Abstract
Mutations in SF3B1 are found in 20% of myelodysplastic syndromes and 5–10% of myeloproliferative neoplasms, where they are considered important for diagnosis and therapy decisions. Sanger sequencing and NGS are the currently available methods to identify SF3B1 mutations, but both are time-consuming and expensive techniques that are not practicable in most small-/medium-sized laboratories. To identify the most frequent SF3B1 mutation, p.Lys700Glu, we developed a novel fast and cheap assay based on PNA-PCR clamping. After setting the optimal PCR conditions, the limit of detection of PNA-PCR clamping was evaluated, and the method allowed up to 0.1% of mutated SF3B1 to be identified. Successively, PNA-PCR clamping and Sanger sequencing were used to blind test 90 DNA from patients affected by myelodysplastic syndromes and myeloproliferative neoplasms for the SF3B1 p.Lys700Glu mutation. PNA-PCR clamping and Sanger sequencing congruently identified 75 negative and 13 positive patients. Two patients identified as positive by PNA-PCR clamping were missed by Sanger analysis. The discordant samples were analyzed by NGS, which confirmed the PNA-PCR clamping result, indicating that these samples contained the SF3B1 p.Lys700Glu mutation. This approach could easily increase the characterization of myelodysplastic syndromes and myeloproliferative neoplasms in small-/medium-sized laboratories, and guide patients towards more appropriate therapy.
Highlights
Introduction published maps and institutional affilSplicing represents the process by which introns are excised from the precursor messenger RNA and contiguous exons are joined together [1]
After signing the informed consent form, 31 bone marrow (BM) and 59 peripheral blood (PB) samples were collected from 90 patients (57 myelodysplastic syndromes (MDS) and 33 myeloproliferative neoplasms (MPN))
The presence of the p.Lys700Glu mutation favors the bond between the genomic DNA sequence (gDNA) and the primer, which is complementary to the mutated sequence, making mutated gDNA amplification possible (Figure 1)
Summary
Splicing represents the process by which introns are excised from the precursor messenger RNA (pre-mRNA) and contiguous exons are joined together [1]. Genes undergo different splicing processes, generating different mRNA isoforms that lead to the translation of proteins with distinct functions [2]. Splicing is catalyzed by the spliceosome, a macromolecule composed of five small nuclear RNA (snRNA), each associated with proteins to form small nuclear ribonucleoproteins (snRNP). The splicing factor 3b subunit 1 (SF3B1) gene is located on chromosome 2 in position. It encodes subunit 1 of the SF3B splice complex, a 146 kDa protein that is essential for the spliceosome function. Mutations in SF3B1 are common in different hematological malignancies, and they were mostly found in myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). MDS are clonal disorders of hematopoietic stem cells, characterized by ineffective hematopoiesis and the risk of evolving into acute myeloid iations
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