Overview of Genomic Technologies in Clinical Diagnostics

Question 1

What are the genomic technologies currently available?

Answer 1

• 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

Question 2

What is PCR?

Answer 2

Enzyme based method used to amplify segments of DNA using a thermal polymerase in a cyclical process

Question 3

When is PCR used?

Answer 3

Fundamental for many DNA applications

PCR used to amplify specific regions of DNA

Primers flank the region you want to amplify

Question 4

What is fragment analysis?

Answer 4

PCR based assay

PCR followed by capillary electrophoresis (bands appear as peaks)

Question 5

What is fragment analysis used for?

Answer 5

To detect repeat expansions or other small size changes (up to a few hundred bp)

Question 6

Give an example of a repeat expansion disease identified using fragment analysis

Answer 6

Huntington’s disease – severe neurodegenerative disorder

Question 7

What causes Huntingtons disease?

Answer 7

Caused by CAG repeat expansion in the Huntingtin (HTT) gene

Expanded protein is toxic and accumulates in neurons causing cell death

Question 8

Describe the normal and pathogenic range of CAG repeats

Answer 8

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

Question 9

What is sanger sequencing?

Answer 9

Cycle Sequencing; based on the same principles as PCR

Question 10

How does sanger sequencing identify base sequences?

Answer 10

Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence

Question 11

What is sanger sequencing used for?

Answer 11

Good for sequencing single exons of genes
Identify SNPs
Up to 800bp of sequence per reaction

Question 12

What are the downfalls of Sanger sequencing?

Answer 12

Slow, low-throughput and costly to perform for large numbers of samples

Question 13

Outline how sanger sequencing is carried out

Answer 13

1. Sample placed on a plate
2. Sample taken up into capillaries
3. Passed through a laser
4. 4 bases show different colours

Question 14

What is FISH?

Answer 14

Fluorescent in situ hybridisation

• Cultured cells, metaphase spread
• Microscopic (5-10Mb)

Question 15

What can we detect using FISH?

Answer 15

Large chromosomal abnormalities

• Extra chromosomes
• Large deleted segments
• Translocations

Question 16

Outline how FISH is carried out

Answer 16

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 complementary target sequence
5. Target fluoresces or lights up

Question 17

How is FISH used for special karyotyping?

Answer 17

Designed lots of probes to mark all chromosomes in a different way to colour coordinate them (barcode)

Question 18

What is array cgh?

Answer 18

Array comparative genomic hybridisation is for detection of submicroscopic chromosomal abnormalities as well as large

Question 19

What is the process of array CGH

Answer 19

1. Patient DNA labelled Green
2. Control DNA labelled Red
3. Mix DNA samples together
4. Hybridise sample to array microchip w/ spots covering
   whole genome on
5. Patient DNA and control DNA bind to spots

Question 20

Describe array cgh results

Answer 20

Increased green signal over a chromosomal segment in patient DNA indicates a gain in the patient sample not present in parents

Red indicates a loss in DNA

Question 21

What is array CGH used for?

Answer 21

Can identify someone’s copy no. status

• Good for analysing pathogenic chromosomal mutations
• Shows structural variety

Question 22

What is MLPA?

Answer 22

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

Question 23

What does each MLPA consist of?

Answer 23

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

Question 24

What can MPLA identify?

Answer 24

MLPA used to detect abnormal copy numbers at specific chromosomal locations
and sub-microscopic (small) gene deletions/partial gene deletions

Question 25

What does each MLPA probe contain?

Answer 25

One probe oligonucleotide contains the sequence recognized by forward primer The other contains the sequence recognized by reverse primer

Question 26

In MLPA when do the 2 separate probes become one probe?

Answer 26

Only when both probe oligonucleotides are hybridized to their respective targets, can they be ligated into a complete probe

Question 27

How do we amplify and visualise the complete probe in MLPA?

Answer 27

Perform fragment analysis (capillary electrophoresis) of MLPA product

Question 28

Describe a use of MLPA

Answer 28

An important use of MLPA is to determine relative ploidy (how many chromosome copies?) as specific locations

Question 29

How can we determine the strength of the probe signals in MLPA?

Answer 29

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 30

What is a commonly used NGS?

Answer 30

Solexa sequencing-by-synthesis (SBS) developed end of 2005 Sequencing market is now dominated by Illumina SBS sequencing

Question 31

What are the benefits of NGS?

Answer 31

NGS is an end to sequential testing - Wider range of tests in a shorter time for less money

Question 32

What are the current strategies and aims of NGS?

Answer 32

Disease panels - Enriching to sequence only known disease genes relevant to phenotype - Panels expandable to include new genes as they are published - Potentially pathogenic variants confirmed by Sanger sequencing

Question 33

How many genes are present in the human genome?

Answer 33

There are ~21,000 genes in the human genome

Question 34

How significant is the exome?

Answer 34

Gene protein coding exons or ‘exome’ represents 1-2% of genome Some ~80% pathogenic mutations are protein coding

Question 35

What is the advantages of exome sequencing?

Answer 35

More efficient to only sequence bits we are interested in, rather than entire genome Costs £1,000 for a genome, but only £200-£300 for an exome

Question 36

What are the uses of exome sequencing?

Answer 36

- Target enrichment - Capture target regions of interest with baits - Potential to capture several Mb genomic regions (typically 30-60 Mb)

Question 37

What is the future of whole genome sequencing WGS?

Answer 37

Universally accepted that genome sequencing will become commonplace in diagnostic genetics

Question 38

What other tests are available other than WGS?

Answer 38

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

Question 39

What is the significance of WGS?

Answer 39

Whole genome sequencing is NOT trivial Interpretation of clinical genomes is currently has a substantial manual component

Question 40

What is the biggest problem with WGS and WES?

Answer 40

Result interpretation is the greatest challenge - 20,000 variants per coding genes ‘exome’ - 3 million variants in a whole human genome

Question 41

What are the ethical considerations of WES and WGS?

Answer 41

- Modified patient consent process - Data analysis pathways – inspect relevant genes first - Strategy for reporting 'incidental' findings

Question 42

What is the NHS Diagnostic Library?

Answer 42

Accredited laboratory: ISO standard 15189 for Medical Laboratories Has scientific, technical and administrative staff

Question 43

What are the roles of the NHS diagnostic library?

Answer 43

Provide clinical and laboratory diagnosis for genetic disorders Liaise with clinicians, nurses and other health professionals Provide genetic advice for sample referrals and results

Question 44

What is the main role of the NHS Diagnostic library?

Answer 44

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

Question 45

How do consultants reach a genetic diagnosis?

Answer 45

Perform specific tests with proven: Clinical Validity - How well the test predicts the phenotype Clinical Utility - How the test adds to patient management

Question 46

Outline the diagnostic test outcomes

Answer 46

- Pathogenic mutation - Normal variation; Polymorphism - Novel variant ; Investigations to establish significance

Question 47

How do we interpret diagnostic test results?

Answer 47

To establish if a mutation is pathogenic figure out the following: - Mode of inheritance - Locus-specific databases of (un)/published data - Nonsense, frameshift, splice site (exon +/-2 bp) mutations - Missense/intronic mutation - In-silico tools for missense + splicing mutations