Overview of genomic technologies in clinical diagnostics

Question 1

List 7 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

Polymerase Chain Reaction (PCR) - use

Answer 2

PCR is used to amplify a specific region of DNA

Question 3

Polymerase Chain Reaction (PCR) - action

Answer 3

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

Question 4

Fragment analysis - define

Answer 4

PCR based assay

PCR followed by capillary electrophoresis

Here we are sizing the PCR product

Question 5

Fragment analysis - use

Answer 5

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

Question 6

Huntington’s disease - define

Answer 6

Huntington’s disease – severe neurodegenerative disorder

Question 7

Huntington’s disease - cause

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

Huntington’s disease - ranges of normal to pathogenic

Answer 8

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

Question 9

Huntington’s disease - diagnosed using

Answer 9

Diagnosed with fragment analysis

Question 10

Sanger Sequencing - define

Answer 10

Cycle Sequencing; based on the same principles as PCR

Question 11

Sanger Sequencing - describe action

Answer 11

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

Question 12

Sanger Sequencing - +ves/-ves

Answer 12

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

Question 13

FISH - to detect

Answer 13

To detect large chromosomal abnormalities

Extra chromosomes

Large deleted segments

Translocations

Question 14

FISH - define

Answer 14

Fluorescent in situ hybridisation

Uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity

Question 15

FISH - describe method

Answer 15

Design Fluorescent probe to chromosomal region of interest

Denature probe and target DNA

Mix probe and target DNA (hybridisation)

Probe binds to target

Target fluoresces or lights up !

Question 16

Array CGH - define

Answer 16

Array comparative genomic hybridisation

ultra-high resolution way of objectively and quantitatively detecting. whether a patient’s DNA has losses (deletions) or gains (duplications, triplications. etc) which are pathogenic

Question 17

Array CGH - use

Answer 17

For detection of sub-microscopic chromosomal abnormalities

Question 18

Array CGH - results

Answer 18

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 19

MLPA - define

Answer 19

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

Question 20

MLPA - action

Answer 20

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

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

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

Question 21

MLPA - use

Answer 21

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

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

Question 22

MLPA - describe action of the product

Answer 22

Perform fragment analysis (capillary electrophoresis) of MLPA product

Question 23

MLPA - describe involvement in ploidy

Answer 23

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 24

Explain the Current strategy: Disease panels

Answer 24

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

Question 25

Explain reason for using exome sequencing

Answer 25

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

Question 26

Exome Sequencing - briefly describe action

Answer 26

Target enrichment Capture target regions of interest with baits - streptavidin coated

Question 27

Exome Sequencing - capacity for results

Answer 27

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

Question 28

Describe which tests will not automatically move to whole genome sequencing

Answer 28

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 29

Explain the challenge of result interpretation in sequencing

Answer 29

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

Question 30

Explain the ethical considerations in sequencing

Answer 30

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

Question 31

Describe a challenge of genome/exome sequencing

Answer 31

Infrastructure and training (particularly IT and clinical scientists)

Question 32

The NHS Diagnostic Laboratory - main role

Answer 32

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

Question 33

The NHS Diagnostic Laboratory - perform specific tests with proven what

Answer 33

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

Question 34

The NHS Diagnostic Laboratory - list 5 roles

Answer 34

Diagnostic (diagnosis, treatment, pathogenicity) Predictive (life choices) Carrier (recessive) Informed consent (counselling/implications) Diagnostic testing is available for all Consultant referrals

Question 35

Diagnostic Testing - list regulations

Answer 35

All referrals via Regional Genetics Centres Close liaison with nurse specialists, genetic counsellors, clinicians during testing Strict international guidelines for predictive testing Follow up at clinics, nurse led clinics, nurse telephone clinics as required

Question 36

Diagnostic Test Outcomes - list

Answer 36

Pathogenic mutation Normal variation Polymorphism Novel variant Investigations to establish significance

Question 37

How to establish if a mutation is pathogenic?

Answer 37

Mode of inheritance Locus-specific databases of published and unpublished data Nonsense, frameshift, splice site (exon+/-2 bp) mutations Missense/intronic mutation - In-silico tools for missense and splicing mutations

Question 38

Interpreting Results - list regu;ations

Answer 38

Do not report known polymorphisms Conservative approach to reporting novel mutations of uncertain pathogenicity - ‘Uncertain significance' - 'Likely to be pathogenic' Request samples from family members Continue testing other genes

Question 39

100,000 genomes project - define

Answer 39

UK Government project that is sequencing whole genomes from National Health Service patients

Question 40

100,000 genomes project - +ves

Answer 40

100,000 genomes project: 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 Personalised medicine

Question 41

100,000 genomes project - Who/what is being sequenced?

Answer 41

Rare diseases – index cases + families | Cancer – germline and tumour samples

Question 42

Explain how the use of the Genomics England Panel App allows us to focus on specific genes in pt's genome we think are important

Answer 42

Genomics England Panel App ‘Experts’ develop lists of possible genes than can cause a specific disease These panels are reviewed by the community Diseases have specific sets of virtual gene panels as a first port-of-call to look for pathogenic mutations Thus we can focus on specific genes of the patients genome we think are important

Question 43

Compare Tier 1-3 variants

Answer 43

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 44

Describe the formation of the Tier 1-3 variant classification system

Answer 44

Classification of mutations by genomics England Variants within virtual panel divided into three tiers Expert review is required