Overview Of Genomic Technologies for Clinical Diagnosis

Question 1

1. Briefly explain what PCR does?

Answer 1

• Used to amplify a specific region of DNA for downstream applications – each cycle doubles the DNA amount.
• Primers flank the region you want to amplify
Process : Denaturation , Annealing , Extension

Question 2

2. Explain how PCR works?

Answer 2

• PCR based assay - PCR followed by capillary electrophoresis to size the PCR product
• Can be used to detect repeat expansions or other small size changes (up to a few hundred bp).

Question 3

3. What is the repeat expansion disease Huntingtons?

Answer 3

•Huntington’s disease (severe neurodegenerative disorder) is caused by CAG repeat expansion in the Huntingtin (HTT) gene.

Question 4

4. How many copies of the CAG codon in a normal, intermediate and pathogenic situation?

Answer 4

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

•Accumulation of expanded toxic protein in neurons causes cell death.

Question 5

5. How does Sanger Sequencing work?

Answer 5

•Each of the 4 DNA nucleotides are labelled with a different dye - determine the nucleotide sequence to look for mutations or single nucleotide polymorphisms (SNPs).

Question 6

6. Is sanger sequencing good for sequencing large numbers or samples or only single exons?

Answer 6

•Up to 800bp of sequence per reaction so it’s good for sequencing single exons of genes but slow, low-throughput and costly to perform for large numbers of samples

Question 7

7. Whats an example of a mutation that causes the disease cutaneous vasculitis ?

Answer 7

R1042G mutation in gene C3 segregates with affected individuals and causes disease cutaneous vasculitis

Question 8

8. What is FISH used to detect?

Answer 8

• Detect large chromosomal abnormalities e.g. extra chromosomes, large deleted segments and translocations.
• Cultured cells, metaphase spread - Microscopic (5-10Mb)

Question 9

9. How does FISH work?

Answer 9

• Design a fluorescent probe to the chromosomal region of interest, denature target DNA and mix probe with it until hybridisation occurs (probe binds to target causing it to fluoresce/light up).
• Can use it for the whole karyotype or a specific chromosome.

Question 10

10. What is Array CGH (comparative genomic hybridisation) used to detect?

Answer 10

• For detection of sub-microscopic chromosomal abnormalities as well as large chromosomal abnormalities.

Question 11

11. How does Array CGH work?

* GO through this properly*

Answer 11

• The patient DNA is labelled green and control DNA is labelled red. They are mixed and allowed to hybridise to an array before scanning to produce a patient array comparative genomic hybridisation profile.
• Increased green signal over a chromosomal segment in the patient DNA indicates a gain in the patient sample chromosomes that is not present in the parents.

Question 12

12. What is a MLPA (Multiplex ligation-dependent probe amplification) used to detect?

Answer 12

• Variation of PCR that permits amplification of multiple targets – used to detect abnormal copy numbers at specific chromosomal locations (relative ploidy) and sub-microscopic (small) gene deletions/partial gene deletions.

Question 13

13. How does an MLPA work?

Answer 13

• Probes consist of two oligonucleotides that recognize adjacent target sites on the DNA – one oligonucleotide is recognised by the forward primer and the other is recognised by the reverse primer.
o When both oligonucleotides hybridize to their respective targets - they can be ligated into a complete probe.
• Perform fragment analysis (capillary electrophoresis) of MLPA product.
• 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 14

14. What has Sanger Sequencing been replaced by?

Answer 14

Next Generation Sequencing

Question 15

15. Which techniques are used in next gen sequencing?

Answer 15

• Solexa sequencing-by-synthesis (SBS) developed end of 2005

* Sequencing market is now dominated by Illumina SBS sequencing

Question 16

16. Whats the major advantage of next generation sequencing?

Answer 16

Wider range of tests in a shorter time for less money (through-put jumped 10 orders of magnitude).

Question 17

17. One current strategy for Next Gen sequencing is “Disease Panel Tests “ how do these works?

Answer 17

o Sequences are enriched for known disease genes relevant to the phenotype and expanded to include new genes as they are published.
o Potentially pathogenic variants confirmed by Sanger sequencing.

Question 18

18. What is useful about exome sequencing?

Answer 18

• Often we are only interested in the exome because most pathogenic mutations (80%) are usually protein coding and it is 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 19

19. How does exome sequencing work?

Answer 19

• Target enrichment allows us to capture specific target regions:
o RNA baits and magnetic beads tagged with biotin (or other markers) that bind to the baits are used to capture specific regions of interest.
o Potential to capture several Mb genomic regions (typically 30-60 Mb)

Question 20

20. What is the future for whole genome sequencing?

Answer 20

• WGS will become commonplace in diagnostic genetics in the future but not all tests will automatically move to whole genome sequencing because some diseases are more suitable to other methods:

Question 21

21. Which diseases may not be suitable for whole genome sequencing -> What would it be preferable to use ?

Answer 21

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

Question 22

22. Is whole genome sequencing easy/quick?

Answer 22

• Data generation and processing are relatively automated/quick but data interpretation of clinical genomes currently has a substantial manual component (most tricky part).

