A Highly Sensitive Genetic Protocol to Detect NF1 Mutations
2011; Elsevier BV; Volume: 13; Issue: 2 Linguagem: Inglês
10.1016/j.jmoldx.2010.09.002
ISSN1943-7811
AutoresM. Carmen Valero, Yolanda Martín, Elisabete Hernández-Imaz, Alba Hernández, Germán Melean, Ana María Valero, Francisco Javier Álvarez Rodríguez, Dolores Tellerı́a, Concepción Hernández-Chico,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoNeurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, the presence of pseudogenes, and the wide variety of possible lesions. We developed a method for detecting germline mutations by combining an original RNA-based cDNA-PCR mutation detection method and denaturing high-performance liquid chromatography (DHPLC) with multiplex ligation-dependent probe amplification (MLPA). The protocol was validated in a cohort of 56 blood samples from NF1 patients who fulfilled NIH diagnostic criteria, identifying the germline mutation in 53 cases (95% sensitivity). The efficiency and reliability of this approach facilitated detection of different types of mutations, including single-base substitutions, deletions or insertions of one to several nucleotides, microdeletions, and changes in intragenic copy number. Because mutational screening for minor lesions was performed using cDNA and the characterization of mutated alleles was performed at both the RNA and genomic DNA level, the analysis provided insight into the nature of the different mutations and their effect on NF1 mRNA splicing. After validation, we implemented the protocol as a routine test. Here we present the overall unbiased spectrum of NF1 mutations identified in 93 patients in a cohort of 105. The results indicate that this protocol is a powerful new tool for the molecular diagnosis of NF1. Neurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, the presence of pseudogenes, and the wide variety of possible lesions. We developed a method for detecting germline mutations by combining an original RNA-based cDNA-PCR mutation detection method and denaturing high-performance liquid chromatography (DHPLC) with multiplex ligation-dependent probe amplification (MLPA). The protocol was validated in a cohort of 56 blood samples from NF1 patients who fulfilled NIH diagnostic criteria, identifying the germline mutation in 53 cases (95% sensitivity). The efficiency and reliability of this approach facilitated detection of different types of mutations, including single-base substitutions, deletions or insertions of one to several nucleotides, microdeletions, and changes in intragenic copy number. Because mutational screening for minor lesions was performed using cDNA and the characterization of mutated alleles was performed at both the RNA and genomic DNA level, the analysis provided insight into the nature of the different mutations and their effect on NF1 mRNA splicing. After validation, we implemented the protocol as a routine test. Here we present the overall unbiased spectrum of NF1 mutations identified in 93 patients in a cohort of 105. The results indicate that this protocol is a powerful new tool for the molecular diagnosis of NF1. Neurofibromatosis type 1 (NF1; OMIM #162200) is one of the most common human autosomal dominant disorders, with an estimated incidence of 1:3500 live births.1Huson S.M. Clark P. Compston D.A.S. Harper P.S. A genetic study of von Recklinghausen neurofibromatosis in South East Wales: prevalence, fitness, mutation rate, and effect of parental transmission on severity.J Med Genet. 1989; 26: 704-711Crossref PubMed Scopus (430) Google Scholar It is clinically characterized by café-au-lait spots, axillary and/or inguinal freckling, cutaneous and plexiform neurofibromas, Lisch nodules of the iris, optic gliomas, specific bone lesions, and an increased risk of malignant tumors.2Huson S.M. Neurofibromatosis: historical perspective, classification and diagnostic criteria.in: Huson S.M. Hughes R.A.C. The Neurofibromatoses: A Pathogenic and Clinical Overview. Chapman & Hall Medical, London1994: 1-22Google Scholar, 3Riccardi V.M. Neurofibromatosis: Phenotype, Natural History, and Pathogenesis.ed 2. Johns Hopkins University Press, Baltimore1992Google Scholar The condition is fully penetrant and has a highly variable expression, even within the same family.4Huson S.M. Harper P.S. Compston D.A. Von Recklinghausen neurofibromatosis A clinical and population study in south-east Wales.Brain. 1988; 111: 1355-1381Crossref PubMed Scopus (565) Google Scholar NF1 is caused by mutations in the neurofibromin gene (NF1; NM_000267.2) on chromosome subband 17q11.2.5Cawthon R.M. O'Connell P. Buchberg A.M. Viskochil D. Weiss R.B. Culver M. Stevens J. Jenkins N.A. Copeland N.G. White R. Identification and characterization of transcripts from the neurofibromatosis 1 region: the sequence and genomic structure of EVI2 and mapping of other transcripts.Genomics. 1990; 7: 555-565Crossref PubMed Scopus (115) Google Scholar, 6Viskochil D. Buchberg A.M. Xu G. Cawthon R.M. Stevens J. Wolff R.K. Culver M. Carey J.C. Copeland N.G. Jenkins N.A. White R. O'Connell P. Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus.Cell. 1990; 62: 187-192Abstract Full Text PDF PubMed Scopus (895) Google Scholar, 7Wallace M.R. Marchuk D.A. Andersen L.B. Letcher R. Odeh H.M. Saulino A.M. Fountain J.W. Brereton A. Nicholson J. Mitchell A.L. et al.Type I neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients [Erratum appeared in Science 1990, 250:1749].Science. 1990; 249: 181-186Crossref PubMed Scopus (1210) Google Scholar The gene spans >350 kb of genomic DNA and comprises 57 constitutive exons and three alternatively spliced exons, encoding an 11- to 13-kb ubiquitous mRNA transcript.8Viskochil D.H. Gene structure and function Neurofibromatosis Type 1: From Genotype to Phenotype.in: Upadhyaya M. Cooper D.N. BIOS Publishers, Oxford1998: 39-56Google Scholar The most common transcript codes for neurofibromin, a 2818-amino-acid polypeptide. A domain in the central region of neurofibromin (exons 20–27a) has structural and functional similarities to the mammalian GTPase-activating protein (GAP)-related protein family, acting as a negative regulator of Ras signaling.9Ballester R. Marchuk D. Boguski M. Saulino A. Letcher R. Wigler M. Collins F. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins.Cell. 1990; 63: 851-859Abstract Full Text PDF PubMed Scopus (652) Google Scholar, 10Martin G.A. Viskochil D. Bollag G. McCabe P.C. Crosier W.J. Haubruck H. Conroy L. Clark R. O'Connell P. Cawthon R.M. Innis M.A. McCormick F. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21.Cell. 1990; 63: 843-849Abstract Full Text PDF PubMed Scopus (737) Google Scholar, 11Xu G.F. Lin B. Tanaka K. Dunn D. Wood D. Gesteland R. White R. Weiss R. Tamanoi F. The catalytic domain of the neurofibromatosis type I gene product stimulates ras GTPase and complement IRA mutants of S. cerevisiae.Cell. 1990; 63: 835-841Abstract Full Text PDF PubMed Scopus (552) Google Scholar NF1 is a tumor suppressor gene. In accordance with Knudson's two-hit hypothesis, inheriting one germline mutation of NF1 gene is not sufficient to cause cancer development. A second hit in the wild-type NF1 allele occurring somatically produces a tumor.12Colman S.D. Williams C.A. Wallace M.R. Benign neurofibromas in type 1 neurofibromatosis (NF1) show somatic deletions of the NF1 gene.Nat Genet. 