CGH and what is it used to detect?

Answer 10

• Array comparative genomic hybridisation
• Used to detect sub-microscopic chromosomal abnormalities (which FISH can’t do)

Question 11

Describe how Array CGH works and the subsequent result scenarios?

Answer 11

1. Patient DNA (green dye) and control DNA (red dye)
2. Applied to microarray and hybridised
3. Microarray measures fluorescent signals and generates plot
4. Green indicates DNA gain, red shows DNA loss (both chromosomal abnroamlities.

Question 12

What is MLPA?

Answer 12

Multiplex ligation-dependent probe amplification (MLPA) is a variation of PCR that permits amplification of multiple targets.

Question 13

What is MLPA used to detect?

Answer 13

• We use MLPA to detect abnormal copy numbers (Repeats of sections of genome) at specific chromosomal locations
• MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions

Question 14

What does each probe consist of in MLPA?

Answer 14

Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA

Question 15

Describe the probes used for MLPA

Answer 15

• Two probes are used
• One probe oligonucleotide contains the sequence recognized by the forward primer
• The other contains the sequence recognized by the reverse primer.

Question 16

Describe the process of MLPA?

Answer 16

1. Hybridisation occurs of both probe oligonucleotides to their respective targets
2. Ligations occurs both probes into complete probe
3. PC amplification occurs of complete probe to form amplified library
4. Perform fragment analysis (capillary electrophoresis) of MLPA product
   a. Allows to see size/dosage of products forms

Question 17

How are results of MLPA worked out?

Answer 17

• 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 18

What are MLPA results used for?

Answer 18

• An important use of MLPA is to determine relative ploidy (how many chromosome copies?) at specific locations.
• For example, probes may be designed to target various regions of chromosome of a human cell

Question 19

What is next gen sequencing used for and why is it used over sequential testing?

Answer 19

• Current strategy: Disease panels
• Disease panal: sequences 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
• Has replaced SS for almost all Sequencing tests in the lab

Question 20

In regards to whole genome sequencing, what will it not replace in 1. cystic fibrosis 2. capillary-based methods 3. large sized chromosla aberrations

Answer 20

• 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

Question 21

What are the common issues surrounding exome and genome sequencing? (PART 1)

Answer 21

1. Result interpretation is the greatest challenge
   - 20,000 genetic variants identified per coding genes ‘exome
   - 3 million variants in a whole human genome
   - Difficult to identify pathogenic mutations due to large number of mutations
2. Ethical considerations
   - Modified patient consent process
   - Data analysis pathways - inspect relevant genes first
   - Strategy for reporting ‘incidental’ findings
   - Incidental findings = identification of pathogenic mutations of other disease not discovered for person - raises ethical issues of whether to tell person

Question 22

What are the common issues surrounding exome and genome sequencing? (PART 2)

Answer 22

3. Infrastructure and training (particularly IT and clinical scientists)
   - For interpretation of genes

Question 23

What stage of whole genome sequencing is the most time-consuming and why?

Answer 23

• 3 steps: data generation, data processing and automated interpretation and manual intrepration
• Interpretation of clinical genomes currently has a substantial manual component - longest step
• Whole genome sequencing is NOT trivial

Question 24

What is the 100,000 genomics project?

Answer 24

• Bring direct benefit of whole genome sequencing and genetics to patients via company genomics england
• Enable new scientific discovery and medical insights
• Personalised medicine

Question 25

State the source of genes for the 100,000 genomics project

Answer 25

- England - wide collection - GMCs (genomic medicine centres)

Question 26

Who/what is being sequenced?

Answer 26

- Rare diseases - index cases (initial patient of disease outbreak) + families - Cancer - germline and tumour samples

Question 27

How are mutations classified by genomics England in the 100,000 genome project?

Answer 27

- Classified as variants within a virtual panel into 3 tiers - Tier 1 variants: Known pathogenic, Protein truncating (shorten length?) - Tier 2 variants: Protein altering (missense), Intronic (splice site) - Tier 3 variants: Loss-of-function variants in genes not on the disease gene panel - Expert review is required for this

Question 28

Revise at flash card 20 What is exome sequencing?

Answer 28

- 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

Question 29

Why is the exome sequencing used over the genome?

Answer 29

- 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

Question 30

Revise at flash card 20 State the general technique for exome sequencing?

Answer 30

- Technique: Target enrichment - Mix with RNA - hybridise - purify via magnetic beads -> capture beads -> wash beads and digest RNA -> Amplify and sequence - Capture target regions of interest with baits - Potential to capture several Mb genomic regions (typically 30-60 Mb).

Question 31

What is the NHS diagnostic laboratory and state its main aim?

Answer 31

- Provide clinical and laboratory diagnosis for genetic disorders - Provide genetic advice for sample referrals and results - The main role of the lab is to help Consultants reach a genetic diagnosis for individuals and families to help guide treatment and clinical management (management plan)

Question 32

What factors are used to measure the effectiveness of NHS diagnostic lab tests and what network has a network of all the genetic disease?

Answer 32

- Perform specific tests with proven: - Clinical Validity: How well the test predicts the phenotype - Clinical Utility: How the test adds to the management of the patient - UKGTN (UK genetic testing network)-approved tests: - In-depth and up-to-date knowledge of the genetic diseases covered

Question 33

What is the NHS diagnostic laboratory used for (5)?

Answer 33

- Diagnostic: Diagnosis! Management and Treatment! Interpretation of pathogenicity - Predictive: Life choices, management - Carrier (recessive): Life choices, management - Diagnostic testing is available for all Consultant referrals - Clinical Geneticists most common referrers - Informed consent: Genetic counselling, Implications for other family members

Question 34

State the 3 possible outcomes of diagnostic tests?

Answer 34

- Pathogenic mutation: The variant is responsible for causing disease - Normal variation: Polymorphism - Novel variant: Investigations to establish clinical significance, Effect of gene is unclear basically

Question 35

How do we establish whether a mutation is pathogenic?

Answer 35

- Mode of inheritance - Genetic databases of published and unpublished data: See previous history of gene - Nonsense, frameshift, splice site (exon+/-2 bp) mutations - Missense intronic mutation!: - In-silico tools for missense and splicing mutations - In genetics, a missense mutation is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. It is a type of nonsynonymous substitution.

Question 36

Describe how diagnostic tests results are intrepreted?

Answer 36

- Do not report known polymorphisms - Have a conservative approach to reporting novel mutations of uncertain pathogenicity: Uncertain significance', ‘Likely to be pathogenic' - Request samples from family members - look for segregation of genes - How pairs of gene variants are separated into reproductive cells - If gene can't be found - Continue testing other genes?