Abstract
Our experience of providing an NF1 gene diagnostic mutation detection service as part of the UK Genetic Testing Network (UKGTN) is presented. A total of 169 unrelated individuals suspected of having neurofibromatosis type I (NF1) were referred for NF1 diagnostic testing over a 2 year period. Mutation analysis of the entire NF1 coding region and the flanking splice sites was carried out, and included the use of a combination of FISH, dHPLC and MLPA. Possible disease causing mutations were identified in 109 (64%) cases. These comprised 88 different sequence alterations, of which 57 were novel. Out of the 169 cases referred, there were 102 patients with reliable clinical data, of whom 78 satisfied the NIH diagnostic criteria for NF1. Within this better defined cohort of NF1 patients, NF1 mutations were identified in 61 individuals (78%), showing the importance of clinical selection on overall test sensitivity, and highlighting the problem of full clinical data collection in the audit of routine services. As mutation detection technologies advance, facilitating direct sequencing of all coding and flanking non-coding regions of the NF1 gene, the development of an even more cost-effective, quick and sensitive diagnostic test for future testing of NF1 is discussed.
Similar content being viewed by others
References
Upadhyaya M, Cooper DN (1998) Neurofibromatosis Type 1 from genotype to phenotype. In: Upadhyaya M, Cooped DN (eds) BIOS Scientific, Oxford
Cichowski K, Jacks T (2001) NF1 tumor suppressor gene function: 1narrowing the GAP. Cell 104(4):593–604
Huson SM, Hughes RAC (1994) The neurofibromatoses: a pathogenetic and clinical overview. Chapman and Hall Medical, London
Ars E, Serra E, Garcia J, Kruyer H, Gaona A, Lazaro C, Estivill X (2000) Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1. Hum Mol Genet 9(2):237–247
Purandare SM, Huntsman Breidenbach H, Li Y, Zhu XL, Sawada S, Neil SM, Brothman A, White R, Cawthon R, Viskochil D (1995) Identification of neurofibromatosis 1 (NF1) homologous loci by direct sequencing, fluorescence in situ hybridization, and PCR amplification of somatic cell hybrids. Genomics 30(3):476–485
Luijten M, Wang Y, Smith BT, Westerveld A, Smink LJ, Dunham I, Roe BA, Hulsebos TJ (2000) Mechanism of spreading of the highly related neurofibromatosis type 1 (NF1) pseudogenes on chromosomes 2, 14 and 22. Eur J Hum Genet 8(3):209–214
Yu H, Zhao X, Su B, Li D, Xu Y, Luo S, Xiao C, Wang W (2005) Expression of NF1 pseudogenes. Hum Mutat 26(5):487–488
Upadhyaya M, Fryer A, MacMillan J, Broadhead W, Huson SM, Harper PS (1992) Prenatal diagnosis and presymptomatic detection of neurofibromatosis type 1. J Med Genet 29(3):180–183
Lazaro C, Ravella A, Casals T, Volpini V, Estivill X (1992) Prenatal diagnosis of sporadic neurofibromatosis 1. Lancet 339(8785):119–20
NIH (1988) NIH Consensus development conference statement. Neurofibromatosis. Arch Neurol 45:575–8
Evans DG, Baser ME, McGaughran J, Sharif S, Howard E, Moran A (2002) Malignant peripheral nerve sheath tumours in neurofibromatosis 1. J Med Genet 39(5):311–314
Easton DF, Ponder MA, Huson SM, Ponder BA (1993) An analysis of variation in expression of neurofibromatosis (NF) type 1 (NF1): evidence for modifying genes. Am J Hum Genet 53(2):305–313