Question 23

23. How many variants are there is a coded exome and in a whole human genome?

Answer 23

20,000 variants per coding genes ‘exome’ and 3 million variants in a whole human genome.

Question 24

24. What ethical considerations need to be considered when exome and genome sequencing?

Answer 24

o Modified patient consent process
o Data analysis pathways – inspect relevant genes first
o Strategy for reporting ‘incidental’ findings

Question 25

25. What is the NHS diagnostics laboratory?

Answer 25

• Accredited laboratory with scientific, technical and administrative staff that provides clinical/laboratory diagnosis for genetic disorders and provides genetic advice for sample referrals/results.

Question 26

26. What is the main role of the NHS diagnostics laboratory?

Answer 26

help consultants reach a genetic diagnosis s to help guide treatment and clinical management
•Perform specific tests with proven :
-clinical validity (how well the test predicts the phenotype) and
-clinical utility (how the test adds to the management of the patient).

Question 27

27. Where could you find up to date in depth knowledge of genetic diseases

Answer 27

•UKGTN (UK genetic testing network)-approved

Question 28

28. What 4 main uses are there of the NHS diagnostic laboratory?

Answer 28

o Diagnostic: Diagnosis, Management and Treatment and Interpretation of pathogenicity
o Predictive: Life choices, management
o Carrier (recessive): Life choices, management
o Informed consent: Genetic counselling and Implications for other family members

Question 29

29. Are all diagnostic testing referrals via Regional Genetics Centres?

Answer 29

Yes

Question 30

30. Who does the patient have close contact with during diagnostic testing?

Answer 30

• Close liaison with nurse specialists, genetic counsellors, clinicians during testing - follow up at clinics, nurse led clinics, nurse telephone clinics as required.

Question 31

31. What can be found during diagnostic testing?

Answer 31

pathogenic mutations, normal variants (polymorphism) and novel variants (investigation required for significance).

Question 32

32. How do we establish if a mutation is pathogenic?

Answer 32

• Mode of inheritance - Request samples from family members.
• Locus-specific databases of published and unpublished data
• Missense/intronic mutation: In-silico tools for missense and splicing mutations
• Conservative approach to reporting novel mutations of uncertain pathogenicity - ‘Uncertain significance’ or ‘Likely to be pathogenic’

Question 33

33. What is Charcot-Marie-Tooth disease type 2 (CMT2)

Answer 33

o Degeneration of the long nerves in legs and arms leading to muscle wasting and sensory defects.
o Onset usually in childhood

Question 34

34. Which mutation causes Charcot-Marie-Tooth disease type 2 (CMT2)

Answer 34

•Mitofusin 2 (MFN2)

Question 35

35. Case study:
    • Two siblings with very severe early-onset CMT2 and parents unaffected.
    • NGS: Gene sequenced and found to be homozygous for c.647T>C p.(Phe216Ser).
    • Parents sequenced, expected them to both be heterozygous but the father was homozygous TT.

Explain why this could be?

Answer 35

o Could be due to non-mendelian inheritance, father not the father, paternal exon deletion etc.

Question 36

36. Which exon deletions were found in both father and son after MLPA?

Answer 36

• MLPA measures dosage of all MFN2 exons and found that the child and father carry MFN2 exons 7-8 deletions.
• Breakpoint sequencing: Deletion of 1,476 bp from 700 bp 3’ of ex 6 to 2.1kb 5’ of ex 9

Question 37

37. What is the use of genomic england (a 100,000 genome project)

Answer 37

Bring direct benefit of genetics to patients, enable new scientific discovery and medical insights, create an ethical and transparent programme based on consent and patient engagement and personalised medicine.

Question 38

38. How do healthcare professionals interpret genomic testing clinically?

Answer 38

• ‘Experts’ develop lists of possible genes than can cause a specific disease - are reviewed by the community.
• Diseases have specific sets of virtual gene panels as a first port-of-call to look for pathogenic mutations so we can focus on specific genes of the patient’s genome we think are important

Question 39

39. • Classification of mutations by genomics England - variants within virtual panel divided into three tiers- Explain these?

Answer 39

o Tier 1 variants: Known pathogenic or Protein truncating.
o Tier 2 variants: Protein altering (missense) or Intronic (splice site).
o Tier 3 variants: Loss-of-function variants in genes not on the disease gene panel.