1995; 11: 90-92Crossref PubMed Scopus (201) Google Scholar Identifying the lesions that cause NF1 presents a complex challenge, given the large size of the NF1 gene, the lack of mutation hotspots, the wide variety of possible lesions, and the presence of pseudogenes.13Messiaen L.M. Wimmer K. NF1 mutational spectrum Neurofibromatoses.in: Kaufmann D. Monographs in Human Genetics 16. Karger, Basel2008: 63-77Google Scholar Most NF1 mutations (∼85% to 90%) are small lesions, such as single-base substitutions, insertions, or deletions. Other mutations are single or multiexon deletions or duplications (∼2%) and microdeletions encompassing NF1 and its neighboring genes (∼5% to 10%).13Messiaen L.M. Wimmer K. NF1 mutational spectrum Neurofibromatoses.in: Kaufmann D. Monographs in Human Genetics 16. Karger, Basel2008: 63-77Google Scholar The NF1 gene is subject to one of highest mutation rates known for human genes, and in ∼50% of all NF1 patients the case is classified as sporadic.2Huson S.M. Neurofibromatosis: historical perspective, classification and diagnostic criteria.in: Huson S.M. Hughes R.A.C. The Neurofibromatoses: A Pathogenic and Clinical Overview. Chapman & Hall Medical, London1994: 1-22Google Scholar Genetic counseling and presymptomatic molecular diagnosis are feasible only when the pathogenic germline mutation can be identified. Although numerous NF1 mutations have been identified, detecting mutations in NF1 has proved challenging, and current protocols are based on combinations of complementary techniques. Among the diverse techniques that have been used to identify minor intragenic lesions are the protein truncation test, single-strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, denaturing high-performance liquid chromatography (DHPLC), and cDNA sequencing, with each method displaying varying degrees of sensitivity.14Messiaen L.M. Callens T. Mortier G. Beysen D. Vandenbroucke I. Van Roy N. Speleman F. De Paepe A. Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.Hum Mutat. 2000; 15: 541-555Crossref PubMed Scopus (418) Google Scholar, 15Ars E. Serra E. García J. Kruyer H. Gaona A. Lázaro C. Estivill X. Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1 [Erratum appeared in Hum Mol Genet 2000;9:659].Hum Mol Genet. 2000; 9: 237-247Crossref PubMed Scopus (278) Google Scholar, 16Pros E. Gómez C. Martín T. Fábregas P. Serra E. Lázaro C. Nature and mRNA effect of 282 different NF1 point mutations: focus on splicing alterations.Hum Mutat. 2008; 29: E173-E193Crossref PubMed Scopus (84) Google Scholar, 17Valero M.C. Velasco E. Moreno F. Hernández-Chico C. Characterization of four mutations in the neurofibromatosis type 1 gene by denaturing gradient gel electrophoresis (DGGE).Hum Mol Genet. 1994; 3: 639-641Crossref PubMed Scopus (36) Google Scholar, 18Fahsold R. Hoffmeyer S. Mischung C. Gille C. Ehlers C. Kücükceylan N. Abdel-Nour M. Gewies A. Peters H. Kaufmann D. Buske A. Tinschert S. Nürnberg P. Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.Am J Hum Genet. 2000; 66: 790-818Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 19De Luca A. Buccino A. Gianni D. Mangino M. Giustini S. Richetta A. Divona L. Calvieri S. Mingarelli R. Dallapiccola B. NF1 gene analysis based on DHPLC.Hum Mutat. 