Detection of <em>SF3B1</em> p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms
Mutations in <i>SF3B1</i> 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 <i>SF3B1</i> m...
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MDPI AG
2022-02-01
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| Series: | Journal of Clinical Medicine |
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| Online Access: | https://www.mdpi.com/2077-0383/11/5/1267 |
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| author | Jessica Petiti Federico Itri Elisabetta Signorino Antonio Frolli Carmen Fava Marco Armenio Silvia Marini Emilia Giugliano Marco Lo Iacono Giuseppe Saglio Daniela Cilloni |
| author_facet | Jessica Petiti Federico Itri Elisabetta Signorino Antonio Frolli Carmen Fava Marco Armenio Silvia Marini Emilia Giugliano Marco Lo Iacono Giuseppe Saglio Daniela Cilloni |
| author_sort | Jessica Petiti |
| collection | DOAJ |
| description | Mutations in <i>SF3B1</i> 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 <i>SF3B1</i> mutations, but both are time-consuming and expensive techniques that are not practicable in most small-/medium-sized laboratories. To identify the most frequent <i>SF3B1</i> 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 <i>SF3B1</i> 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 <i>SF3B1</i> 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 <i>SF3B1</i> 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. |
| first_indexed | 2024-03-09T20:35:11Z |
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| issn | 2077-0383 |
| language | English |
| last_indexed | 2024-03-09T20:35:11Z |
| publishDate | 2022-02-01 |
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| series | Journal of Clinical Medicine |
| spelling | doaj.art-a16cd92e2df143c5bf534c4f5b9b54fc2023-11-23T23:13:20ZengMDPI AGJournal of Clinical Medicine2077-03832022-02-01115126710.3390/jcm11051267Detection of <em>SF3B1</em> p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative NeoplasmsJessica Petiti0Federico Itri1Elisabetta Signorino2Antonio Frolli3Carmen Fava4Marco Armenio5Silvia Marini6Emilia Giugliano7Marco Lo Iacono8Giuseppe Saglio9Daniela Cilloni10Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, ItalyDepartment of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, ItalyDepartment of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, ItalyDepartment of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, ItalyDepartment of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, ItalyDepartment of Molecular Biotechnology and Health Sciences, University of Turin, 10126 Turin, ItalyDivision of Internal Medicine and Hematology, San Luigi Gonzaga Hospital, 10043 Orbassano, ItalyDivision of Internal Medicine and Hematology, San Luigi Gonzaga Hospital, 10043 Orbassano, ItalyDepartment of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, ItalyDepartment of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, ItalyDepartment of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, ItalyMutations in <i>SF3B1</i> 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 <i>SF3B1</i> mutations, but both are time-consuming and expensive techniques that are not practicable in most small-/medium-sized laboratories. To identify the most frequent <i>SF3B1</i> 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 <i>SF3B1</i> 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 <i>SF3B1</i> 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 <i>SF3B1</i> 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.https://www.mdpi.com/2077-0383/11/5/1267<i>SF3B1</i> p.Lys700GluMDSMPNPNA-PCR clamping |
| spellingShingle | Jessica Petiti Federico Itri Elisabetta Signorino Antonio Frolli Carmen Fava Marco Armenio Silvia Marini Emilia Giugliano Marco Lo Iacono Giuseppe Saglio Daniela Cilloni Detection of <em>SF3B1</em> p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms Journal of Clinical Medicine <i>SF3B1</i> p.Lys700Glu MDS MPN PNA-PCR clamping |
| title | Detection of <em>SF3B1</em> p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms |
| title_full | Detection of <em>SF3B1</em> p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms |
| title_fullStr | Detection of <em>SF3B1</em> p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms |
| title_full_unstemmed | Detection of <em>SF3B1</em> p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms |
| title_short | Detection of <em>SF3B1</em> p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms |
| title_sort | detection of em sf3b1 em p lys700glu mutation by pna pcr clamping in myelodysplastic syndromes and myeloproliferative neoplasms |
| topic | <i>SF3B1</i> p.Lys700Glu MDS MPN PNA-PCR clamping |
| url | https://www.mdpi.com/2077-0383/11/5/1267 |
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