Castle B, Baser ME, Huson SM, Cooper DN, Upadhyaya M (2003) Evaluation of genotype-phenotype correlations in neurofibromatosis type 1. J Med Genet 40(10):e109
Viskochil DH (1998) Gene structure and expression. In: Upadhyaya M, Cooper DN (eds) Neurofibromatosis Type 1 from genotype to phenotype. BIOS Scientific, Oxford
Verlinsky Y, Rechitsky S, Verlinsky O, Chistokhina A, Sharapova T, Masciangelo C, Levy M, Kaplan B, Lederer K, Kuliev A (2002) Preimplantation diagnosis for neurofibromatosis. Reprod Biomed Online 4(3):218–222
Spits C, De Rycke M, Van Ranst N, Joris H, Verpoest W, Lissens W, Devroey P, Van Steirteghem A, Liebaers ISermon K (2005) Preimplantation genetic diagnosis for neurofibromatosis type 1. Mol Hum Reprod 11(5):381–387
Upadhyaya M, Han S, Consoli C, Majounie E, Horan M, Thomas NS, Potts C, Griffiths S, Ruggieri M, von Deimling A, Cooper DN (2004) Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors. Hum Mutat 23(2):134–146
Wu R, Lopez-Correa C, Rutkowski JL, Baumbach LL, Glover TW, Legius E (1999) Germline mutations in NF1 patients with malignancies. Genes Chromosomes Cancer 26(4):376–380
Fahsold R, Hoffmeyer S, Mischung C, Gille C, Ehlers C, Kucukceylan N, Abdel-Nour M, Gewies A, Peters H, Kaufmann D, Buske A, Tinschert S, Nurnberg P (2000) 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 66(3):790–818
Messiaen LM, Callens T, Mortier G, Beysen D, Vandenbroucke I, Van Roy N, Speleman F, Paepe AD (2000) Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum Mutat 15(6):541–555
Ars E, Kruyer H, Morell M, Pros E, Serra E, Ravella A, Estivill X, Lazaro C (2003) Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients. J Med Genet 40(6):e82
Han SS, Cooper DN, Upadhyaya MN (2001) Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 (NF1) gene. Hum Genet 109(5):487–497
Oguzkan S, Cinbis M, Ayter S, Anlar B, Aysun S (2003) Molecular analysis of neurofibromatosis type 1 in Turkish families using polymorphic markers. Turk J Pediatr 45(3):192–7
Lopez Correa C, Brems H, Lazaro C, Estivill X, Clementi M, Mason S, Rutkowski JL, Marynen P, Legius E (1999) Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions. Hum Mutat 14(5):387–393
Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 30(12):e57
Kluwe L, Friedrich RE, Korf B, Fahsold R, Mautner VF (2002 Mar) NF1 mutations in neurofibromatosis 1 patients with plexiform neurofibromas. Hum Muta 19(3):309
Mattocks C, Baralle D, Tarpey P, ffrench-Constant C, Bobrow M, Whittaker J (2004) Automated comparative sequence analysis identifies mutations in 89% of NF1 patients and confirms a mutation cluster in exons 11–17 distinct from the GAP related domain. J Med Genet 41(4):e48
Toliat MR, Erdogan F,Gewies A, Fahsold, Buske A, Tinschert S, Nurnberg P (2000) Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence of mutations in exon 4b. Electrophoresis 21(3):541–544
Heim RA, Kam-Morgan L, N.Binnie CG, Corns DD, Cayouette MC, Farber RA, Aylsworth AS, Silverman LM, Luce MC (1995) Distribution of 13 truncating mutations in the neurofibromatosis 1 gene. Hum Mol Genet 4(6):975–981
Park VM, Pivnick EK (1998) Neurofibromatosis type 1 (NF1): a protein truncation assay yielding identification of mutations in 73% of patients. J Med Genet 35(10):813–820