2003; 21: 171-172Crossref PubMed Scopus (37) Google Scholar, 20Griffiths S. Thompson P. Frayling I. Upadhyaya M. Molecular diagnosis of neurofibromatosis type 1: 2 years experience.Fam Cancer. 2007; 6: 21-34Crossref PubMed Scopus (70) Google Scholar, 21Upadhyaya M. Han S. Consoli C. Majounie E. Horan M. Thomas N.S. Potts C. Griffiths S. Ruggieri M. von Deimling A. Cooper D.N. Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors.Hum Mutat. 2004; 23: 134-146Crossref PubMed Scopus (86) Google Scholar Furthermore, other tests must be applied to identify entire-gene deletions and/or variation in intragenic copy number; these include fluorescence in situ hybridization, Southern blotting, array comparative genomic hybridization, and multiplex ligation-dependent probe amplification (MLPA).22Wu B.L. Austin M.A. Schneider G.H. Boles R.G. Korf B.R. Deletion of the entire NF1 gene detected by the FISH: four deletion patients associated with severe manifestations.Am J Med Genet. 1995; 59: 528-535Crossref PubMed Scopus (81) Google Scholar, 23Riva P. Corrado L. Natacci F. Castorina P. Wu B.L. Schneider G.H. Clementi M. Tenconi R. Korf B.R. Larizza L. NF1 microdeletion syndrome: refined FISH characterization of sporadic and familial deletions with locus-specific probes.Am J Hum Genet. 2000; 66: 100-109Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 24Shen M.H. Mantripragada K. Dumanski J.P. Frayling I. Upadhyaya M. Detection of copy number changes at the NF1 locus with improved high resolution array CGH.Clin Genet. 2007; 72: 238-244Crossref PubMed Scopus (6) Google Scholar, 25Pasmant E. Sabbagh A. Masliah-Planchon J. Haddad V. Hamel M.J. Laurendeau I. Soulier J. Parfait B. Wolkenstein P. Bièche I. Vidaud M. Vidaud D. Detection and characterization of NF1 microdeletions by custom high resolution array CGH.J Mol Diagn. 2009; 11: 524-529Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 26Wimmer K. Yao S. Claes K. Kehrer-Sawatzki H. Tinschert S. De Raedt T. Legius E. Callens T. Beiglböck H. Maertens O. Messiaen L. Spectrum of single- and multiexon NF1 copy number changes in a cohort of 1,100 unselected NF1 patients.Genes Chromosomes Cancer. 2006; 45: 265-276Crossref PubMed Scopus (121) Google Scholar, 27De Luca A. Bottillo I. Dasdia M.C. Morella A. Lanari V. Bernardini L. Divona L. Giustini S. Sinibaldi L. Novelli A. Torrente I. Schirinzi A. Dallapiccola B. Deletions of NF1 gene and exons detected by multiplex ligation-dependent probe amplification.J Med Genet. 2007; 44: 800-808Crossref PubMed Scopus (63) Google Scholar On the other hand, screening for NF1 mutations has revealed a high proportion of mutations affecting the correct splicing of the gene, even in cases in which canonical splicing sequences were not affected, and this circumstance highlights the importance of mRNA studies.13Messiaen L.M. Wimmer K. NF1 mutational spectrum Neurofibromatoses.in: Kaufmann D. Monographs in Human Genetics 16. Karger, Basel2008: 63-77Google Scholar, 15Ars E. Serra E. García J. Kruyer H. Gaona A. Lázaro C. Estivill X. Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1 [Erratum appeared in Hum Mol Genet 2000;9:659].Hum Mol Genet. 2000; 9: 237-247Crossref PubMed Scopus (278) Google Scholar, 16Pros E. Gómez C. Martín T. Fábregas P. Serra E. Lázaro C. Nature and mRNA effect of 282 different NF1 point mutations: focus on splicing alterations.Hum Mutat. 2008; 29: E173-E193Crossref PubMed Scopus (84) Google Scholar, 18Fahsold R. Hoffmeyer S. Mischung C. Gille C. Ehlers C. Kücükceylan N. Abdel-Nour M. Gewies A. Peters H. Kaufmann D. Buske A. Tinschert S. Nürnberg P. Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.Am J Hum Genet. 2000; 66: 790-818Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 21Upadhyaya M. Han S. Consoli C. Majounie E. Horan M. Thomas N.S. Potts C. Griffiths S. Ruggieri M. von Deimling A. Cooper D.N. Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors.Hum Mutat. 2004; 23: 134-146Crossref PubMed Scopus (86) Google Scholar, 28Wimmer K. Roca X. Beiglböck H. Callens T. Etzler J. Rao A.R. Krainer A.R. Fonatsch C. Messiaen L. Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5′ splice-site disruption.Hum Mutat. 2007; 28: 599-612Crossref PubMed Scopus (102) Google Scholar In the present study, our principal aim was to develop a sensitive genetic protocol to identify germline NF1 mutations and to assess its sensitivity. Accordingly, the entire NF1 coding region was analyzed using an original RNA-based assay by complementing denaturing high-performance liquid chromatography (DHPLC) to conduct mutational screening of NF1 cDNA with MLPA to identify microdeletions and alterations in intragenic copy number. This protocol provides a simple, semiautomated, and cost-effective way to detect NF1 mutations from blood samples. Indeed, when we validated the method in a cohort of 56 NF1 patients, the pathogenic mutation was identified in 53 patients, demonstrating high sensitivity (95%) for the protocol. Thus, we have developed a routine and feasible molecular diagnosis protocol, which we have implemented for routine testing of NF1 mutations. To validate the protocol, 56 independent NF1 cases were enrolled, involving 66 patients and 124 relatives. The patients were seen in our laboratory after having undergone clinical evaluation at the Neurological and Pediatric Departments of several hospitals in Spain. All of the patients in this cohort met the NIH clinical diagnostic criteria for NF1. Of the 56 patients, 46 presented as sporadic cases and 10 as familial cases. Later, 49 more cases arrived at our laboratory for molecular diagnosis, having been diagnosed with NF1 or due to the clinical suspicion of NF1. These cases provided additional study material after the protocol had been validated and sensitivity demonstrated. The present study was approved by the Medical Ethical Committee of the Universitary Hospital Ramón y Cajal, and written informed consent was obtained from all of the participants. Genomic DNA was purified from peripheral blood collected in EDTA by the salting-out method. Haplotype analysis was performed with five polymorphic intragenic markers: IVS26.2.3, ALU, IVS27AC28.4, ACI27.2, and IVS38GTI.29Fang L. Chalhoub N. Li W. Feingold J. Ortenberg J. Lemieux B. Thirion J.P. Genotype analysis of the NF1 gene in the French Canadians from the Quebec population.Am J Med Genet. 2001; 104: 189-198Crossref PubMed Scopus (4) Google Scholar, 30Xu G.F. Nelson L. O'Connell P. White R. An Alu polymorphism intragenic to the neurofibromatosis type 1 gene (NF1).Nucleic Acids Res. 1991; 19: 3764Crossref PubMed Scopus (75) Google Scholar, 31Lazaro C. Gaona A. Estivill X. Two CA/GT repeat polymorphisms in intron 27 of the human neurofibromatosis (NF1) gene.Hum Genet. 1994; 93: 351-352Crossref PubMed Scopus (55) Google Scholar, 32Valero M.C. Velasco E. Valero A. Moreno F. Hernández-Chico C. Linkage disequilibrium between four intragenic polymorphic microsatellites of the NF1 gene and its implications for genetic counselling.J Med Genet. 1996; 33: 590-593Crossref PubMed Scopus (11) Google Scholar, 33Lázaro C. Gaona A. Xu G. Weiss R. Estivill X. A highly informative CA/GT repeat polymorphism in intron 38 of the human neurofibromatosis type 1 (NF1) gene.Hum Genet. 