Overview of Genomic Technologies in Genetic Diagnosis

Question 1

Genomic Technologies in Genetic Diagnosis include…

Answer 1

• PCR
• Fragment Analysis
• Sanger Sequencing
• Fluorescence in situ hybridisation (FISH)
• Array CGH
• Multiplex ligation-dependant probe amplification (MLPA)
• Next-Generation Sequencing

Question 2

What is PCR?

Answer 2

A technique used to amplify a specific region of DNA

Question 3

How does PCR work?

Answer 3

Primers flank the region you want to amplify and DNA Polymerase use that primer to extend nucleotides.

Each cycle doubles the amount of DNA copies of your target sequence

Question 4

What is fragment analysis?

Answer 4

Fragment analysis is a technique to separate the DNA fragments produced in PCR by capillary electrophoresis

-sizing the PCR product

Question 5

Uses of fragment analysis

Answer 5

Used to detect:

• repeat expansions
• other small size changes (up to a few hundred base pairs)

Question 6

Example of a repeat expansion disease

Answer 6

Huntington’s disease

Question 7

What is Huntington’s disease and what is it caused by?

Answer 7

A severe neurodegenerative disorder
-caused by CAG repeat expansion in the Huntington (HTT) gene, resulting in an expanded protein toxic to neurones causing cell death

Normal (<27 copies)
Intermediate (26-35 copies)
Pathogenic (>35 copies)

The more repeats you have, the more severe the disease

Question 8

How is Huntington’s disease diagnosed?

Answer 8

Fragment analysis

Question 9

What is Sanger Sequencing?

Answer 9

Target DNA is copied many times, making fragments of different lengths (same principles as PCR)

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

Question 10

Features of sanger sequencing

Answer 10

Sequences up to 800bp of sequence per reaction (good for sequencing single exons of genes)

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

Question 11

What can Sanger Sequencing be used for?

Answer 11

Identification of single nucleotide polymorphisms (SNPs) or mutations

Question 12

Fluorescence in situ hybridization (FISH)

Answer 12

A fluorescence-labelled probe detects specific nucleotide sequences within cultured cells on a microscopic slide (in metaphase spread).

1) Design fluorescent probe to the chromosomal region of interest
2) Denature probe and target DNA
3) Mix probe and target DNA (hybridisation)
4) Target fluoresces and lights up
5) Can observe it under a microscope

Question 13

Uses of FISH

Answer 13

Used to detect large chromosomal abnormalities:

• extra chromosomes (e.g. trisomy 21)
• large deleted segments
• translocations

Question 14

Array Comparative Genomic Hybridization (CGH)

Answer 14

Microarray technique used to identify copy number variations (CNVs)- duplications & deletions

Patient DNA labelled green
Control DNA labelled red

If there is equal hybridisation between patient and control, the green and red cancel each other out and we get no signal.

Green signal= more patient DNA
Red signal= loss of patient DNA

Question 15

Multiplex ligation-dependent probe amplification (MLPA)

Answer 15

Variation of PCR which permits amplification of multiple DNA targets using a single probe which consists of two oligonucleotides

oligonucleotides recognise adjacent target sites on the DNA

Question 16

Use of MLPA

Answer 16

To detect abnormal copy numbers at specific chromosomal locations
-relative ploidy (how many chromosome copies at specific locations)
To detect sub-microscopic gene deletions/partial gene deletions

Question 17

How does MLPA work?

Answer 17

Denaturation of dsDNA

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

Only when both probe nucleotides are hybridised to their respective targets, can they be ligated into a complete probe.

Then we can perform an amplification step.

Then you perform fragment analysis (capillary electrophoresis) of MLPA product, and 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 18

Next Generation Sequencing

Features of NGS

Answer 18

group of automated techniques used for rapid DNA sequencing
-replaced Sanger sequencing for almost all sequencing tests in the lab

Wider range of tests in a shorter time for less money

Question 19

Uses of Next Generation Sequencing

Answer 19

Disease Panel Test

• 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 are confirmed by Sanger sequencing

Question 20

Exome sequencing (NGS)

Answer 20

A strategy of sequencing only the coding regions of a genome

More efficient to only sequence exome because ~80% of pathogenic mutations are protein coding

Costs £1000 for a genome, but only £200-£300 for an exome

Question 21

How does exome sequencing work?

Answer 21

Target Enrichment

1) We make NGS library and hybridise it with baits which are RNA sequences complimentary to the gene exons
2) In the hybridisation reactions, any sequences which are complementary will bind to one another
3) Baits with biotin tags on them can capture these specific DNA sequences with streptavidin coated magnetic beads
4) Using a magnet, we can pull out those specific fragments
5) We can then wash away the genomic DNA we don’t want, resulting in an enriched library of our exons

Question 22

Whole Genome Sequencing (WGS)- NGS

Answer 22

Universally accepted that genome sequencing will become commonplace in diagnostic genetics

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 23

Disadvantage of NGS

Answer 23

Result interpretation of clinical genomes (exome and whole) currently has a substantial manual component:

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

Infrastructure and training needed (particularly IT and clinical scientists)

Whole genome sequencing is NOT trivial.

Question 24

Ethical considerations for NGS (exome and genome sequencing)

Answer 24

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

Question 25

What is the main role of the lab?

Answer 25

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 26

Possible diagnostic test outcomes

Answer 26

Pathogenic Mutation

Normal Variation (e.g. polymorphism)

Novel Variant
-further investigations needed to establish significance

Question 27

How do we establish if a mutation is pathogenic?

Answer 27

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

Question 28

Reporting genetic results in the lab

Answer 28

A genetic lab would not report known polymorphisms. They would only report things where they have definitely found a mutation.

Conservative approach to reporting novel mutations of uncertain pathogenicity:

• ‘Uncertain significance’
• ‘Likely to be pathogenic’

Request samples from family members

Continue testing other genes