Gasparini P, D’Agruma L, Pio de Cillis G, Balestrazzi P, Mingarelli R, Zelante L (1996) Scanning the first part of the neurofibromatosis type 1 gene by RNA-SSCP: identification of three novel mutations and of two new polymorphisms. Hum Genet 97(4):492–495
Upadhyaya M, Maynard J, Osborn M, Harper PS (1997) Six novel mutations in the neurofibromatosis type 1 (NF1) gene. Hum Mutat 10(3):248–250
Valero MC, Velasco E, Moreno F, Hernandez-Chico C (1994) Characterization of four mutations in the neurofibromatosis type 1 gene by denaturing gradient gel electrophoresis (DGGE). Hum Mol Genet 3(4):639–641
Zatkova A, Messiaen L, Vandenbroucke I, Wieser R, Fonatsch C, Krainer AR, Wimmer K (2004) Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1. Hum Mutat 24(6):491–501
Hudson J, Wu CL, Tassabehji M, Summers EM, Simon S, Super M, Donnai D, Thakker N (1997) Novel and recurrent mutations in the neurofibromatosis type 1 (NF1) gene. Hum Mutat 9(4):366–367
Cawthon RM, Weiss R, Xu GF, Viskochil D, Culver M, Stevens J, Robertson M, Dunn D, Gesteland R, O’Connell P (1990) A major segment of the neurofibromatosis type 1 gene: cDNA sequence genomic structure and point mutations. Cell 62(1):193–201
Robinson PN, Boddrich A, Peters H, Tinschert S, Buske A, Kaufmann D, Nurnberg P (1995) Two recurrent nonsense mutations and a 4 bp deletion in a quasi-symmetric element in exon 37 of the NF1 gene. Hum Genet 96(1):95–98
Messiaen L, Callens T, De Paepe A, Craen M, Mortier G (1997) Characterisation of two different nonsense mutations C6792A and C6792G causing skipping of exon 37 in the NF1 gene. Hum Genet 101(1):75–80
Origone P, Bellini C, Sambarino D, Banelli B, Morcaldi G, La Rosa C, Stanzial F, Castellan C, Coviello DA, Garre C, Bonioli E (2003) Neurofibromatosis type 1 (NF1): Identification of eight unreported mutations in NF1 gene in Italian patients [corrected]. Hum Mutat 22(2):179–180
Mantripragada KK, Thuresson AC, Piotrowski A, Diaz de Stahl T, Menzel U, Grigelionis G, Ferner RE, Griffiths S, Bolund L, Mautner V, Nordling M, Legius E, Vetrie D, Dahl N, Messiaen L, Upadhyaya M, Bruder CE, Dumanski JP (2006 Jan) Identification of novel deletion breakpoints bordered by segmental duplications in the NF1 locus using high-resolution array-CGH. J Med Genet 43(1):28–38
Hoffmeyer S, Assum G, Griesser J, Kaufmann D, Nürnberg P, Krone W (1995) On unequal allelic expression of the neurofibromin gene in neurofibromatosis type 1. Hum Mol Genet 4:1267–1272
Upadhyaya M, Osborn M, Cooper DN (2003) Detection of NF1 mutations utilizing the protein truncation test (PTT). Methods Mol Biol 217:315–27
Kluwe L, Siebest R, Gesk S, Fridrich RE, Tinschert S, Kehrer-Sawatzki H, MautnerVF (2004) Screening 500 unselected neurofibromatosis 1 patients for deletions of the NF1 gene. Hum Mut 23(2):111–116
Korf BR (1998) The NF1 genetic analysis consortium. In: Upadhyaya M, Cooper DN (eds) Neurofibromatosis Type 1 from genotype to phenotype. BIOS Scientific, Oxford
Messiaen LM, Wimmer K (2005) Pitfalls of automated comparative sequence analysis as a single platform for routine clinical testing for NF1. J Med Genet 42(5):e25
Baralle M, Baralle D, De Conti L, Mattocks C, Whittaker J, Krezevich A, ffrench-constant C, Baralle E (2003) Identification of a mutation that perturbs NF1 gene splicing using genomic DNA samples and a minigene assay. J Med genet 40:220–222
