The SALSA MLPA
Probemix P420 MPN mix 1 is a research use only (RUO)
assay for detection of eight different mutations frequently found in MPNs in the JAK2, MPL, CALR
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 systemic mastocytosis (SM).
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
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. P420-B1 MPN mix 1 probemix 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
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 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.
The SALSA MLPA Probemix P420-B1 MPN mix 1 contains 25 MLPA probes with amplification products between 115 and 338 nucleotides (nt). This includes eight mutation-specific probes for the following mutations:
p.V617F = c.1849G>T
p.E543_D544del = c.1627_1632delGAAGAT
p.N542_E543del = c.1624_1629delAATGAA
p.W515L = c.1544G>T
p.W515K = c.1543_1544TG>AA
p.D816V = c.2447A>T
p.L367fs*46 = c.1092_1143del52
p.K385fs*47 = c.1154_1155insTTGTC
These above mentioned eight probes will only generate a signal when the specific mutation is present. In addition, 17 reference probes are included that target relatively copy number stable regions in various cancer types including MPNs. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com
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
SALSA Binning DNA SD069
The SD069 Binning DNA provided with this probemix can be used for binning of all probes including the following 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). SD069 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains target sequences detected by the above mentioned probes. Inclusion of one reaction with 5 μl SD069 Binning 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. 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). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signal(s). 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 SD069 Binning DNA product description, available online: www.mrcholland.com
This product is for research use only (RUO).
Sample DNA developed for this product: