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

Primers 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

(Denaturation, Annealing, Extension)

Question 3

What are the key points of Fragment Analysis?

Answer 3

PCR based assay

PCR followed by capillary electrophoresis (separating molecules by size)

Here we are sizing the PCR product

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 Huntingtin (HTT) gene

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

Expanded protein is toxic and accumulates in neurons causing cell death

Diagnosed with fragment analysis

Question 5

What are the key points of Sanger sequencing?

Answer 5

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

reading 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 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 chromosomal abnormalities

Extra chromosomes

Large deleted segments

Translocations

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 !

Question 8

What are the key points to Array CGH?

Answer 8

Array comparative genomic hybridisation

For detection of sub-microscopic chromosomal abnormalities

Patient DNA labelled Green

Control DNA labelled Red

Patient array comparative genomic hybridisation profile

Increased green signal over a chromosomal segment in the patient DNA

Indicates a gain in the patient sample not present in the parents

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

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

We use MLPA to detect abnormal copy numbers at specific chromosomal locations

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

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)

What has NGS replaced?

Answer 10

An end to sequential testing

Wider range of tests in a shorter time for less money

Current strategy: Disease panels
Enriching to sequence 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

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

One of the most common NGS technique is EXOME SEQUENCING

Question 11

What does Exome sequencing involve?

Answer 11

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

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

Target enrichment

Capture target regions of interest with baits

Potential to capture several Mb genomic regions (typically 30-60 Mb

Question 12

What is whole Genome sequencing?

Answer 12

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 13

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

Answer 13

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

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

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

Question 14

What is the The 100,000 Genomes Project?

Answer 14

100,000 genomes project
Bring direct benefit of whole genome sequencing and genetics to patients
Enable new scientific discovery and medical insights
Personalised medicine

England – wide collection
GMCs (genomic medicine centres)
Who/what is being sequenced?
Rare diseases – index cases + families
Cancer – germline and tumour samples 

Classification of mutations by genomics England:

Variants within virtual panel divided into three tiers

(Expert review is required )

Tier 1 variants
Known pathogenic
Protein truncating

Tier 2 variants 
Protein altering (missense)
Intronic (splice site)

Tier 3 variants
Loss-of-function variants in genes not on the disease gene panel

Question 15

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

Answer 15

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:
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 16

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

Answer 16

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

Potential outcomes it can provide:
Pathogenic mutation

Normal variation
Polymorphism

Novel variant
Investigations to establish clinical significance…

Question 17

How can you establish if a mutation is pathogenic?

Question 18

Describe the basic principles of PCR

Answer 18

o Fundamental for any DNA application
o PCR is used to amplify a specific region of DNA;.
o Primers flank the region you want to amplify.
o Each cycle doubles the amount of DNA copies of your target sequence
o Amplify enough DNA molecules so that we have sufficient material to sequence or for other applications
o Most widely used technique in labs
o Amplifies a region of interest
o Have to design the primers

Question 19

Describe the basic principles of fragment analysis

Answer 19

o Fundamental for any DNA application
o PCR is used to amplify a specific region of DNA;.
o Primers flank the region you want to amplify.
o Each cycle doubles the amount of DNA copies of your target sequence
o Amplify enough DNA molecules so that we have sufficient material to sequence or for other applications
o Most widely used technique in labs
o Amplifies a region of interest
o Have to design the primers

Question 20

What is Huntington’s disease?

What causes it?

How is it identified?

Answer 20

o Huntington’s disease – severe neurodegenerative disorder
o Caused by CAG repeat expansion in the Huntingtin (HTT) gene
o Normal < 27 copies; Intermediate 27-35; Pathogenic > 35
o Expanded protein is toxic and accumulates in neurons causing cell death
o Diagnosed with fragment analysis
o The more repeats you have, the more toxic the protein is

Question 21

Describe the principles of sanger sequencing

Answer 21

o Cycle Sequencing; based on the same principles as PCR
o Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence.
o Up to 800 bp of sequence per reaction
o One reaction = one sequence
o Slow and low-throughput
o Costly to perform ££££
o Most widely used
o Nucleotides are dyed different colours, you can read the dyes to obtain the DNA sequence
o We can identify single nucleotide polymorphisms (SNPs), or mutations in this way. A star can be used to show that the patient below has a mutation in that position.
o After the PCR, the product is run on capillary electrophoresis
o The sequence is built up based on the colours
o The sample are loaded on the plates below and then sucked up into the capillaries
o They then pass through a laser beam. The laser beam will shine the different colour of each base and that’s how you get the sequence.
o Detection of a mutation in a family by use of Sanger Sequencing
o R1042G mutation in gene C3 segregates with affected individuals
o Mutation causes disease cutaneous vasculitis
o Individuals in black are affected

Question 22

What is FISH?

