Overview of genomic technologies in clinical diagnostics

Question 1

List some examples of Genomic Technologies

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 are the key points of Polymerase Chain Reaction (PCR)?

Answer 2

→ Fundamental for many DNA applications
→ PCR is used to amplify a specific region of DNA that we know
→ Primers (oligonucleotide that is complementary to the region to be amplified) 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

There is a 3 step process:
1. Denaturation
2. Annealing
3. Extension

Question 3

What are the key points of Fragment Analysis?

Answer 3

→ It is used to analyse of the amplified PCR product
→ PCR based assay
→ PCR followed by capillary electrophoresis (separating molecules by size)
→ Can be used to detect repeat expansions or other small size changes (up to a few hundred base pairs)

Question 4

What are some examples of Repeat Expansion Diseases?

Answer 4

→ Huntington’s disease – severe neurodegenerative disorder
→ Caused by CAG repeat expansion in the Huntington (HTT) gene
→ Normal < 27 copies; Intermediate 27-35 copies; Pathogenic > 35 copies (more than 35)
→ Expanded protein is toxic and accumulates in neurons causing cell death
→ Can be diagnosed with fragment analysis

Question 5

What are the key points of Sanger sequencing?

Answer 5

→ Sanger sequencing is for detecting mutation

→ Cycle Sequencing; based on the same principles as PCR

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

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

→ Slow, low-throughput and costly to perform for large numbers of samples (you can read the dyes to obtain the DNA sequence)
→ We can identify single nucleotide polymorphisms (SNPs), or mutations

→ Detection of a mutation in a family by use of Sanger Sequencing
→ R1042G (arginine to glycine) mutation in gene C3 segregates with affected individuals
→ Mutation causes disease cutaneous vasculitis

Question 6

What are the key points of Fluorescence in situ hybridisation (FISH)?

Answer 6

→ To detect large (microscopic) chromosomal abnormalities:
1. Extra chromosomes
2. Large deleted segments
3. Translocations - moving of chromosomes
4. Chromosome abnormalities

Question 7

What are the steps in FISH?

Answer 7

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 !

An example is seeing trisomy on chromosome 21 to diagnose Down Syndrome

Question 8

What are the key points to Array CGH (comparative genomic hybridisation)?

Answer 8

→ For detection of sub-microscopic (too small to be seen w/ FISH) chromosomal abnormalities

→ Patient DNA labelled Green
→ Control DNA labelled Red
→ DNA mixed and hybridised to a microarray
→ Signals detected from platform

Outcomes:
1. DNA dosage gain
2. DNA dosage loss
3. Equal hybridisation

→ Patient array CGH profile
→ Increased/ excess green signal over a chromosomal segment in the patient DNA
→ Indicates a gain in the patient sample not present in the parents
→ Chromosomal abnormality in this region

Question 9

What is Multiplex ligation-dependent probe amplification (MPLA) and what is it used for?

Answer 9

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

→ We use MLPA to detect abnormal copy numbers at specific chromosomal locations, not genome wide

→ MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions

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

→ Perform fragment analysis (capillary electrophoresis) of MLPA product
→ An important use of MLPA is to determine relative ploidy (how many chromosome copies?) as specific locations
→ For example, probes may be designed to target various regions of chromosome of a human cell
→ 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 10

What does Next Generation Sequencing involve (NGS)?

Answer 10

→ An end to sequential testing
→ Wider range of tests in a shorter time for less money
→ Current strategy: Disease panels

a. Enriching to sequence only the known disease genes relevant to the phenotype
b. Panels expandable to include new genes as they are published
c. Potentially pathogenic variants confirmed by Sanger sequencing

Question 11

What has NGS replaced?

Answer 11

Next Generation Sequencing has replaced Sanger sequencing for almost all sequencing tests in the lab

Question 12

What is Exome sequencing and why is it used?

Answer 12

→ Most common technique used in NGS for:

a. diagnosing genetic disease
b. discovering disease causing genes

→ There are ~21,000 genes in the human genome and 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
→ Therefore, 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 13

What does Exome sequencing involve?

Answer 13

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

More specifically:

1. DNA library from patient sample
2. Incubate library with RNA baits
3. Hybridation step
4. Exons baited
5. Purification column based on magnetic beads 				attached to streptavidin that recognises biotin on RNA 		baits	
6. Exon fragments caught with magnet
7. Wash away unbound fragments that we don't want to sequence (introns etc)
8. Left with enriched library that can be sequenced

Question 14

What is whole Genome sequencing?