Wimmer K, Eckart M, Rehder H, Fonatsch C (2000) Illegitimate splicing of the NF1 gene in healthy individuals mimics mutation induced splicing alterations in NF1 patients. Hum Genet 106(3):311–313
Horan MP, Cooper DN, Upadhyaya M (2000) Hypermethylation of the neurofibromatosis type 1 (NF1) gene promoter is not a common event in the inactivation of the NF1 gene in NF1-specific tumours. Hum Genet 107(1):33–39
Harder A, Rosche M, Reuss DE, Holtkamp N, Uhlmann K, Friedrich R, Mautner VF, von Deimling A (2004) Methylation analysis of the neurofibromatosis type 1 (NF1) promoter in peripheral nerve sheath tumours. Eur J Cancer 40(18):2820–2828
Fishbein L, Eady B, Sanek N, Muir D, Wallace MR (2005) Analysis of somatic NF1 promoter methylation in plexiform neurofibromas and Schwann cells. Cancer Genet Cytogenet 157(2):181–186
Ebinger M, Senf L, Wachowski O, Scheurlen W (2005) No aberrant methylation of neurofibromatosis 1 gene (NF1) promoter in pilocytic astrocytoma in childhood. Pediatr Hematol Oncol 22(1):83–87
Baralle D, Mattocks C, Kalidas K, Elmslie F, Whittaker J, Lees M, Ragge N, Patton MA, Winter RM, ffrench-Constant C (2003) Different mutations in the NF1 gene are associated with Neurofibromatosis-Noonan syndrome (NFNS). Am J Med Genet 119(1):1–8
DeLuca A, Bottillo I, Sarkozy A, Carta C, Neri C, Bellacchio E, Schirinzi A, Conti E, Zampino G, Battaglia A, Majore S, Rinaldi MM, Cavella M, Marion B, Pizzuti A, Digilio MC, Tartaglia M, Dallapiccola B (2005 Dec NF1 gene mutations represent the major molecular event underlying Neurofibromatosis Noonan syndrome. Am J Hum Genet 77(6):1092–1101
Lamlum H, Ilyas M, Rowan A, Clark S, Johnson V, Bell J, Frayling I, Efstathiou J, Pack K, Payne S, Roylance R, Gorman P, Sheer D, Neale K, Phillips R, Talbot I, Bodmer W, Tomlinson I (1999) The type of somatic mutation at APC in familial adenomatous polyposis is determined by the site of the germline mutation: a new facet to Knudson’s ‘two-hit’ hypothesis. Nat Med 5(9):1071–1075
Kayes LM, Burke W, Riccardi VM, Bennett R, Ehrlich P, Rubenstein A, Stephens K (1994) Deletions spanning the neurofibromatosis 1 gene: identification and phenotype of five patients. Am J Hum Genet 54(3):424–436
Upadhyaya M, Ruggieri M, Maynard J, Osborn M, Hartog C, Mudd S, Penttinen M, Cordeiro I, Ponder M, Ponder BA, Krawczak M, Cooper DN (1998) Gross deletions of the neurofibromatosis type 1 (NF1) gene are predominantly of maternal origin and commonly associated with a learning disability, dysmorphic features and developmental delay. Hum Genet 102(5):591–597
Acknowledgments
We thank all the clinicians for their help in providing the clinical information. We are grateful to the Wales Gene Park for their financial support, and the staff of the Molecular Genetics Laboratory of the All-Wales Genetics Laboratory Service. We thank Dr Nick Thomas for his help with the splice prediction program.We wish to thank Dr Susan Huson for her support in initially setting up a diagnostic service, Linda Selwood for her administrative support and Andrew Wallace for developing MLPA data analysis spreadsheets.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Griffiths, S., Thompson, P., Frayling, I. et al. Molecular diagnosis of neurofibromatosis type 1: 2 years experience. Familial Cancer 6, 21–34 (2007). https://doi.org/10.1007/s10689-006-9001-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10689-006-9001-3