Answer 22

o Fluorescent in situ hybridisation
o Cultured cells, metaphase spread
o Microscopic (5-10Mb)
o To detect large chromosomal abnormalities
o Extra chromosomes
o Large deleted segments
o Translocations
o Old technique
1. Used to look at chromosomes in cells
2. Design Fluorescent probe to chromosomal region of interest, has a dye at the end
3. Denature probe and target DNA
4. Mix probe and target DNA (hybridisation) to the chromosome
5. Probe binds to target
6. Target fluoresces or lights up !

Question 23

Describe the principles of array CGH

Answer 23

o Array comparative genomic hybridisation
o For detection of sub-microscopic chromosomal abnormalities

o Patient DNA labelled Green
o Control DNA labelled Red
o More advanced technology than FISH
o Patient DNA  labelled green
o Control DNA (unrelated person)  la belled red
1. Mixed together in equal proportions
2. Hybridise to an array
3. Scan the array and look at the signal
- Control DNA labelled Red
- If there is a loss of DNA in the patient sample, we will see a red signal
- Patient DNA labelled Green
- If there is a gain, we will see more of the green signal
o Easy to see a loss or gain of chromosomes
o Resolution is very low so easy to pick up very small chromosomal abnormalities (e.g. small single gene deletions)

o Patient array comparative genomic hybridisation profile
o Increased green signal over a chromosomal segment in the patient DNA
o Indicates a gain in the patie nt sample not present in the parents
o Increase green signal in the patient but not the parents hence  de novo
o Used to diagnose severe congenital problems

These microarrays are created by the deposit and immobilization of small amounts of DNA (known as probes) on a solid support, such as a glass slide, in an ordered fashion. Probes vary in size from oligonucleotides manufactured to represent areas of interest (25–85 base pairs) to genomic clones such as bacterial artificial chromosomes (80,000–200,000 base pairs). Because probes are several orders of magnitude smaller than metaphase chromosomes, the theoretical resolution of aCGH is proportionally higher than that of traditional CGH. The level of resolution is determined by considering both probe size and the genomic distance between DNA probes. For example, a microarray with probes selected from regions across the genome that are 1 Mb apart will be unable to detect copy number changes of the intervening sequence.

Question 24

What is MPLA?

Answer 24

o Multiplex ligation-dependent probe amplification (MLPA) is a variation of PCR that permits amplification of multiple targets with only a single primer pair
o Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA

o We use MLPA to detect abnormal copy numbers at specific chromosomal locations
o MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions

o Based on PCR
o Used specifically for chromosomal abnormalities
o Looks for losses or gains
o Only used to detect things that you may have prior knowledge could be missing.
o Need to know what your target is before you do it

o One probe oligonucleotide contains the sequence recognized by the forward primer, the other contains the sequence recognized by the reverse primer.
o Only when both probe oligonucleotides are hybridized to their respective targets, can they be ligated into a complete probe
o Hybridise patient sample to oligonucleotides
o The amount of product is proportional to the amount of DNA  this is because the complete probe being formed is dependent on the ligation reaction.

o Perform fragment analysis of MLPA product
o An important use of MLPA is to determine relative ploidy (how many chromosome copies?) as specific locations
o For example, probes may be designed to target various regions of chromosome of a human cell
o The signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome
o Run products on capillary electrophoresis and shows if we have a loss or gain of a gene.
o We can see that there is an exon missing on this gene

Question 25

Describe the principles of NGS

Answer 25

o Next Generation Sequencing has replaced Sanger sequencing for almost all sequencing tests in the lab
o Technological advances since the end of the human genome project

o Decrease in the cost of DNA sequencing. Advances in technology  the cost has decreased as well.
o Since the end of 2007, the cost has dropped at a rate faster than that of Moore’s law
o Development of new NGS methods began 13 years ago with 454 pyrosequencing
o DNA sequencing throughput jumped 10 orders of magnitude
o Solera sequencing-by-synthesis (SBS) developed end of 2005
o Sequencing market is now dominated by Illumina SBS sequencing

o An end to sequential testing
o Wider range of tests in a shorter time for less money
o Current strategy: Disease panels
- Enriching to sequence 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
o e.g. for parkinsons disease
o It is 10% genetic so you can see if it caused due to genes. 20 genes are involved in Parkinson’s and we can sequence all of these in one go in NGS
o Still check mutations with sanger sequencing

Question 26

What is exome sequencing?

