Cargando…

Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Petiti, Jessica, Itri, Federico, Signorino, Elisabetta, Frolli, Antonio, Fava, Carmen, Armenio, Marco, Marini, Silvia, Giugliano, Emilia, Lo Iacono, Marco, Saglio, Giuseppe, Cilloni, Daniela
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8911290/
https://www.ncbi.nlm.nih.gov/pubmed/35268357
http://dx.doi.org/10.3390/jcm11051267
_version_ 1784666759786135552
author Petiti, Jessica
Itri, Federico
Signorino, Elisabetta
Frolli, Antonio
Fava, Carmen
Armenio, Marco
Marini, Silvia
Giugliano, Emilia
Lo Iacono, Marco
Saglio, Giuseppe
Cilloni, Daniela
author_facet Petiti, Jessica
Itri, Federico
Signorino, Elisabetta
Frolli, Antonio
Fava, Carmen
Armenio, Marco
Marini, Silvia
Giugliano, Emilia
Lo Iacono, Marco
Saglio, Giuseppe
Cilloni, Daniela
author_sort Petiti, Jessica
collection PubMed
description 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.
format Online
Article
Text
id pubmed-8911290
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-89112902022-03-11 Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms Petiti, Jessica Itri, Federico Signorino, Elisabetta Frolli, Antonio Fava, Carmen Armenio, Marco Marini, Silvia Giugliano, Emilia Lo Iacono, Marco Saglio, Giuseppe Cilloni, Daniela J Clin Med Article 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. MDPI 2022-02-25 /pmc/articles/PMC8911290/ /pubmed/35268357 http://dx.doi.org/10.3390/jcm11051267 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Petiti, Jessica
Itri, Federico
Signorino, Elisabetta
Frolli, Antonio
Fava, Carmen
Armenio, Marco
Marini, Silvia
Giugliano, Emilia
Lo Iacono, Marco
Saglio, Giuseppe
Cilloni, Daniela
Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms
title Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms
title_full Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms
title_fullStr Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms
title_full_unstemmed Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms
title_short Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms
title_sort detection of sf3b1 p.lys700glu mutation by pna-pcr clamping in myelodysplastic syndromes and myeloproliferative neoplasms
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8911290/
https://www.ncbi.nlm.nih.gov/pubmed/35268357
http://dx.doi.org/10.3390/jcm11051267
work_keys_str_mv AT petitijessica detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT itrifederico detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT signorinoelisabetta detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT frolliantonio detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT favacarmen detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT armeniomarco detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT marinisilvia detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT giuglianoemilia detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT loiaconomarco detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT sagliogiuseppe detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms
AT cillonidaniela detectionofsf3b1plys700glumutationbypnapcrclampinginmyelodysplasticsyndromesandmyeloproliferativeneoplasms