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SALSA MLPA Probemix P520 MPN mix 2

Myeloproliferative neoplasms

Region: JAK2 9p24.1; MPL 1p34.2; CALR 19p13.13; KIT 4q12

General information
The SALSA MLPA Probemix P520 MPN mix 2 is a research use only (RUO) assay for detection of eight different mutations frequently found in MPNs in JAK2, MPL, CALR and KIT genes.

MPNs are clonal hematopoietic stem cell malignancies, characterized by excessive production of blood cells. MPNs are subdivided in polycytemia vera (PV), essential thrombocytemia (ET), primary myelofibrosis (PMF) and less common conditions like chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia (CEL), hypereosinophilic syndrome (HES) and mastocytosis.

Discovery of a frequent JAK2 mutation (9p24.1), common to classic MPNs (PV, ET and PMF), has linked these diseases on a molecular level. The current WHO diagnostic criteria for classic MPNs include presence of JAK2, CALR or MPL mutations. The JAK2 V617F point mutation is detected in ~98% of PV patients, and in ~60% of patients with ET and PMF, whereas other JAK2 exon 12 mutations are commonly found in V617F negative PV patients. P520-A2 MPN mix 2 contains three mutation-specific JAK2 probes: one probe for V617F and two probes for the most common exon 12 mutations N542_E543del and E543_D544del.

Mutations in the MPL gene (1p34.2) are found in 4-11% of JAK2 V617F negative ET and PMF patients. This problemix contains two mutation-specific probes for MPL, W515K and W515L, that are diagnostically relevant in PV, ET and PMF according to the WHO classification.

The discovery of novel CALR gene (19p13.13) mutations in ET and PMF provides additional diagnostic tools for MPNs. Patients with ET and PMF but negative for JAK2 and MPL mutations, have been reported to harbour somatic insertions and deletions in exon 9 of the CALR gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2) are the most common mutations found in the CALR gene (53% and 32%, respectively). These mutations result in a frameshift to an alternative reading frame (Klampfl et al. 2013, Nangalia et al. 2013). CALR mutation-specific probes for the 52-bp deletion (L367fs*46, type 1) and 5-bp insertion (K385fs*47, type 2) are included in this probemix.

In addition, a probe specific for the D816V mutation in the KIT gene (4q12) is present. This is the most common KIT mutation and is present in >90% of patients with systemic mastocytosis (SM). Consequently, the presence of this mutation is considered a diagnostic criterion of SM according to the WHO classification.

This SALSA MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.

Probemix content
The SALSA MLPA Probemix P520-A2 MPN mix 2 contains 25 MLPA probes with amplification products between 115 and 338 nucleotides (nt). This probemix contains 8 probes specific for the JAK2, MPL, CALR and KIT mutations which will only generate a signal when the mutation is present (see details of these mutations detected below).

JAK2 p.V617F = c.1849G>T
JAK2 p.N542_E543del = c.1624_1629delAATGAA
JAK2 p.E543_D544del = c.1627_1632delGAAGAT
MPL p.W515K = c.1543_1544TG>AA
MPL p.W515L = c.1544G>T
CALR p.L367fs*46 = c.1092_1143del52
CALR p.K385fs*47 = c.1154_1155insTTGTC
KIT p.D816V = c.2447A>T

This probemix is modified to allow higher detection sensitivity for mutations. Only >1% allele burden is needed for reliable detection of the above mentioned mutations.

Finally, 17 reference probes are included in P520-A2 probemix, detecting different autosomal chromosomal locations which are relatively stable in MPNs. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com).

In mutation-negative samples, only the 17 reference probes are expected to generate a signal. The remaining eight probes will only generate a signal when the corresponding mutation is present in the test sample. Please note that, however, mutation-specific probes can generate a low background signal in normal samples.

This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mrcholland.com, and specifically for this probemix in Figure 1 of this product description.

SALSA Binning DNA SD057
The SD057 Binning DNA provided with this probemix can aid the binning and serve as a mutation calling threshold sample for the eight mutation-specific probes: CALR L367fs*46 (S0999-L26702; 124 nt), CALR K385fs*47 (S1001-L26517; 130 nt), JAK2 N542_E543del (16924-L21237; 167 nt), JAK2 E543_D544del (16924-L21238; 172 nt), MPL W515K (S1048-SP0405-L29870; 181 nt), MPL W515L (S1048-SP0405-L29871; 186 nt), KIT D816V (17722-SP0542-L23707; 200 nt) and JAK2 V617F (13190-L21572; 240 nt).

SD057 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe.

NOTE that this SD057 sample DNA contains an estimated 1% burden of the above mentioned mutation-specific sequences.

Inclusion of one reaction with 5 μl SD057 (20 ng/μl) Binning DNA with total of 100 ng of DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software and as an artificial positive control for the specific point mutations. For reliable threshold determination and subsequently mutation calling, it is highly recommended to include three reactions of SD057 in each MLPA experiment (please see Interpretation of results for mutation calls section for more information).

Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD057 Binning DNA product description, available online: www.mrcholland.com.

This product is for research use only (RUO).

Sample DNA
Sample DNA developed for this product:

Order Items

Probemix

Item no.
Description
Technology
Price
P520-025R
SALSA MLPA Probemix P520 MPN mix 2 – 25 rxn
€ 281.00
P520-050R
SALSA MLPA Probemix P520 MPN mix 2 – 50 rxn
€ 550.00
P520-100R
SALSA MLPA Probemix P520 MPN mix 2 – 100 rxn
€ 1075.00

Required Reagents (Sold Separately)

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA Reagent Kit – 100 rxn – FAM
€ 341.00
EK1-Cy5
SALSA MLPA Reagent Kit – 100 rxn – Cy5
€ 341.00
EK5-FAM
SALSA MLPA Reagent Kit – 500 rxn – FAM
€ 1571.00
EK5-Cy5
SALSA MLPA Reagent Kit – 500 rxn – Cy5
€ 1571.00
EK20-FAM
SALSA MLPA Reagent Kit – 2000 rxn – FAM
€ 6037.00

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