L6: Genomic Technologies In Clinical Diagnostics Flashcards
What are the genomic technologies?
→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
Why is DNA amplified in PCR?
→so that we have sufficient material for downstream applications
How is fragment analysis carried out?
→PCR based assay
→PCR followed by capillary electrophoresis
→sizing the PCR product
What else can PCR be used for?
→to detect repeat expansions or other small size changes (up to a few hundred bp)
What is an example of a repeat expansion diseases?
→Huntington’s disease – severe neurodegenerative disorder
What is Huttington’s disease caused by?
→Caused by CAG repeat expansion in the Huntingtin (HTT) gene
When are repast expansions pathogenic for Huntington’s?
→Pathogenic > 35 copies
What is normal expansion copies?
→Normal < 27 copies
Why are 35 copies pathogenic in Huntington’s?
→Expanded protein is toxic and accumulates in neurons causing cell death
How is Huntingtion’s diagnosed?
→fragment analysis
What are the characteristics of Sanger Sequencing?
→Slow, low-throughput
→costly to perform for large numbers of samples
What is FISH?
→Fluorescent in situ hybridisation
What size molecules is FISH used for?
→Microscopic (5-10Mb)- large abnormalities
What is FISH used for?
→To detect large chromosomal abnormalities
→Extra chromosomes
→Large deleted segments
→Translocations
Describe the FISH process
→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
What are two types of FISH?
→spectral karyotyping
→target specific FISH
What is spectral karyotyping?
→homologous pairs of chromosomes are manipulated in such a way that they have distinctive colors.
→ detect chromosomal abnormalities
What condition can be detected with FISH?
→Trisomy 21
What is array CGH?
→Array comparative genomic hybridisation
What is a-CGH used for?
→detection of sub-microscopic chromosomal abnormalities
Describe a-CGH process
→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
What is MLPA?
→Multiplex ligation-dependent probe amplification
→a variation of PCR that permits amplification of multiple targets
What can MLPA detect?
→abnormal copy numbers at specific chromosomal locations
→detect sub-microscopic (small) gene deletions/partial gene deletions
Describe the MLPA process
→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
What happens after PCR of probe in MLPA?
→Perform fragment analysis (capillary electrophoresis) of MLPA product
What can MLPA determine?
→relative ploidy (how many chromosome copies) as specific locations
What are the signal strengths of the probes compared with?
→those obtained from a reference DNA sample known to have two copies of the chromosome
How is Sanger sequencing still involved in NGS?
→Potentially pathogenic variants confirmed by Sanger sequencing
Describe exome sequencing
→Target enrichment
→Capture target regions of interest with baits
What is used for cystic fibrosis diagnosed?
→Panels/single gene tests
What are the ethical considerations of exome and genome sequecing?
→Modified patient consent process
→Data analysis pathways – inspect relevant genes first
→Strategy for reporting ‘incidental’ findings
What are the 3 tiers of variants in clinicl interpretation?
→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
What is the difference between clinical validity and clinical utility?
→Clinical Validity: How well the test predicts the phenotype
→Clinical Utility: How the test adds to the management of the patient
What are the three diagnostic test outcomes?
→Pathogenic mutation
→Normal variation
Polymorphism
→Novel variant
How to establish if a mutation is pathogenic?
→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
What does Mitofusin2 cause?
→Charcot-Marie-Tooth disease type 2 (CMT2)
What type of disease is 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
What is MFN2 sequenced by?
→next generation sequencing
