L6: Genomic Technologies In Clinical Diagnostics

Question 1

What are the 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

Why is DNA amplified in PCR?

Answer 2

→so that we have sufficient material for downstream applications

Question 3

How is fragment analysis carried out?

Answer 3

→PCR based assay

→PCR followed by capillary electrophoresis

→sizing the PCR product

Question 4

What else can PCR be used for?

Answer 4

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

Question 5

What is an example of a repeat expansion diseases?

Answer 5

→Huntington’s disease – severe neurodegenerative disorder

Question 6

What is Huttington’s disease caused by?

Answer 6

→Caused by CAG repeat expansion in the Huntingtin (HTT) gene

Question 7

When are repast expansions pathogenic for Huntington’s?

Answer 7

→Pathogenic > 35 copies

Question 8

What is normal expansion copies?

Answer 8

→Normal < 27 copies

Question 9

Why are 35 copies pathogenic in Huntington’s?

Answer 9

→Expanded protein is toxic and accumulates in neurons causing cell death

Question 10

How is Huntingtion’s diagnosed?

Answer 10

→fragment analysis

Question 11

What are the characteristics of Sanger Sequencing?

Answer 11

→Slow, low-throughput

→costly to perform for large numbers of samples

Question 12

What is FISH?

Answer 12

→Fluorescent in situ hybridisation

Question 13

What size molecules is FISH used for?

Answer 13

→Microscopic (5-10Mb)- large abnormalities

Question 14

What is FISH used for?

Answer 14

→To detect large chromosomal abnormalities

→Extra chromosomes

→Large deleted segments

→Translocations

Question 15

Describe the FISH process

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

What are two types of FISH?

Answer 16

→spectral karyotyping

→target specific FISH

Question 17

What is spectral karyotyping?

Answer 17

→homologous pairs of chromosomes are manipulated in such a way that they have distinctive colors.
→ detect chromosomal abnormalities

Question 18

What condition can be detected with FISH?

Answer 18

→Trisomy 21

Question 19

What is array CGH?

Answer 19

→Array comparative genomic hybridisation

Question 20

What is a-CGH used for?

Answer 20

→detection of sub-microscopic chromosomal abnormalities

Question 21

Describe a-CGH process

Answer 21

→patient and control DNA are labelled with fluorescent dye and applied to the microarray
→patient and control DNA compete to hybridise to the microarray
→microarray scanner measures the fluorescent signals
→software analyses data

Question 22

What is MLPA?

Answer 22

→Multiplex ligation-dependent probe amplification

→a variation of PCR that permits amplification of multiple targets

Question 23

What can MLPA detect?

Answer 23

→abnormal copy numbers at specific chromosomal locations

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

Question 24

Describe the MLPA process

Answer 24

→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

→PCR amplification of probe

Question 25

What happens after PCR of probe in MLPA?

Answer 25

→Perform fragment analysis (capillary electrophoresis) of MLPA product

Question 26

What can MLPA determine?

Answer 26

→relative ploidy (how many chromosome copies) as specific locations

Question 27

What are the signal strengths of the probes compared with?

Answer 27

→those obtained from a reference DNA sample known to have two copies of the chromosome

Question 28

How is Sanger sequencing still involved in NGS?

Answer 28

→Potentially pathogenic variants confirmed by Sanger sequencing

Question 29

Describe exome sequencing

Answer 29

→Target enrichment

→Capture target regions of interest with baits

Question 30

What is used for cystic fibrosis diagnosed?

Answer 30

→Panels/single gene tests

Question 31

What are the ethical considerations of exome and genome sequecing?

Answer 31

→Modified patient consent process

→Data analysis pathways – inspect relevant genes first

→Strategy for reporting ‘incidental’ findings

Question 32

What are the 3 tiers of variants in clinicl interpretation?

Answer 32

→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 33

What is the difference between clinical validity and clinical utility?

Answer 33

→Clinical Validity: How well the test predicts the phenotype

→Clinical Utility: How the test adds to the management of the patient

Question 34

What are the three diagnostic test outcomes?

Answer 34

→Pathogenic mutation

→Normal variation
Polymorphism

→Novel variant

Question 35

How to establish if a mutation is pathogenic?

Answer 35

→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

Question 36

What does Mitofusin2 cause?

Answer 36

→Charcot-Marie-Tooth disease type 2 (CMT2)

Question 37

What type of disease is Charcot-Marie-Tooth disease type 2 (CMT2)?

Answer 37

→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

Question 38

What is MFN2 sequenced by?

Answer 38

→next generation sequencing