1993; 92: 429-430Crossref PubMed Scopus (66) Google Scholar To identify complete or partial NF1 gene deletions/duplications, MLPA assays were performed using the SALSA MLPA P081/P082 NF1 kit to screen for single- and multiexon deletions/duplications, or the SALSA MLPA P122-C1 NF1 AREA kit to detect gross deletions in the NF1 chromosomal region, according to the manufacturer's recommendations (MRC-Holland, Amsterdam, The Netherlands). The SALSA P081/P082 kit contains two reaction mixes with probes for all of the constitutive NF1 exons except exons 5, 7, 17, 19a, 45 and 47, as well as a probe for a sequence located 17 kb downstream of the NF1 gene and two probes specific for the OMG gene (NM_002544.4) embedded in intron 27b of NF1. As a control, 22 probes for other human genes located on different chromosomes are included. The SALSA P122-C1 kit contains five intragenic NF1 probes in exons 1, 12b, 23–2, 40, and 48, as well as 23 probes for 15 genes located close to the NF1 gene: nine centromeric genes or pseudogenes [TRAF4 (NM_004295.3), SSH2 (NM_033389.2), BLMH (NM_000386.2), CPD (NM_001304.3), SUZ12P (NG_005675.2), CRLF3 (NM_015986.3), ATAD5 (NM_024857.3), ADAP2 (previously CENTA2) (NM_018404.2), and RNF135 (NM_197939.1)] and six telomeric genes to NF1 [UTP6 (NM_018428.2), SUZ12 (NM_015355.2), LRRC37B (NM_052888.2), ZNF207 (NM_003457.3), PSMD11 (NM_002815.2), and MYO1D (NM_015194.1). The kit also contains 11 control probes. Briefly, denatured genomic DNA (150 ng) was added to the MLPA mix and the probes were allowed to anneal overnight before the subsequent ligation reaction was performed. PCR was performed with 6-carboxyfluorescein (FAM)-labeled primers using 5 μL of the ligation reaction as the template. The PCR products were then separated on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA), including two normal DNA samples in each batch of the MLPA assays for external normalization of MLPA products. The peak areas were measured with GeneScan analysis software (Applied Biosystems), and the data were analyzed with Coffalyser MLPA analysis software version 6 (MRC-Holland). Each MLPA product was normalized by dividing its peak area by the combined peak areas of the control probes (internal normalization) to get the relative peak area (RPA) values. The RPA value of each probe was then compared to that from the two reference controls (external normalization) to obtain the RPA ratios. Each patient's DNA was tested at least three times in independent assays to obtain average RPA ratios. These calculations were expected to yield a RPA value of 1.0 for each probe. Any decrease or increase in the RPA to values 1.3 was considered to be indicative of a deletion or a duplication, respectively. Total RNA was extracted from fresh peripheral blood collected in EDTA using a QIAamp RNA blood kit following the manufacturer's instructions (Qiagen, Valencia, CA). To prevent illegitimate splicing, blood samples were processed after venipuncture with a maximum delay of 4 hours and they were not stored at 4°C.15Ars E. Serra E. García J. Kruyer H. Gaona A. Lázaro C. Estivill X. Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1 [Erratum appeared in Hum Mol Genet 2000;9:659].Hum Mol Genet. 2000; 9: 237-247Crossref PubMed Scopus (278) Google Scholar, 34Wimmer K. Eckart M. Rehder H. Fonatsch C. Illegitimate splicing of the NF1 gene in healthy individuals mimics mutation-induced splicing alterations in NF1 patients.Hum Genet. 