The P520-A2 MPN mix 2 is an MLPA probemix that allows higher detection sensitivity for mutations. Only 1% allele burden is required for detection of the eight different mutations frequently found in MPN samples.
With each P520 probemix order, an artificial DNA sample (SD057) with an estimated 1% allele burden is supplied for data binning in the fragment analysis procedure, and as an artificial positive control for all the mutation-specific probes. This SD057 should be used in each P520 experiment for correct data binning and mutation calling.
Please note that this probemix should be used only as a qualitative tool (mutation present or absent) and NOT as a quantitative tool for measuring exact mutation burden. In addition, internal or external reference standards should be included both in the validation phase and in experiments.
This SALSA® MLPA® probemix is intended for research use only (RUO) purposes.
Note 1: The use of this product requires two modifications as compared to the General MLPA Protocol. First, the PCR reaction should be prolonged from 35 to 40 cycles. Second, we strongly recommend to use ≥100 ng of patient DNA per MLPA reaction (maximum 400 ng of DNA) for most optimal and robust mutation detection with the P520 probemix. Customers are strongly advised not to make any additional modifications to the MLPA protocol, as this could potentially lead to false positive or false negative results for the mutation calling.
Note 2: Probemix P420 MPN mix 1 can be used for patient samples with a >10% allele burden, which will generate a saturated mutation-specific signal when using P520.
Myeloproliferative neoplasms (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. P520-A2 MPN mix 2 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 T et al. 2013, N Engl J Med. 369:2379-90; Nangalia J et al. 2013, N Engl J Med. 369:2391-405). 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.
Finally, 17 reference probes are included in the P520-A2 probemix, detecting different autosomal chromosomal locations which are relatively stable in MPNs. Please note that the probe signals of the reference probes in this P520 probemix show much greater variation as compared to standard MLPA products. This is due to the nature of the high sensitivity of this assay.
This SALSA® MLPA® probemix is designed to detect the presence of the aforementioned mutations in a DNA sample. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals can be more sensitive to sample purity and small changes in experimental conditions.
This MLPA probemix cannot detect any mutations that are outside the target sequences of the MLPA probes. Neither can it be used for copy number detection of the aforementioned target genes. Even when MLPA analysis did not detect any aberrations, the possibility remains that changes in the corresponding gene(s) do exist but remain undetected. This probemix covers only the most frequent mutations of clinical and diagnostic relevance in MPNs. Rare or infrequent mutations are not covered. If mutation calling with MLPA is negative for a patient sample, we recommend to perform sequencing of the JAK2, MPL and CALR genes to detect the rare or unknown mutations possibly present in a patient sample.
SD057 Sample DNA
The SD057 Sample DNA, provided with each P520-A2 probemix vial, is an aid for binning of 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; 179 nt), MPL W515L (S1048-SP0405-L29871; 185 nt), KIT D816V (17722-SP0542-L23707; 200 nt) and JAK2 V617F (13190-L21572; 240 nt).
NOTE that this SD057 sample DNA contains an estimated 1% burden of the above mentioned mutation-specific sequences. Inclusion of one reaction with SD057 DNA in MLPA experiments is recommended 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. SD057 DNA consists of female DNA mixed with a plasmid DNA that contains the target sequences detected by the above mentioned probes. MLPA reactions for binning purposes should be performed with 5 μl of SD057 (20 ng/μl) with total of 100 ng of DNA. This product is for research use only (RUO).
Sample DNA developed for this product: