Overview of Genomic Technologies in Genetic Diagnostics

Question 1

What is PCR?

Answer 1

Polymerase Chain Reaction (PCR)
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

Question 2

What is fragment analysis?

Answer 2

PCR based assay
PCR followed by capillary electrophoresis, where we are separating out the PCR 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 bp)

Question 3

How is Huntington’s a repeat expansion disease?

Answer 3

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 4

What is sanger sequencing?

Answer 4

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

Question 5

What can FISH be used to detect?

Answer 5

FISH- fluorescent in situ hybridisation
To detect large chromosomal abnormalities
We can look for:
	- Extra chromosomes
	- Large deleted segments
	- Translocations

Question 6

How is FISh carried out?

Answer 6

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 7

What is array CGH?

Answer 7

Array comparative genomic hybridisation
For detection of sub-microscopic chromosomal abnormalities
Patient DNA labelled Green
Control DNA labelled Red
Equal hybridisation means the patient DNA is the same as control
Red means DNA dosage loss
Green means DNA dosage gain

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 8

What is MLPA and how does it occur?

Answer 8

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
Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA
MLPA can detect sub-microscopic (small) gene deletions/partial gene deletions

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 9

What is next generation sequencing?

Answer 9

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

Question 10

What’s the purpose of whole-genome sequencing?

Answer 10

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 11

What are some challenges around exome and genome sequencing?

Answer 11

Result interpretation is the greatest challenge
- 20,000 genetic variants identified per coding genes ‘exome’
- 3 million variants in a whole human genome
Ethical considerations
- Modified patient consent process
- Data analysis pathways – inspect relevant genes first
- Strategy for reporting ‘incidental’ findings
Infrastructure and training (particularly IT and clinical scientists)

Question 12

How do you establish if a mutation is pathogenic?

Answer 12

Mode of inheritance
Genetic 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

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

Question 13

What is MFN2?

Answer 13

Mitofusin 2 (MFN2) causes Charcot-Marie-Tooth disease type 2 (CMT2)
- Degeneration of the long nerves in legs and arms leading to muscle wasting and sensory defects.
- Onset usually in childhood
- Autosomal Dominant and Autosomal Recessive
Two siblings with very severe early-onset CMT2.
Parents unaffected
MFN2 sequenced by next generation sequencing
- Apparently homozygous for c.647T>C p.(Phe216Ser) mutation
- Parents sequenced, expected them to both be heterozygous