Answer 26

o There are ~21,000 genes in the human genome
o Often we are only interested in the gene protein coding exons or ‘exome’ represents 1-2% of the genome

o Some ~80% pathogenic mutations are protein coding
o More efficient to only sequence the bits we are interested in, rather than the entire genome
o Costs £1,000 for a genome, but only £200-£300 for an exome

o Target enrichment
o Capture target regions of interest with baits
o Potential to capture several Mb genomic regions (typically 30-60 Mb
o Capture gene exons with baits
o Once we’ve made a library of patient DNA, we incubate it with the RNA baits that are complementary to the genes. Perform hybridization. Capture the fragments that are hybridized and wash away the bits of DNA that we are not interested in.
o We capture the fragments using magnetic beads and now have an enriched sample of sequence

Question 27

Describe the principles of whole-genome sequencing?

Answer 27

o Universally accepted that genome sequencing will become commonplace in diagnostic genetics
o Not all tests will automatically move to whole exome sequencing / whole genome sequencing

• Panels/single gene tests may still be more suitable for some diseases.
• Capillary-based methods: Repeat expansions, MLPA, Family mutation Sanger sequencing
• Array-CGH: large sized chromosomal aberrations
  o However we would only use fragment analysis for Huntington’s (it would be unwise inefficient to sequence the whole genome)
  o Interpretation of clinical genomes is currently has a substantial manual component
  o Whole genome sequencing is NOT trivial

Question 28

What role does NHS labs play?

Answer 28

o Accredited laboratory: ISO standard 15189 for Medical Laboratories.
o Scientific, technical and administrative staff.

o Provide clinical and laboratory diagnosis for inherited disorders.
o Liaise with clinicians, nurses and other health professionals
o Provide genetic advice for sample referrals and results
o Carry out translational research for patient benefit
o Train clinical research fellows, clinical scientists, post- and undergraduate students
o Strict guidelines to be followed and they have to be accredited.

o The main role of the lab is to help Consultants reach a genetic diagnosis for individuals and families to help guide treatment and management.
o 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
o UKGTN (UK genetic testing network)-approved tests
o In-depth and up-to-date knowledge of the genetic diseases covered
o Translational Research

o Genetic diseases run in families so whole families can be tested.
o Children might want to be tested to see if they will have a condition.

Question 29

How does diagnostic testing take place?

Answer 29

o All referrals via Regional Genetics Centres
o Close liaison with nurse specialists, genetic counsellors, clinicians during testing

o International guidelines for predictive testing
o Follow up at clinics, nurse led clinics, nurse telephone clinics as required
o Pathogenic mutation
o Normal variation
 Polymorphism – Common in the population but is not pathogenic
o Novel variant
 Investigations to establish significance
 Have to do some detective work to see if a mutation is pathogenic or not
o Previously published/detected variant of uncertain significance…

Question 30

What is c.647T>C p.(Phe216Ser)?

Answer 30

o Sanger confirmation / validation
o Father appeared homozygous WT
o SNP under primer, UPD, paternal deletion?
o Base 647 is in exon 7
o Non Mendelian inheritance
o What is going on here ?
o Father appeared homozygous WT
o SNP under primer, UPD, paternal deletion?
o Base 647 is in exon 7
o Mother has passed on the mutation
o Father is T/T which is surprising as the kids are homozygous

Question 31

What is Del Ex7-8?

Answer 31

o MLPA measures dosage of all MFN2 exons
o Breakpoint sequencing:

o Deletion of 1,476 bp from 700 bp 3’ of ex 6 to 2.1kb 5’ of ex 9
o Children and father all carry deletion of MFN2 exons 7-8
o Hemizygous at base 647

o A deletion in the gene as well
o The father has the deletion rather than a C allele
o Each child has inherited a mutation and a deletion .
o So it looks like the child is homozygous but they actually have two defective alleles.
o RHS  agarose gel confirming the deletion .
o The 100,000 Genomes Project