title: Application of COLD-PCR for improved detection of NF2 mosaic mutations.
abstract: Somatic mosaicism represents the coexistence of two or more cell populations with different genotypes in one person, and it is involved in >30 monogenic disorders. Somatic mosaicism characterizes approximately 25% to 33% of patients with de novo neurofibromatosis type 2 (NF2). The identification of mosaicism is crucial to patients and their families because the clinical course of the disease and its transmission risk is influenced by the degree and distribution of mutated cells. Moreover, in NF2, the capability of discriminating patients with mosaicism is especially important to make differential diagnosis with schwannomatosis. However, the identification of mosaic variants is considerably difficult, and the development of specific molecular techniques to detect low levels of unknown molecular alterations is required. Co-amplification at lower denaturation temperature (COLD)-PCR has been described as a powerful method to selectively amplify minority alleles from mixtures of wild-type and mutation-containing sequences. Here, we applied COLD-PCR to molecular analysis of patients with NF2 mosaicism. With the use of COLD-PCR, followed by direct sequencing, we were able to detect NF2 mutations in blood DNA of three patients with NF2 mosaicism. Our study has shown the capability of COLD-PCR in enriching low-represented mutated allele in blood DNA sample, making it usable for molecular diagnosis of patients with mosaicism.
authors.affiliation: Medical Genetic Unit, Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Paganini
authors.affiliation: Clinical Biochemistry Unit, Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Mancini
authors.affiliation: Medical Genetic Unit, Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Baroncelli
authors.affiliation: Medical Genetic Unit, Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy.
authors.forename: Guido
authors.initials: G
authors.lastname: Arena
authors.affiliation: Medical Genetic Unit, Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Gensini
authors.affiliation: Medical Genetic Unit, Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Papi
authors.affiliation: Medical Genetic Unit, Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy. Electronic address: r.sestini@dfc.unifi.it.
authors.forename: Roberta
authors.initials: R
authors.lastname: Sestini
mesh_terms: D003080:Cold Temperature; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D016515:Genes, Neurofibromatosis 2; D006801:Humans; D009030:Mosaicism; D016518:Neurofibromatosis 2 / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D025581:Neurofibromin 2 / Q000097:blood / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D025581:Neurofibromin 2