2000; 106: 311-313Crossref PubMed Scopus (49) Google Scholar (Blood samples collected in PAXgene tubes (Qiagen) or equivalent, stored at room temperature, and processed 24 to 36 hours later are also suitable for this mutational analysis protocol.) Reverse transcription was performed using 500 ng of total RNA isolated and random hexamers with a first-strand cDNA synthesis kit for RT-PCR (AMV) (Roche Applied Science, Indianapolis, IN). The entire coding region of the NF1 gene was amplified in 23 overlapping fragments by PCR in a 25 μL final reaction mix containing 1.5 μL cDNA as the template, 5 pmol each primer, 200 μmol/L dNTPs, and 1× reaction buffer with 1.5 mmol/L MgSO4 and 1.25 U Optimase polymerase (Transgenomic, Crewe, UK; Santa Clara, CA). Oligo 6.1 software was used to design primers (Table 1). DHPLC was performed on a WAVE DNA fragment analysis system using a DNAsep column (Transgenomic).Table 1Primer Sequences and Conditions for PCR Amplification and Denaturing High-Performance Liquid Chromatography AnalysisFragmentPrimer sequenceSize (bp)ExonsPositionAnnealing temperature (°C)PCRDHPLCF15′-GAGGACATGGCCGCGCACA-3′4371–4a−6_4315755.75′-GAAAATAAAACCCCAGAGGCAGAA-3′F25′-TCTGCCATTTTCTTCACACCT-3′4464a–6350_7955556.55′-TGCTGCTTTACGTTTGGTGCT-3′F35′-GAAAAGCTATTTGACTTGGTGG-3′4336–8739_11715557.25′-TGTTGTGAGGGCTTATACGA-3′F45′-TGGATCTAATGATTGACTGCCT-3′4948–10c1118_16115557.45′-GTGTGACTGAGGGACCAGTTG-3′F55′-AAACCCAAGGCAGTACAGCAG-3′46010c–131550_20095656.85′-GCACTATCCATAGAGGAGTTCCC-3′F65′-CTACGTACTCCTGGAGCCTCT-3′44312b–151951_23935760.25′-TTGGCTTTTGGATAGTTAAGGAT-3′F75′-CTGAGGCTTGGGAAGATACAC-3′44014–162321_27605757.8/595′-CAGATCCTTAACATTGGTCCG-3′F85′-CTGTTGTCCTTAATGGTGTGT-3′48916–19a2692_31805856.15′-ATCATCATCTGCTGCTTGGT-3′F95′-GACCTCTCATTTTGCCAAGAGAT-3′35218–203082_34335657.2/585′-TACAGTGCCTCAGTGATGCC-3′F105′-GCAAACAGGTGGCAGGAAAC-3′45120–223375_382554–5558.85′-GAAGAGAGTCTGCATGGAGTCT-3′F115′-TCTCGGCATTTACTCTACCAAC-3′36522–23.23745_41095657.85′-TGGTATAAACAGTGGCACACAC-3′F125′-TTAGAACCATCAGAGAGCCTT-3′422⁎PCR size of isoform without alternatively spliced exon 23a.23.2–263976_43975557.5/595′-GGACAATCAGATGCTATATCAA-3′F135′-TTGTGAAAAGCAACTTTGATG-3′45825–284337_47945458.35′-ATTGATTTGACCAGTTTTGAA-3′F145′-TATTTTCTACCAAGCTGGGAC-3′46027b–284710_51695556.75′-GAGAGCATTGTGGAATACCTT-3′F155′-ACATAGAGCATGAACAACAGAAA-3′48628–305090_55755657.8/59.85′-AGGCACACAGAAGATTATAGGCA-3′F165′-CTGGGACACTGCTCAATATCG-3′44829–315489_59365656.4/57.25′-AGGCTTCCCCATATTTTTGCTT-3′F175′-TGACAAGCTGATAACAATGACC-3′48831–335862_63495557.65′-GTGAACAAGTACACAGAGAGTGAA-3′F185′-TCTCCCTTAGAGCTTCCACAC-3′50133–376284_67845657.5/59.55′-CAGGTCCTTTTAAGCAACTCTC-3′F195′-ATGGGCAGATAAAGCAGATAAT-3′52836–406719_72465556.7/595′-CCACGCTCTGTGTATTCACTT-3′F205′-ATCCTTCACCTGCTATTGTTG-3′46640–437136_76015656.6/575′-TTAGGAGCCTTTGTGTCTGATA-3′F215′-CTGGACATGGGGCAACCTTCT-3′39142–457507_78975757.5/585′-AGACTTTGGGAAACACAACACTGG-3′F225′-TACTTACTGATCCGAAGATCCA-3′39344–487763_81555457.35′-CAATCAAGGCATCAAGAAACTTA-3′F235′-GCAGGACCGTTTTCAAAGCAA-3′48247–3′8080_8457 + 1045858.8/60.25′-GGAAGTGCAGCATTACAACATGG-3′Exon 15′-CAGACCCTCTCCTTGCCTCTT-3′439162665′-GGATGGAGGGTCGGAGGCTG-3′ PCR size of isoform without alternatively spliced exon 23a. Open table in a new tab Heteroduplex formation was enhanced by subjecting the PCR product to denaturation at 95°C for 10 minutes and then decreasing the temperature by 1.5°C/min until a temperature of 25°C was reached. The PCR products were analyzed for heteroduplexes by subjecting 3 to 7 μL of the reaction product to a 2% linear acetonitrile gradient at a flow rate of 0.9 ml/min. The optimal running conditions (starting buffer B concentration, oven temperature, and time shift) were selected using Transgenomic WAVEMAKER software version 4.1. Depending on the melting profile given by the software, DHPLC analysis was performed at one or two different temperatures (Table 1). The PCR products were checked by agarose gel electrophoresis before DHPLC was performed, and any amplicon with an altered mobility in agarose gel electrophoresis or with a variant DHPLC profile was characterized by sequencing a second RT-PCR product using an ABI Prism 3100 genetic analyzer (Applied Biosystems). Primers for cDNA fragment 12 (F12) amplified two mRNA species, with and without exon 23a. Sequencing of the F12 products was performed with PCR primers (Table 1) and additionally with the following primers: NF_Ex23aU, 5′-GCAACTTGCCACTCCCTACT-3′ and NF_Ex23aL 5′-TGATTTTTTGTTTTCCTTTT-3′. The mutations identified were confirmed in genomic DNA by direct cycle sequencing using primers and conditions for genomic sequence amplification as described previously,18Fahsold R. Hoffmeyer S. Mischung C. Gille C. Ehlers C. Kücükceylan N. Abdel-Nour M. Gewies A. Peters H. Kaufmann D. Buske A. Tinschert S. Nürnberg P. Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.Am J Hum Genet. 2000; 66: 790-818Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 35Han S. Cooper D.N. Upadhyaya M.N. Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 (NF1) gene.Hum Genet. 2001; 109: 487-497Crossref PubMed Scopus (56) Google Scholar except that for amplification of certain exons at least one primer had to be redesigned (primer sequences and conditions are available on request). A complementary screening of the genomic sequence of exon 1 was also performed. PCR reactions were set up in a final volume of 25 μL with 75 ng of genomic DNA, 10 pmol each primer,18Fahsold R. Hoffmeyer S. Mischung C. Gille C. Ehlers C. Kücükceylan N. Abdel-Nour M. Gewies A. Peters H. Kaufmann D. Buske A. Tinschert S. Nürnberg P. Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.Am J Hum Genet. 2000; 66: 790-818Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar 200 μmol/L dNTPs, 1 mmol/L MgCl2, 1× reaction buffer, and 1 U FastStart Taq DNA polymerase (Roche Applied Science). The reactions were cycled through an initial denaturation at 94°C for 12 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 62°C for 45 seconds, and extension at 72°C for 45 seconds, with a final 7 minutes extension at 72°C. PCR products were analyzed by DHPLC (Table 1). The mutations and their putative effect at the protein level are named according to Human Genome Variation Society guidelines.36Den Dunnen J.T. Antonarakis S.E. Mutation nomenclature.Curr Protoc Hum Genet. 2003; (Chapter 7:Unit 7.13)PubMed Google Scholar Mutation numbering is based on the NF1 mRNA sequence from GenBank (NM_000267.2), with the A of the translation start codon considered as nucleotide number 1. Exons are not named consecutively, but according to the accepted nomenclature used by researchers in the field. We have developed a new genetic protocol for molecular diagnosis of NF1 and have validated it in a cohort of 56 unrelated NF1 patients. All of the cases presented at least two diagnostic criteria for NF1, and a molecular diagnosis was not available for any of them. The protocol is based on the combination of MLPA and RNA-based mutation detection by RT-PCR and DHPLC. First, all of the patient samples were tested for single and multiexon deletion/duplication by MLPA with the SALSA MLPA P081/P082 assay. Complete gene deletions were corroborat
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