Testing

Question 1

Genes tested for suspected MPN?

Answer 1

JAK2, CALR, MPL

Question 2

Capillary Electrophoresis

Answer 2

seperates ionic fragments by size, no volume exchanged

Question 3

CALR Testing

Answer 3

PB, BM
looking for 52 bp deletion or 5 bp insertion in exon 9
Fluorescent PCR

Question 4

CALR

Answer 4

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

Question 5

JAK2

Answer 5

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

Question 6

LOH at 1p and 19q

Answer 6

Common in oligodendrogliomas
Emerged as a predictor of chemosensitivity
May be independent of tumor grade

Question 7

LOH at 17p

Answer 7

More common in astrocytomoas (including some glioblastomas)

Question 8

LOH at 10q

Answer 8

Frequently seen in high-grade astrocytomas and glioblastoma multiforme
Is an independent, adverse prognostic marker

Question 9

All or part of chromosome 10 is deleted in ___

Answer 9

30-70% of anaplastic astrocytomas and 60-95% of glioblastomas

Question 10

Huntington’s

Answer 10

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

Question 11

HD Testing

Answer 11

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).

Question 12

Fragile X

Answer 12

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

Question 13

Premutation carriers of Fragile X

Answer 13

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 %

Question 14

Fragile X testing

Answer 14

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

Question 15

Complex Diseases

Answer 15

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

Question 16

Thrombophilia

Answer 16

Complex disease
An imbalance in naturally occurring clotting factors. This can increase risk of developing blood clots

Question 17

FV gene

Answer 17

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.

Question 18

Heterozygous FV gene c.1691G>A carriers

Answer 18

Have a lifelong 10-fold increased relative risk of venous thrombosis

Question 19

Homozygous FV gene c.1691G>A individuals

Answer 19

Have an 80-fold increased relative risk of venous thrombosis

Question 20

FV Leiden

Answer 20

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

Question 21

Mean age of onset of symptoms for people w/thrombosis

Answer 21

44 yo for heterozygotes
31 yo for homozygotes

Question 22

Provoked thromboembolism

Answer 22

One or more predisposing risk factors

Question 23

Unprovoked thromboembolism

Answer 23

Precipitating cause is undetermined

Question 24

What can lead to a false negative on a coag test? How can this be avoided?

Answer 24

Patients taking FIIa or FVa oral inhibitors
Avoid by doing DNA testing rather than a functional assay

Question 25

BRAF testing

Answer 25

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

Question 26

BRAF

Answer 26

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.

Question 27

FLT3

Answer 27

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.

Question 28

FLT3 testing

Answer 28

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.

Question 29

SNaPshot (5 steps)

Answer 29

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

Question 30

LOH Testing

Answer 30

* 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).

Question 31

NPM1

Answer 31

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.