COLD-PCR and innovative microarray substrates for detecting and genotyping MPL exon 10 W515 substitutions.
BACKGROUND
Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in exon 10 of the MPL (myeloproliferative leukemia virus oncogene) gene, mainly substitutions encoding W515 variants, have recently been described in a minority of patients with ET or PMF. We optimized analytically sensitive methods for detecting and genotyping MPL variants.


METHODS
We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells.


RESULTS
Mutations that were present at very low levels in patients who had previously been scored as having an MPL variant by HRM and as wild type by direct sequencing were successfully identified in granulocyte DNA. Notably, the microarray approach displayed analytical sensitivities of 0.1% to 5% mutant allele, depending on the particular mutation. This analytical sensitivity is similar to that obtained with COLD-PCR. The assay requires no enrichment strategy and allows both the characterization of each variant allele and the evaluation of its proportion in every patient.


CONCLUSIONS
These procedures, which are transferable to clinical diagnostic laboratories, can be used for detecting very low proportions of minority mutant alleles that cannot be identified by other, conventional methods.
San Raffaele Scientific Institute, Genomic Unit for the Diagnosis of Human Pathologies, Center for Translational Genomics and Bioinformatics, Milan, Italy. brisci.angela@hsr.it
Angela
A
Brisci
Francesco
F
Damin
Daniela
D
Pietra
Silvia
S
Galbiati
Sabrina
S
Boggi
Ilaria
I
Casetti
Elisa
E
Rumi
Marcella
M
Chiari
Mario
M
Cazzola
Maurizio
M
Ferrari
Laura
L
Cremonesi
D017931:DNA Primers; D005091:Exons; D005838:Genotype; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D055728:Primary Myelofibrosis / Q000235:genetics; D013920:Thrombocythemia, Essential / Q000235:genetics; D013926:Thrombopoietin / Q000235:genetics*
D017931:DNA Primers; D013926:Thrombopoietin