Testing Flashcards
Genes tested for suspected MPN?
JAK2, CALR, MPL
Capillary Electrophoresis
seperates ionic fragments by size, no volume exchanged
CALR Testing
PB, BM
looking for 52 bp deletion or 5 bp insertion in exon 9
Fluorescent PCR
CALR
19p13.2-p13.3
Mutated in ~67% of JAK2/MPL- MPNs
Not seen in polycythemia
Most somatic mutations are frameshift
In-frame deletions are normal SNPS
JAK2
9p24
Linked to anti-apoptotic signaling via BCL2 production in hematopoietic cells
V617F mutation (exon 14) is found in > 95% PV, 40-50% ET, 45-50% CIMF
Exon 10 mutation is clinically relevant but less common
LOH at 1p and 19q
Common in oligodendrogliomas
Emerged as a predictor of chemosensitivity
May be independent of tumor grade
LOH at 17p
More common in astrocytomoas (including some glioblastomas)
LOH at 10q
Frequently seen in high-grade astrocytomas and glioblastoma multiforme
Is an independent, adverse prognostic marker
All or part of chromosome 10 is deleted in ___
30-70% of anaplastic astrocytomas and 60-95% of glioblastomas
Huntington’s
4p16.3, Auto/dom, CAG repeat in exon 1 of the HD gene
10-26 repeats = normal
27-35 = intermediate/mutable
36-39 = reduced penetrance
>40 = HD
HD Testing
PB
Fluorescent PCR using oligonucleotide primers specific for the CAG repeat
Limitations: hairpin loops can form during expansion decreasing peak height. If suspected, examine larger alleles at a lower sensitivity (zoom).
Fragile X
Xq27.3, CGG repeat in the 5’UTR in exon 1 of FMR1 gene and hypermethylation coupled w/histone deacetylation of this region and adjacent CpG island
Results in loss of function in the FMR protein (FMRP), which suppresses translation of certain mRNAs.
2nd most common form of ID
Symptoms are often milder in females b/c of random X inactivation and normal X expression in 1/2 their cells.
5-45 repeats = normal
46-54 = gray zone
55-200 = premutation
> 200 = full mutation expansion
Premutation carriers of Fragile X
Do not have fragile X
Females: ~ 20% have primary ovarian insufficiency w/cessation of menstruation before 40 yo (FXPOF, this does not occur in full mutation females)
Males: ~ 33% exhibit FX-associated tremor/ataxia syndrome (FXTAS)
FXTAS can be observed in females but at a lower %
Fragile X testing
PB, CVS, amniotic fluid
Souther blot of PB can detect majority of FX cases
PCR w/capillary electrophoresis can provide the precise CGG-repeat #, which is useful when determining the risk of affected offspring of a premutation female
AmplideX FMR1 assay by Asuragen uses fluorescent PCR and CE
Complex Diseases
Multifactorial inheritance pattern suggests interaction of 1 or more genes in combination w/lifestyle and one or more environmental factors.
Does not follow a typical Mendelian inheritance pattern.
ex. thrombophilia, hereditary pancreatitis
Thrombophilia
Complex disease
An imbalance in naturally occurring clotting factors. This can increase risk of developing blood clots
FV gene
1q24.2
In the coag pathway, FV is converted to its activated form, FVa by thrombin - peptide bonds are cleaved, inactivating FV and reducing the conversion of prothrombin to thrombin. This shifts the balance of hemostasis to favor coag and increases thrombin production.
Heterozygous FV gene c.1691G>A carriers
Have a lifelong 10-fold increased relative risk of venous thrombosis
Homozygous FV gene c.1691G>A individuals
Have an 80-fold increased relative risk of venous thrombosis
FV Leiden
G to A substitution in the FV protein at codon 506
Common in the white population of N. European descent w/~3-5% frequency
Absent in African & SE Asian populations
Does not confer increased risk for arterial thrombosis
Mean age of onset of symptoms for people w/thrombosis
44 yo for heterozygotes
31 yo for homozygotes
Provoked thromboembolism
One or more predisposing risk factors
Unprovoked thromboembolism
Precipitating cause is undetermined
What can lead to a false negative on a coag test? How can this be avoided?
