Application of COLD-PCR for Improved Detection of NF2 Mosaic Mutations
2014; Elsevier BV; Volume: 16; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2014.02.007
ISSN1943-7811
AutoresIrene Paganini, Irene Mancini, Marta Baroncelli, Guido Arena, Francesca Gensini, Laura Papi, Roberta Sestini,
Tópico(s)Bone Tumor Diagnosis and Treatments
ResumoSomatic 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. 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. The term mosaicism implies the presence of two or more genetically distinct cell lines in a single organism. Somatic mosaicism has been implicated in >30 monogenic disorders, showing variable expressivity.1Youssoufian H. Pyeritz R.E. Mechanisms and consequences of somatic mosaicism in humans.Nat Rev Genet. 2002; 3: 748-758Crossref PubMed Scopus (294) Google Scholar The frequency of mosaicism might depend on the particular disorder, tissue origin, or selective pressure, and it is a frequent phenomenon in disorders that exhibit a high mutation rate. Ascertainment bias can cause an underestimation of the true frequency of mosaicism; because molecular diagnosis frequently begins by the analysis of blood cell DNA, a low level of mosaicism in these cells often remains undetected. Somatic mosaicism is common in neurofibromatosis type 2 (NF2; MIM 101000).2Kluwe L. Mautner V. Heinrich B. Dezube R. Jacoby L.B. Friedrich R.E. MacCollin M. Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas.J Med Genet. 2003; 40: 109-114Crossref PubMed Scopus (126) Google Scholar, 3Moyhuddin A. Baser M.E. Watson C. Purcell S. Ramsden R.T. Heiberg A. Wallace A.J. Evans D.G. Somatic mosaicism in neurofibromatosis 2: prevalence and risk of disease transmission to offspring.J Med Genet. 2003; 40: 459-463Crossref PubMed Scopus (103) Google Scholar, 4Evans D.G. Ramsden R.T. Shenton A. Gokhale C. Bowers N.L. Huson S.M. Pichert G. Wallace A. Mosaicism in neurofibromatosis type 2: an update of risk based on uni/bilaterality of vestibular schwannoma at presentation and sensitive mutation analysis including multiple ligation-dependent probe amplification.J Med Genet. 2007; 44: 424-428Crossref PubMed Scopus (117) Google Scholar NF2 is an inherited tumor syndrome that predisposes to the development of nervous system tumors.5Evans D.G. Huson S.M. Donnai D. Neary W. Blair V. Teare D. Newton V. Strachan T. Ramsden R. Harris R. A genetic study of type 2 neurofibromatosis in the United Kingdom. I. Prevalence, mutation rate, fitness, and confirmation of maternal transmission effect on severity.J Med Genet. 1992; 29: 841-846Crossref PubMed Scopus (411) Google Scholar Bilateral vestibular schwannomas are pathognomonic of the disorder; furthermore, schwannomas at other locations, meningiomas, ependymomas, and juvenile posterior subcapsular lenticular opacities are often associated with the disease.5Evans D.G. Huson S.M. Donnai D. Neary W. Blair V. Teare D. Newton V. Strachan T. Ramsden R. Harris R. A genetic study of type 2 neurofibromatosis in the United Kingdom. I. Prevalence, mutation rate, fitness, and confirmation of maternal transmission effect on severity.J Med Genet. 1992; 29: 841-846Crossref PubMed Scopus (411) Google Scholar, 6Parry D.M. Eldridge R. Kaiser-Kupfer M.I. Bouzas E.A. Pikus A. Patronas N. Neurofibromatosis 2 (NF2): clinical characteristics of 63 affected individuals and clinical evidence for heterogeneity.Am J Med Genet. 1994; 52: 450-461Crossref PubMed Scopus (336) Google Scholar, 7Mautner V.F. Lindenau M. Baser M.E. Hazim W. Tatagiba M. Haase W. Samii M. Wais R. Pulst S.M. The neuroimaging and clinical spectrum of neurofibromatosis 2.Neurosurgery. 