title: Development of a high-resolution melting curve analysis screening test for SRSF2 splicing factor gene mutations in myelodysplastic syndromes.
abstract: Somatic mutations of the spliceosome machinery have been recently identified by whole genome analysis in hematologic diseases, such as myelodysplastic syndrome, chronic lymphocytic leukemia, myeloproliferative neoplasms, acute myeloid leukemia, and advanced forms of mastocytosis, and also in nonhematologic conditions. SRSF2 is a member of the serine/arginine-rich family pre-mRNA splicing factors that plays a role in mRNA export from the nucleus and translation. We describe a high-resolution melting (HRM) curve analysis to screen for SRSF2 hotspot mutations in a fast, sensitive, and reliable way. Fifty bone marrow samples from patients with myelodysplastic syndrome were analyzed by the HRM assay and by direct sequencing. HRM screening identified four melting patterns corresponding to a negative (wild-type) group and three different mutated groups. Each mutated group was identified according to the positive control used: P95H, P95L, and P95R, respectively. An HRM mutated pattern was identified in seven patients. Positive and negative results from HRM were compared with direct sequencing results with a sensitivity and specificity of 100% (95% CI, 0.56-1, and 95% CI, 0.89-1, respectively). Analytical sensitivity analysis revealed a detection threshold of up to 1:9 (mutated/wild type) dilution. This rapid screening method may provide useful information for clinical decision making and be helpful to optimize laboratory resources and reduce turnaround time.
authors.affiliation: Laboratory of Neuro-Oncoematology, Santa Lucia Foundation, Rome, Italy; Laboratorios Dr. Moreira, Monterrey, Mexico; Department of Biopathology, Tor Vergata University, Rome, Italy.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Garza
authors.affiliation: Department of Hematology, Universita' Cattolica Sacro Cuore, Rome, Italy.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Fabiani
authors.affiliation: Laboratory of Neuro-Oncoematology, Santa Lucia Foundation, Rome, Italy; Department of Biopathology, Tor Vergata University, Rome, Italy; Hematology Department of Chemical Biochemistry, Rosario National University, Rosario, Argentina.
authors.forename: Nelida
authors.initials: N
authors.lastname: Noguera
authors.affiliation: Policlinico Foundation, Tor Vergata, Rome, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Panetta
authors.affiliation: Laboratory of Neuro-Oncoematology, Santa Lucia Foundation, Rome, Italy; Department of Biopathology, Tor Vergata University, Rome, Italy.
authors.forename: Maria L
authors.initials: ML
authors.lastname: Piredda
authors.affiliation: Laboratory of Neuro-Oncoematology, Santa Lucia Foundation, Rome, Italy; Department of Biopathology, Tor Vergata University, Rome, Italy.
authors.forename: Loredana
authors.initials: L
authors.lastname: Borgia
authors.affiliation: Department of Biopathology, Tor Vergata University, Rome, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Maurillo
authors.affiliation: Department of Biopathology, Tor Vergata University, Rome, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Catalano
authors.affiliation: Department of Hematology, Universita' Cattolica Sacro Cuore, Rome, Italy.
authors.forename: Maria T
authors.initials: MT
authors.lastname: Voso
authors.affiliation: Laboratory of Neuro-Oncoematology, Santa Lucia Foundation, Rome, Italy; Department of Biopathology, Tor Vergata University, Rome, Italy. Electronic address: francesco.lo.coco@uniroma2.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Lo-Coco
mesh_terms: D000465:Algorithms; D019943:Amino Acid Substitution; D001853:Bone Marrow / Q000378:metabolism / Q000473:pathology; D006801:Humans; D009154:Mutation; D009190:Myelodysplastic Syndromes / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D000068103:Serine-Arginine Splicing Factors
chemical_list: D009687:Nuclear Proteins; D012261:Ribonucleoproteins; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors