Clinical Molecular Testing for Genetic Conditions Flashcards
(44 cards)
1
Q
primary types of molecular testing
A
- genotyping (targeted variant testing)
- sequencing of entire genes
- deletion/duplication analysis of genes
- genome-scale sequencing
- DNA/protein analysis
2
Q
silent mutations
A
- results in codon with the same protein code
3
Q
missense mutation
A
- results in codon with different protein code
4
Q
nonsense mutation
A
- inserts stop codon
5
Q
frameshift deltion
A
- deletion/insertion that takes you out of frame
- when you go out of frame, you typically run into a stop codon at some point
- truncates message
6
Q
robust PCR
A
- only a small amount of DNA requires
- from a single cel you can amplify billions of copies of the region of interest
7
Q
how PCR works
A
- double stranded DNA is separated
- forward and reverse primers anneal to homologous DNA
- polymerase adds dNTPs
8
Q
molecular genetic testing on cystic fibrosis
A
- carrier testing
- prenatal testing
- neonatal screening
- diagnosis
9
Q
cystic fibrosis genetics
A
- autosomal recessive disease
- common among caucasians of Northern European ancestry
- Ashkenazi Jews
10
Q
cause of cystic fibrosis
A
- mutation in CTFR gene
- over 1000 different mutations identified
11
Q
cystic fibrosis results
A
- defective chloride conductive results in thick mucus buildup In lungs,
- GI tract
- increase salt in sweat
12
Q
ACOG/ACMG recommendations for cystic fibrosis testing
A
- recommend mutation panel
13
Q
mutation panel
A
- includes 23 mutations in CTFR gene
14
Q
when is the CTFR mutation panel recommended
A
- CF carrier screening in women
- if positive, test partner
15
Q
why not sequence entire CTFR gene?
A
- cost/time
- interpretation of clinical significance of sequence variants
16
Q
interpretation of clinical significance of sequence variants
A
- novel sequence variants
- variants of unknown significance
- not all CTFR mutations are associated with classical cystic fibrosis
- some mutations are associated with milder symptoms, atypical pancreatitis, or isolated CBAVD
17
Q
hereditary breast and ovarian cancer syndrome genetics
A
- autosomal dominant
- incomplete penetrance
18
Q
hereditary breast and ovarian cancer syndrome risk
A
- hereditary mutations in BRCA1 and BRCA 2 gene
- risk of familial breast cancer (>50% risk)
- risk of ovarian cancer (20-40% risk)
19
Q
incomplete penetrance
A
- not everyone who has a deleterious variant manifests disease
20
Q
what reduces hereditary breast and ovarian cancer syndrome
A
- prophylactic bilateral mastectomy (by 90%)
- oophorectomy (by 50%)
21
Q
hereditary break and ovarian cancer syndrome sequencing
A
- need to sequence entire coding regions of these two genes to identify disease causing pathogenic mutations in presenting family member
- subsequent testing may be targeted to a specific variant identified in the family
22
Q
Sanger/Dideoxy sequencing
A
- PCR product is the template for the sequencing reaction
- a fraction of dNTPs are fluorescently labelled ddNTPs, causing chain termination when incorporated
- products are generated that are terminated at every nucleotide position
- products are run on capillary gel electrophoresis instrument
- software converts fluorescent signal to peaks for comparison to reference sequence
23
Q
benefits of Sanger sequencing
A
- allows examination of DNA nucleotide sequence up to 500 bases
- detects single base changes as well as insertions and deletions
24
Q
targeted analysis
A
- mutations are known
- few mutations are associated with disease
- can test for individual mutations or sequence selected exons
- faster, more economical
- interpretation is more clear-cut
25
gene sequencing
- mutations are unknown
- mutations occur throughout the gene
- targeted mutation analysis is negative
- more expensive
- interpretation can be more complicated
26
when to use PCR based methods
- single nucleotide variants
| - small insertions and deletions
27
when to use deletion/duplication analysis
- larger indels
| - whole or partial gene deletions/duplications
28
when to use cytogenetic karyotyping, FISH
- genomic rearrangements
29
clinical applications of massively parallel sequencing
- genetic panels for specific conditions with multiple genes involved
- whole exam sequencing
- whole genome sequencing
30
genetic panels for specific conditions with multiple genes involved
- development delay
- hearing loss
- hereditary cancer syndromes
- somatic mutations in tumors
31
whole exome sequencing
- sequencing of the coding region of all known human genes
32
whole genome sequencing
- sequencing of the entire human genome
33
variants of uncertain significance
- rare variants are common
- just because we can detect a variant, doesn't mean we know what it means
- most variants have little to no clinical effect
34
5-class scale for pathogenicity
- benign
- likely benign
- variant of uncertain significance
- likely pathogenic
- pathogenic
35
benign until proven otherwise
- do not institute medical management
- testing of family members not recommended
- unless this would help with interpretation
36
likely pathogenic/pathogenic
- provided diagnosis and/or indication for medical management
- targeted testing of family members can be performed
37
what to consider when ordering molecular testing
- why the test is being done
- the best method for testing and limitations of available methods
- discuss testing with geneticist or clinical lab director
38
genetic linkage
- violation of Mendel's law of independent assortment when genetic loci are in close proximity
39
linkage disequilibrium
- refers to non-random association of alleles within a population
40
clinical utility of linkage
- use of segregation analysis to localize genes for condition
- genetic testing for tightly-linked variants can be used when causative variant is not known
- most variants identified by genome-wide association studies are not causative but are liked to causative alleles
41
electrophoresis and blotting
- applies a charge difference to induce movement through gel
- move by size
- smaller faster
- proteins denatured to give more charge to mass ratio
- blotting involves transfer from cell to more stable membrane, then use of targeted probe to visualize certain molecules
42
southern blot
- DNA probe to find DNA in sample
43
northern blot
- RNA probe to find RNA in sample
44
western blot
- antibody to find protein in sample