1996; 38: 880-885Crossref PubMed Scopus (237) Google Scholar Approximately one-half of patients with NF2 have no family history for the disease, representing de novo mutations. The disease is caused by alterations in the NF2 tumor-suppressor gene, located in 22q12.8Trofatter J.A. MacCollin M.M. Rutter J.L. Murrell J.R. Duyao M.P. Parry D.M. Eldridge R. Kley N. Menon A.G. Pulaski K. Haase V.H. Ambrose C.M. Munroe D. Bove C. Haines J.L. Martuza R.L. MacDonald M.E. Seizinger B.R. Short M.P. Buckler A.J. Gusella J.F. A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor.Cell. 1993; 72: 791-800Abstract Full Text PDF PubMed Scopus (1095) Google Scholar, 9Rouleau G.A. Merel P. Lutchman M. Sanson M. Zucman J. Marineau C. Hoang-Xuan K. Demczuk S. Desmaze C. Plougastel B. Pulst S.M. Lenoir G. Bijlsma E. Fahsold R. Dumanski J. de Jong P. Parry D. Eldridge R. Aurias A. Delattre O. Thomas G. Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2.Nature. 1993; 363: 515-521Crossref PubMed Scopus (1194) Google Scholar Since its identification, different germline mutations have been found in up to 91% of patients with NF2 with a positive family history, using exon scanning-based methods and multiplex ligation-dependent probe amplification to detect genomic rearrangements.4Evans D.G. Ramsden R.T. Shenton A. Gokhale C. Bowers N.L. Huson S.M. Pichert G. Wallace A. Mosaicism in neurofibromatosis type 2: an update of risk based on uni/bilaterality of vestibular schwannoma at presentation and sensitive mutation analysis including multiple ligation-dependent probe amplification.J Med Genet. 2007; 44: 424-428Crossref PubMed Scopus (117) Google Scholar, 10Kluwe L. Nygren A.O. Errami A. Heinrich B. Matthies C. Tatagiba M. Mautner V. Screening for large mutations of the NF2 gene.Genes Chromosomes Cancer. 2005; 42: 384-391Crossref PubMed Scopus (46) Google Scholar Somatic mosaicism has been confirmed in 25% to 33% of patients with de novo NF2,2Kluwe L. Mautner V. Heinrich B. Dezube R. Jacoby L.B. Friedrich R.E. MacCollin M. Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas.J Med Genet. 2003; 40: 109-114Crossref PubMed Scopus (126) Google Scholar, 3Moyhuddin A. Baser M.E. Watson C. Purcell S. Ramsden R.T. Heiberg A. Wallace A.J. Evans D.G. Somatic mosaicism in neurofibromatosis 2: prevalence and risk of disease transmission to offspring.J Med Genet. 2003; 40: 459-463Crossref PubMed Scopus (103) Google Scholar, 4Evans D.G. Ramsden R.T. Shenton A. Gokhale C. Bowers N.L. Huson S.M. Pichert G. Wallace A. Mosaicism in neurofibromatosis type 2: an update of risk based on uni/bilaterality of vestibular schwannoma at presentation and sensitive mutation analysis including multiple ligation-dependent probe amplification.J Med Genet. 2007; 44: 424-428Crossref PubMed Scopus (117) Google Scholar and it may be even more common in NF2 sporadic patients with mild phenotypes.4Evans D.G. Ramsden R.T. Shenton A. Gokhale C. Bowers N.L. Huson S.M. Pichert G. Wallace A. Mosaicism in neurofibromatosis type 2: an update of risk based on uni/bilaterality of vestibular schwannoma at presentation and sensitive mutation analysis including multiple ligation-dependent probe amplification.J Med Genet. 2007; 44: 424-428Crossref PubMed Scopus (117) Google Scholar The identification of mosaicism in patients with NF2 is clinically relevant and has several important implications for genetic counseling. In addition, the identification of mosaic mutations is crucial to patients and their families because the degree and distribution of mutated cells can influence both the clinical course of the disease and its transmission risk. However, the identification of mosaic variants is considerably difficult, and there is a high need to develop specific molecular techniques to detect low levels of unknown molecular alterations.11Kehrer-Sawatzki H. Cooper D.N. Mosaicism in sporadic neurofibromatosis type 1: variations on a theme common to other hereditary cancer syndromes?.J Med Genet. 2008; 45: 622-631Crossref PubMed Scopus (68) Google Scholar Indeed, mutation screening in blood DNA often does not reveal the causative mutation in patients with mosaic NF2. The only approach currently available to make a molecular diagnosis of mosaic NF2 is to perform mutation screening in at least two tumors from the same patient; the presence of the same mutation in different tumors allows to make NF2 diagnosis even if the mutation is not present in blood DNA. We have previously reported that high-resolution melting analysis (HRMA) represents a sensitive approach for the detection of a low percentage of mutated cells in blood DNA, and it can be used as a scanning method in the molecular analysis of patients with NF2.12Sestini R. Provenzano A. Bacci C. Orlando C. Genuardi M. Papi L. NF2 mutation screening by denaturing high-performance liquid chromatography and high-resolution melting analysis.Genet Test. 2008; 12: 311-318Crossref PubMed Scopus (18) Google Scholar However, the low level of mutated alleles in somatic mosaicisms makes the characterization of the mutation difficult with the use of the Sanger sequencing method. The recent emergence of next-generation sequencing (NGS) is revolutionizing medical sciences. In fact, these approaches have brought remarkable advances in a wide range of medical research areas, such as studies of rare Mendelian disorders. Because NGS can analyze millions of DNA fragments simultaneously and independently, low abundance mutations have now become readily detectable.13Narumi S. Matsuo K. Ishii T. Tanahashi Y. Hasegawa T. Quantitative and sensitive detection of GNAS mutations causing mccune-albright syndrome with next generation sequencing.PLoS One. 2013; 8: e60525Crossref PubMed Scopus (28) Google Scholar Actually, all sequence variants identified with NGS have to be validated with Sanger sequencing. Although Sanger sequencing represents the criterion standard for characterization of the mutation, it is typically able to detect DNA variants present at moderate-to-high abundance, approximately 10% or greater.14Vogelstein B. Kinzler K.W. Digital PCR.Proc Natl Acad Sci U S A. 1999; 96: 9236-9241Crossref PubMed Scopus (1318) Google Scholar 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 (WT) and mutation-containing sequences. The PCR reaction is performed with a critical denaturation temperature (Tc), specific for each amplicon, allowing the selective amplification of low-level mutated alleles.15Li J. Wang L. Mamon H. Kulke M.H. Berbeco R. Makrigiorgos G.M. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.Nat Med. 2008; 14: 579-584Crossref PubMed Scopus (320) Google Scholar Therefore, COLD-PCR provides a general platform to improve the sensitivity of technologies for DNA variation detection, including HRMA and Sanger sequencing.16Luthra R. Zuo Z. COLD-PCR finds hot application in mutation analysis.Clin Chem. 2009; 55: 2077-2078Crossref PubMed Scopus (18) Google Scholar Mutation enrichment by using COLD-PCR can be achieved by two protocols: full COLD-PCR and fast COLD-PCR. In full COLD-PCR an intermediate annealing temperature is used to allow heteroduplex formation, which generally melts at lower denaturation temperatures than homoduplexes. Fast COLD-PCR is rapid and, compared with full COLD-PCR, results in higher enrichment of mutated alleles to determine a reduction of the melting temperature (Tm-reducing mutations). However, the full COLD-PCR protocol is required to enrich for all possible mutations, including small deletions or insertions that cause the increase of the Tm (Tm-increasing mutations).15Li J. Wang L. Mamon H. Kulke M.H. Berbeco R. Makrigiorgos G.M. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.Nat Med. 2008; 14: 579-584Crossref PubMed Scopus (320) Google Scholar Here, we evaluated the application of COLD-PCR for NF2 molecular diagnosis in blood DNA samples of patients with mosaicism. We analyzed five patients with NF2 mosaic mutations and four tumors from three of these patients. Constitutional DNA samples were obtained from peripheral blood leukocytes. DNA was also extracted from two fresh-frozen tumors and two paraffin-embedded tumors from three different patients after provision of informed consent. DNAs from blood and fresh-frozen tumor samples were isolated with the use of standard procedures with phenol/chloroform extraction and ammonium acetate/ethanol precipitation. DNA from paraffin-embedded tumor tissue was extracted with the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). PCR reactions were performed in 20 μL, containing 1× PCR buffer II (Life Technologies, Applied Biosystems, Branchburg, NJ), 0.25 mmol/L of each dNTP, 1.5 mmol/L MgCl2, 0.5 mmol/L of forward and reverse primers, 0.75 U of AmpliTaq DNA polymerase (Life Technologies, Applied Biosystems), and 50 ng of genomic DNA. SYTOs 9 (Invitrogen, Eugene, OR) was used as the intercalating dye. PCR condition consisted of an initial denaturation step at 95°C for 3 minutes, followed by 40 cycles of denaturing at 95°C for 15 seconds, 1 minute at annealing temperature, and elongation at 72°C for 1 minute. The final extension was performed at 72°C for 20 minutes. HRM analysis was performed on the Rotor Gene 6000 Instrument (Corbett Research, Sydney, Australia). Amplified products were denaturated at 95°C for 1 minute and then rapidly cooled to 40°C for 1 minute to facilitate heteroduplex formation. Melt curve data for each PCR product were acquired in a wide temperature range (75°C to 95°C), at a ramping rate of 0.1°C/second. Results were analyzed as fluorescence versus temperature graphs.17Krypuy M. Newnham G.M. Thomas D.M. Conron M. Dobrovic A. High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer.BMC Cancer. 2006; 6: 295Crossref PubMed Scopus (259) Google Scholar To design a COLD-PCR assay that selectively amplifies the minority mutant alleles, a new reduced denaturation temperature for the reactions was determined (Tc). The Tm of a given amplicon was first identified via HRMA of conventional PCR in the presence of an intercalating dye (the Tc is usually 1°C below the experimentally derived Tm18Milbury C.A. Li J. Makrigiorgos G.M. COLD-PCR-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations.Clin Chem. 2009; 55: 2130-2143Crossref PubMed Scopus (63) Google Scholar). Subsequently, a set of COLD-PCR reactions at graded temperatures below the Tm were performed to identify the optimal Tc.15Li J. Wang L. Mamon H. Kulke M.H. Berbeco R. Makrigiorgos G.M. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.Nat Med. 2008; 14: 579-584Crossref PubMed Scopus (320) Google Scholar Fast COLD-PCR cycling conditions were as follow: 95°C for 3 minutes; 20 cycles of 95°C for 15 seconds, primer annealing temperature for 30 seconds, 72°C for 30 seconds; then 35 cycles of Tc for 3 seconds, primer annealing temperature for 30 seconds, 72°C for 30 seconds; and final extension at 72°C for 20 minutes. COLD-PCR assay was performed with the same final reagent concentration used in standard PCR. Full COLD-PCR protocol provides denaturation at 95°C for 3 minutes, followed by 25 cycles of 95°C for 15 seconds, primers annealing temperature for 30 seconds, 72°C for 1 minute; then 30 cycles of 95°C for 15 seconds, 70°C for 8 minutes, Tc for 3 seconds, primers annealing temperature for 30 seconds, 72°C for 1 minute; and final extension at 72°C for 20 minutes. Primers and PCR annealing temperature are reported in Table 1.Table 1Primer Sequences and COLD-PCR ConditionsExonPrimer sequenceTa, °CTc, °CAmplicon size, bp2Forward: 5′-TGGAAAGGGAAGGACCTCTT-3′Reverse: 5′-TAGCCCAACCTTCTTGTCCA-3′62851296Forward: 5′-ATCAGATGACTCCGGAAATGTG-3′Reverse: 5′-AAGCCATAAAGGAATGTAAACC-3′588512213Forward: 5′-GGCCAAAGAGGCAGATCA-3′Reverse: 5′-AGGCTCACCGGGTACGTG-3′5989114Ta, annealing temperature; Tc, critical denaturation temperature. Open table in a new tab Ta, annealing temperature; Tc, critical denaturation temperature. PCR and COLD-PCR products were purified with QIAquick PCR purification kit (Qiagen). Sequencing analysis was performed on both strands with the use of the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies Applied Biosystems, Austin, TX) and a model 310 automated sequencer (Life Technologies Applied Biosystems). Mutation nomenclature follows the Human Genome Variation Society recommendations (http://www.hgvs.org, last accessed January 21, 2014). The DNA mutation numbering is based on the NF2 cDNA sequences (GenBank accession number NM_181832.2) with the A of the ATG translation-initiation codon numbered as +1. Amino acid numbering starts with the translation initiator methionine as +1. To evaluate the diagnostic sensitivity of the COLD-PCR amplification protocol and its ability to identify mosaicism in DNA samples compared with standard PCR, we performed serial dilutions of a WT DNA with genomic DNA derived from patients with nonmosaic NF2 carrying the following mutation: c.169C>T (exon 2). Dilutions were made by mixing patient DNA and normal DNA as follows: 1:1, 1:4, 1:10, 1:20, and 1:100, respectively, corresponding to 25%, 6.25%, 2.5%, 1.25%, and 0.25% of mutated allele. In our experimental conditions, COLD-PCR protocol followed by Sanger sequencing was able to detect the c.169C>T mutation until a dilution of 1:20, in which the mutant allele was present at 1.25% relative to the normal allele. Standard PCR followed by direct sequencing detected the c.169C>T mutation until dilution 1:4 (6.25% of the mutant allele) (Figure 1). Similar results were obtained by analyzing different mutations. COLD-PCR protocol followed by Sanger sequencing allowed us to increase the mutation detection rate compared with conventional PCR of 5% to 10%, depending of mutation type and localization (data not shown). The potential enrichment of mutated allele by COLD-PCR was first evaluated in patient 241 who had a mild form of NF2. HRM analysis in genomic DNA showed an alteration of NF2 exon 6 melting profile, but sequencing analysis did not identify any sequence variant. Two different tumor specimens (T653 and T654) were available for this patient. Sanger sequencing in tumor DNAs showed the same nonsense mutation in exon 6 of the NF2 gene, c.592C>T (p.Arg198Ter), confirming the constitutional mutation and the mosaic condition in this patient. The c.592C>T mutation causes a reduction of the Tm of the PCR amplicon; therefore, we used a fast COLD-PCR protocol to increase the mutation allele percentage in genomic DNA. COLD-PCR protocol allowed the identification of the c.592C>T variant in the blood sample by Sanger sequencing (Figure 2, A and B ). In patient 144 we found a melting profile alteration of NF2 exon 6 at HRM analysis. Direct sequencing of the genomic DNA did not reveal any DNA alteration; however, we detected a c.586C>T variant in a single tumor sample available from this patient. We used the COLD-PCR protocol previously validated in blood DNA from patient 144 to evaluate if the enrichment of minority-represented alleles was adequate to enable mutation identification and characterization. Indeed, the COLD-PCR approach allowed us to selectively amplify the mutated allele as much as necessary to detect the mutation by using sequencing analysis (Figure 3, A and B ). The DNA from the blood of patient 65 showed a melting profile alteration in exon 2 of the NF2 gene, but no sequence variant was identified by direct sequencing. HRM and sequencing analysis of the single tumor sample available from the patient detected c.193C>T mutation. This sequence variant is predicted to cause a reduction of the amplicon Tm; therefore, we applied the fast COLD-PCR protocol, followed by HRMA and sequencing, to both blood and tumor DNAs. Again, the increased amplification of low-level mutated allele allowed us to detect the c.193C>T mutation in the blood sample DNA (Figure 3). Finally, we applied the COLD-PCR protocol to identify an unknown alteration in blood DNA of a patient with NF2 mosaicism with no tumors available for DNA analysis. Patient 295 showed a melting profile alteration in exon 13 of the NF2 gene, but, again, sequencing analysis was not able to detect any mutation. Because the type of mutation was unknown, we did not know if the Tm was increased or reduced. For this reason, we developed a full COLD-PCR protocol for this amplicon. Our strategy allowed us to detect, by sequencing analysis, the molecular alteration in NF2 exon 13 (c.1396C>T) by increasing the amplification of the mutated allele (Figure 4). Mosaicism has been recognized in several hereditary tumor syndromes with a relatively high frequency of new mutations.1Youssoufian H. Pyeritz R.E. Mechanisms and consequences of somatic mosaicism in humans.Nat Rev Genet. 2002; 3: 748-758Crossref PubMed Scopus (294) Google Scholar, 19Hes F.J. Nielsen M. Bik E.C. Konvalinka D. Wijnen J.T. Bakker E. Vasen H.F. Breuning M.H. Tops C.M. Somatic APC mosaicism: an underestimated cause of polyposis coli.Gut. 2008; 57: 71-76Crossref PubMed Scopus (118) Google Scholar, 20Aretz S. Stienen D. Friedrichs N. Stemmler S. Uhlhaas S. Rahner N. Propping P. Friedl W. Somatic APC mosaicism: a frequent cause of familial adenomatous polyposis (FAP).Hum Mutat. 2007; 28: 985-992Crossref PubMed Scopus (107) Google Scholar Persons with mosaicism are often characterized by mild phenotypes,2Kluwe L. Mautner V. Heinrich B. Dezube R. Jacoby L.B. Friedrich R.E. MacCollin M. Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas.J Med Genet. 2003; 40: 109-114Crossref PubMed Scopus (126) Google Scholar, 20Aretz S. Stienen D. Friedrichs N. Stemmler S. Uhlhaas S. Rahner N. Propping P. Friedl W. Somatic APC mosaicism: a frequent cause of familial adenomatous polyposis (FAP).Hum Mutat. 2007; 28: 985-992Crossref PubMed Scopus (107) Google Scholar as a result of the timing of mutations that arise during postzygotic development. In fact, the mutation can be present in one or more of the germinal layers or organ systems. In NF2, a mosaic condition has been reported in approximately one-fourth of sporadic cases. 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. However, identification of unknown mosaic mutations is significantly challenging because of the low levels of molecular alterations.11Kehrer-Sawatzki H. Cooper D.N. Mosaicism in sporadic neurofibromatosis type 1: variations on a theme common to other hereditary cancer syndromes?.J Med Genet. 2008; 45: 622-631Crossref PubMed Scopus (68) Google Scholar In NF2, the capability of discriminating patients with mosaicism is especially important to make a differential diagnosis with schwannomatosis. Actually, persons with two or more nonvestibular schwannomas should be assessed for both schwannomatosis and mosaic NF2, and molecular diagnosis only could solve the question. Indeed, the identification of the same NF2 mutation in different schwannomas could ascertain the NF2 diagnosis. However, mosaic NF2 diagnosis becomes difficult and, sometimes, impossible if multiple tumor samples are unavailable. In this case, specific molecular analysis techniques to detect low levels of unknown molecular alterations are needed. COLD-PCR allows one to artificially increase the percentage of mutated alleles, which are often less represented than WT alleles in the biological sample. COLD-PCR is applicable to the characterization of any type of mutation, regardless of the position within the genomic sequence. In fact, two different COLD-PCR protocols have been developed: full COLD-PCR and fast COLD-PCR. Fast COLD-PCR results in higher enrichments than full COLD-PCR for Tm-reducing mutations. Full COLD-PCR is really time consuming, but it is able to enrich all possible mutations, including the Tm-increasing ones. Up to now, the COLD-PCR approach has been applied to detect known somatic mutations in tumor samples.21Pang B. Durso M.B. Hamilton R.L. Nikiforova M.N. A novel COLD-PCR/FMCA assay enhances the detection of low-abundance IDH1 mutations in gliomas.Diagn Mol Pathol. 2013; 22: 28-34Crossref PubMed Scopus (14) Google Scholar, 22Pennycuick A. Simpson T. Crawley D. Lal R. Santis G. Cane P. Tobal K. Spicer J. 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Here, we suggest the use of COLD-PCR protocols to characterize unknown NF2 mutations in patients with mosaicism. First, we evaluated the usefulness of this approach, analyzing blood DNA of a patient with known NF2 mosaicism in which the disease-causing mutation (c.592C>T) was already identified in two different tumor samples. Afterward, we analyzed two patients and showed, at HRM analysis, a melting profile alteration in blood DNAs in exons 6 and 2 of the NF2 gene, respectively. Only one tumor was available for each patient. Fast COLD-PCR protocol was therefore applied in blood DNA samples of both patients to confirm the presence of the same mutations found in the tumor samples. We used a fast COLD-PCR protocol because both mutations were C>T substitutions, therefore reducing the Tm of the corresponding amplicons. In both cases, the COLD-PCR approach was able to enrich the mutated allele enough to detect the mutations by sequencing analysis. Finally, we applied the COLD-PCR amplification protocol to blood DNA of a patient with NF2 mutations, showing a melting profile alteration in exon 13 of the NF2 gene, without evidence of any sequence alteration, and for which no tumor samples were available. Full COLD-PCR protocol was developed to detect the unknown mutation in this amplicon, because we did not know the effect of the mutation on the Tm. This approach allowed us to identify the c.1396C>T mutation in blood DNA. All of the patients with mosaicism with samples available in our laboratory showed Tm-increasing mutations (C>T substitutions) which allow a high enrichment by using any COLD-PCR protocol. The enrichment of Tm-equivalent and Tm-reducing variants is generally modest and could be obtained with full COLD-PCR only. Milbury et al29Milbury C.A. Li J. Makrigiorgos G.M. Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations.Nucleic Acids Res. 2011; 39: e2Crossref PubMed Scopus (89) Google Scholar described an update of COLD-PCR which enables an improved and complete enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of the previous format. This novel platform incorporates a synthetic reference-engineered sequence such that it matches the WT sequence during PCR reaction, obstructing WT allele amplification. The achievement of ice-COLD-PCR is independent from substitution type, but it is applicable to known mutations only. In fact, the reference sequence must include the mutation site, preferentially in central position, to obtain the major enrichment. Therefore, ice-COLD-PCR is not applicable for analysis of genes without hotspot mutations such as NF2. Recently, detecting the low abundance of mutated alleles with NGS is becoming a robust analytic tool.13Narumi S. Matsuo K. Ishii T. Tanahashi Y. Hasegawa T. Quantitative and sensitive detection of GNAS mutations causing mccune-albright syndrome with next generation sequencing.PLoS One. 2013; 8: e60525Crossref PubMed Scopus (28) Google Scholar However, sequence variants identified with NGS have to be validated with Sanger sequencing that does not allow confirmation of any mutations less than approximately 10% of mutated alleles.14Vogelstein B. Kinzler K.W. Digital PCR.Proc Natl Acad Sci U S A. 1999; 96: 9236-9241Crossref PubMed Scopus (1318) Google Scholar COLD-PCR protocol could be used to verify the presence of DNA alteration, regardless of the prescreening method used. In conclusion, data reported here indicate, for the first time, that the COLD-PCR approach is an efficient method for the enrichment of unknown mutant alleles poorly represented in blood DNA samples and that it can be applied to characterize mosaic mutations in genomic samples.
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