Patients taking FIIa or FVa oral inhibitors
Avoid by doing DNA testing rather than a functional assay
BRAF testing
PB, BM, DNA from PET
Fluorescent, allele-specific PCR to to detect the presence of mutation c.1799T>A (assoc w/p.V600E) in the BRAF gene
BRAF
7q34
Can be present in malignant melanoma, sporadic colorectal tumors showing mismatch repair defects in microsatellites, low-grade ovarian serous carcinoma, and thyroid papillary cancer
Proto-oncogene in the serine/threonine kinase family & amp; a member of the RAF subfamily together with ARAF and RAF1 genes.
Involved in the transduction of mitogenic signals from the cell membrane to the nucleus via the RAS/RAF/MEK/ERK/MAPK pathway.
~80% of these mutations correspond to the transversion mutation T1799A, which causes aa substitution V600E. The other 20% accounts for an unpredictable range of missense mutations, all of which reside in the glycines of the G-loop in exon 11 or in the activation segment in exon 15 near V600.
FLT3
13q12
Preferentially expressed on hematopoietic stem cells. Its ligand, FL, is preferentially expressed on bone marrow stroma cells and the FLT3-FL interaction plays an important role for survival, proliferation, and differentiation into the B-lymphoid and myeloid lineages in early hematopoiesis.
Duplicated sequences of the juxtamembrane domain-coding sequences in exon 14,
but sometimes involving intron 14 and exon 15, of the gene have been identified in 20-30% of AML patients. These duplicated sequences vary in their location
and length from sample to sample but always result in in-frame to produce an abnml protein.
FLT3 ITD assoc w/unfavorable prognosis and is the strongest prognostic factor for overall survival.
FLT3 testing
BM, PB, DNA
Fluorescent PCR + capillary electrophoresis is used to detect the patient-specific duplication(s) within exons 14-15 in FLT3
Interp based is based on the presence or absence of peaks greater than 330 bp.
This procedure applies to FLT3-ITD testing and cannot exclude the presence of other common FLT3 mutations that could be present in this gene.
SNaPshot (5 steps)
Tests for SNVs in IDH1/2, FLT3, and BRAF
* Multiplex PCR (3 panels)
* PCR cleanup (SAP & Exo1)
* Single-base extension using fluorescent dNTPs
* SAP treatment (2nd cleanup)
* Capillary electrophoresis
LOH Testing
- Glioma test using DNA from both nml & tumor samples on same pt to evaluate for LOH at 1p, 10q, 17p, or 19q
- These regions are amplified with fluorescently tagged microsatellite repeat markers.
- If the patient’s constitutive alleles are hom, the marker isn’t informative, and other loci will be tested to
complete the analysis. Only markers that yield het alleles in the constitutive DNA will be informative. * When LOH is present, the tumor DNA LOH
will lack one of the alleles present in the normal DNA,and therefore will yield a hom pattern (aka LOH).
NPM1
5q35 (exon 12)
Detected via fluorescent PCR
Encodes a multifunctional protein that is involved in DNA repair, modulation of chromatin condensation, response to cellular stress, transport of pre-ribosomal
particles, & regulates activity and stability of several tumor suppressor proteins.
The most frequently altered gene in AML (~35% of all AML patients & ~50-60% of AML patients with nml cyto).
Often confers a favorable response to induction chemo as long as it does not co-occur w/FLT3-ITD
A 4 bp duplication or insertion in exon 12 is most commonly seen in AML.
An insertion if the 4 bps inserted aren’t a perfect match to the preceding 4 bps and is a duplication if the 4 bases are a perfect match to the preceding 4 bases
(majority of mutations). Both result in a frameshift in the C-terminal DNA binding domain of the protein, whereby several amino acid residues critical for nuclear localization are lost and residues required for a c-terminus export signal are gained. This results in the aberrant cytoplasmic localization of the nucleophosmin protein causing increased cell survival & decreased apoptosis.