Answer 14

→ NOT all tests will automatically move to whole genome sequencing:

a. Panels/single gene tests may still be more suitable for some diseases, e.g. cystic fibrosis which is caused by a single gene

b. Capillary-based methods:
Repeat expansions, MLPA, family mutation confirmation Sanger sequencing

c. Array-CGH: large sized chromosomal aberrations

Question 15

What are some ethical considerations for exome and Genome Sequencing? What is the greatest challenge?

Answer 15

→ Result interpretation is the greatest challenge
→ 20,000 genetic variants identified per coding genes ‘exome’ and 3 million variants in a whole human genome
→ Therefore, it is hard to interpret in terms of finding pathogenic mutations

There are ethical considerations:
→ Modified patient consent process
→ Data analysis pathways – inspect relevant genes first (don’t want to look at places not of interest)
→ Strategy for reporting ‘incidental’ findings

→ Also when analysing findings etc, you need
Infrastructure and training (particularly IT and clinical scientists).

Question 16

What is The 100,000 Genomes Project? What are the main aims? Who/ what is being sequenced and how?

Answer 16

1)
→ Bring direct benefit of whole genome sequencing and genetics to patients

2)
→ Enable new scientific discovery and medical insights
→ Personalised medicine

3)
→ Rare genetic diseases – index cases (affected sample) + families
→ Cancer – germline (from blood) and tumour samples (DNA sample from tumour itself)
→ England – wide collection by GMCs (genomic medicine centres)

Question 17

How are classification of mutations by genomics done in England?

Answer 17

→ For each disease, there is a virtual gene panel
→ Variants within virtual panel divided into three tiers
→ There is a list of genes known to cause the disease/ phenotype (Expert review is required)
→ Moving down from tier 1 - 3, there is less certainty

a. Tier 1 variants
→ Known pathogenic - previously reported
→ Protein truncating - protein shorter than it should be, a known mechanism of disease

b. Tier 2 variants
→ Protein altering (missense)
→ Intronic (splice site)
→ There is less confidence that these are disease causing
→ Not reported back to patient

c. Tier 3 variants
→ Loss-of-function variants in genes not on the disease gene panel
→ Not reported back to patient

Question 18

What is the NHS Diagnostic Laboratory and what does it do?

Answer 18

→ Accredited laboratory: ISO standard 15189 for Medical Laboratories
→ Scientific, technical and administrative staff
→ Provide clinical and laboratory diagnosis for genetic disorders
→ Liaise with clinicians, nurses and other health professionals
→ 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

→ Perform specific tests with proven:
a. Clinical Validity:
How well the test predicts the phenotype

b. 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 19

What sorts of tests/info does the NHS laboratory provide?

Answer 19

→ Diagnostic
a. Diagnosis
b. Management and Treatment
c. Interpretation of pathogenicity - is the mutation pathogenic?

→ Predictive
a. Life choices, management

→ Carrier (recessive)
a. Life choices, management

→ Diagnostic testing is available for all Consultant referrals
a. Clinical Geneticists most common referrers

→ Informed consent
a. Genetic counselling
b. Implications for other family members

Question 20

What are the potential outcomes the NHS laboratory can provide?

Answer 20

→ Pathogenic mutation
→ Normal variation, polymorphism - isn’t disease causing, no impact on clinical phenotype
→ Novel variant - Investigations to establish clinical significance, most common

Question 21

What are the 4 ways you can establish if a mutation is pathogenic?

Answer 21

1. Mode of inheritance - is it dominant, recessive, X-linked etc ie does it follow the correct pattern of inheritance?
2. Genetic databases of published and unpublished data - i.e. previous reports
3. Nonsense, frameshift, splice site (exon+/-2 bp) mutations - functional predictions, model effect of mutation on gene
4. Missense/intronic mutation - In-silico tools for missense and splicing mutations, can predict and model if changes to protein are damaging

Question 22

Do clinicians report all findings? When there is no clear pathogenic mutation, what do clinicians tell patients?

Answer 22

→ Do not report known polymorphisms

→ Conservative approach to reporting novel mutations of uncertain pathogenicity
a. ‘Uncertain significance’
b. ‘Likely to be pathogenic’

→ Request samples from family members
→ Continue testing other genes ?