The P420-B1 MPN mix 1 contains eight MLPA probes allowing the detection diagnostically relevant mutations frequently detected in MPN samples. A minimum of 10-20% allelic burden in the patient sample is required for detection of these mutations. For lower allelic burden detection of these mutations P520-A2 MPN mix 2 is available, containing the same mutation-specific probes as the P420-B1 probemix. P520-A2 allows higher detection sensitivity for point mutations, as low as 1% allelic burden, for reliable detection of these eight specific mutations.
For data binning in the fragment analysis procedure, and as an artificial positive control for all the mutation-specific probes, an artificial DNA sample (SD069) is supplied with each P420-B1 order.
This SALSA® MLPA® probemix is intended only for research purposes (RUO).
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 detection of a clonal marker e.g. JAK2 V617F mutation in exon 14 or a JAK2 exon 12 mutation. The V617F point mutation is detected in ~97% of PV patients, and in ~60% of patients with ET and PMF, whereas JAK2 exon 12 mutations are commonly found in V617F negative PV patients. P420-B1 MPN mix 1 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. P420-B1 MPN mix 1 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 (19p13.2) mutations in ET and PMF provide 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 >97% of patients with systemic mastocytosis (SM) (Erben P et al. 2014, Ann Hematol. 93:81-8). Consequently, the presence of this mutation is considered a diagnostic criterion of SM according to the WHO classification.
Finally, 17 reference probes have been included in P420-B1 probemix, detecting different autosomal chromosomal locations which are relatively stable in MPNs.
SD069 Sample DNA
This SD069 sample DNA is provided with each probemix vial and can be used in data binning in the fragment analysis and as a positive control for the mutation-specific probes (see product description).
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 P420 MLPA probemix is negative for a patient sample, we recommend first to repeat analysis with P520 probemix, and then if still negative to perform sequencing of the JAK2, MPL and CALR genes to detect the rare or unknown mutations possibly present in a patient sample.
Sample DNA developed for this product: