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Analysis of NF1 somatic mutations in cutaneous neurofibromas from patients with high tumor burden

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Abstract

Neurofibromatosis type 1, (NF1) is a complex, autosomal dominant disorder characterized by benign and malignant tumors which result from NF1 gene mutations. The molecular mechanisms that underlie NF1 tumorigenesis are still poorly understood although inactivation of other modifying loci in conjunction with NF1 mutations is postulated to be involved. These modifying loci may include deficiencies in mismatch repair genes and elements involved in cell cycle regulation (TP53, RB1, and CDKN2A). We have analyzed the somatic mutations in 89 cutaneous neurofibromas derived from three unrelated NF1 patients with high tumor burden, by loss of heterozygosity (LOH) analysis of the NF1, TP53, RB1, and CDKN2A genes, by assessing microsatellite instability (MSI), by direct sequencing of the NF1, TP53, and several mismatch repair (MMR) genes and by multiplex ligation-dependent probe amplification of the NF1 and TP53 genes. The aim was both to assess the possible clonality of these tumors and also to assess the involvement of other potential genetic loci in the development of these neurofibromas. Somatic NF1 mutations were identified in 57 (64%) of neurofibroma samples. Each mutation was distinct demonstrating the independent origin of each tumor. While somatic LOH of the TP53 gene was identified in four tumors, no specific deletions or sequence variations were identified. LOH of markers flanking the RB1 gene was also found in one tumor but no CDKN2A mutations were detected. Although evidence of MSI was seen in 21 tumors, no MMR gene alterations were identified. The identification of LOH involving TP53 and RB1 loci is a novel finding in benign cutaneous neurofibromas possibly demonstrating an alternative underlying molecular mechanism associated with the development of these benign tumors from this cohort of patients.

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References

  1. Huson S (2008) The neurofibromatoses: classification, clinical features and genetic counselling. In: Kaufmann D (ed) Neurofibromatoses (monographs in human genetics) vol 16, 1st edn. S Karger AG, Switzerland, pp 1–20

    Google Scholar 

  2. Bennett E, Thomas N, Upadhyaya M (2009) Neurofibromatosis type 1: its association with the Ras/MAPK pathway syndromes. J Pediatr Neurol 7(2):105–115

    CAS  Google Scholar 

  3. Ferner RE, Huson SM, Thomas N, Moss C, Willshaw H, Evans DG, Upadhyaya M, Towers R, Gleeson M, Steiger C, Kirby A (2007) Guidelines for the diagnosis and management of individuals with neurofibromatosis 1. J Med Genet 44:81–88

    Article  CAS  PubMed  Google Scholar 

  4. Upadhyaya M (2008) NF1 gene structure and NF1 genotype/phenotype correlations. In: Kaufmann D 2008. Neurofibromatoses (Monographs in Human Genetics) 16:46–62

  5. Cichowski K, Jacks T (2001) NF1 tumor suppressor gene function: narrowing the GAP. Cell 104:593–604

    Article  CAS  PubMed  Google Scholar 

  6. Arun D, Gutmann DH (2004) Recent advances in neurofibromatosis type 1. Curr Opin Neurol 17:101–105

    Article  CAS  PubMed  Google Scholar 

  7. Gottfried ON, Viskochil DH, Fults DW, Couldwell WT (2006) Molecular, genetic, and cellular pathogenesis of neurofibromas and surgical implications. Neurosurgery 58(1):1–16

    Article  PubMed  Google Scholar 

  8. Carroll SL, Ratner N (2008) How does the Schwann cell lineage form tumors in NF1? Glia 1;56(14):1590–1605

    Article  Google Scholar 

  9. Wimmer K, Etzler J (2008) Constitutional mismatch repair-deficiency syndrome: have we so far seen only the tip of an iceberg? Hum Genet 24:105–122

    Article  Google Scholar 

  10. Wang Q, Montmain G, Ruano E, Upadhyaya M, Dudley S, Liskay RM, Thibodeau SN, Puisieux A (2003) Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type. Hum Genet 112(2):117–123

    CAS  PubMed  Google Scholar 

  11. Spurlock G, Griffiths S, Uff J, Upadhyaya M et al (2007) Somatic alterations of the NF1 gene in an NF1 individual with multiple benign tumors and malignant tumor types. Familial Cancer 6:463–471

    Article  CAS  PubMed  Google Scholar 

  12. Serra E, Pros E, García C, López E, Lluïsa Gili M, Gómez C, Ravella A, Capellá G, Blanco I, Lázaro C (2007) Tumor LOH analysis provides reliable linkage information for prenatal genetic testing of sporadic NF1 patients. Genes, Chromosomes Cancer 46:820–827

    Article  CAS  PubMed  Google Scholar 

  13. De Raedt T, Maertens O, Chmara M, Brems H, Heyns I, Sciot R, Majounie E, Upadhyaya M, De Schepper S, Speleman F, Messiaen L, Vermeesch JR, Legius E (2006) Somatic loss of wild type NF1 allele in neurofibromas: comparison of NF1 microdeletion and non-microdeletion patients. Genes, Chromosomes Cancer 45:893–904

    Article  PubMed  Google Scholar 

  14. Ottini L, Esposito DL, Richetta A, Carlesimo M, Palmirotta R, Verí MC, Battista P, Frati L, Caramia FG, Calvieri S et al (1995) Alterations of microsatellites in neurofibromas of von Recklinghausen's disease. Cancer Res 1;55(23):5677–5680

    Google Scholar 

  15. Serra E, Puig S, Otero D, Gaona A, Kruyer H, Ars E, Estivill X, Lázaro C (1997) Confirmation of a double-hit model for the NF1 gene in benign neurofibromas. Am J Hum Genet 61(3):512–519

    Article  CAS  PubMed  Google Scholar 

  16. Feitsma H, Kuiper RV, Korving J, Nijman IJ, Cuppen E (2008) Zebrafish with mutations in mismatch repair genes develop neurofibromas and other tumors. Cancer Res 1;68(13):5059–5066

    Article  Google Scholar 

  17. Reitmair AH, Redston M, Cai JC, Chuang TC, Bjerknes M, Cheng H, Hay K, Gallinger S, Bapat B, Mak TW (1996) Spontaneous intestinal carcinomas and skin neoplasms in Msh2-deficient mice. Cancer Res 56:3842–3849

    CAS  PubMed  Google Scholar 

  18. Chen P, Dudley S, Hagen W, Dizon D, Paxton L, Reichow D, Yoon S, Yang K, Arnheim N, Liskay RM, Lipkin SM (2005) Contributions by MutL homologues Mlh3 and Pms2 to DNA mismatch repair and tumor suppression in the mouse. Cancer Res 1;65(19):8662–8670

    Article  Google Scholar 

  19. Kourea HP, Orlow I, Scheithauer BW, Cordon-Cardo C, Woodruff JM (1999) Deletions of the INK4A gene occur in malignant peripheral nerve sheath tumors but not in neurofibromas. Am J Pathol 155:1855–1860

    CAS  PubMed  Google Scholar 

  20. Nielsen GP, Stemmer-Rachamimov AO, Ino Y, Moller MB, Rosenberg AE, Louis DN (1999) Malignant transformation of neurofibromas in neurofibromatosis 1 is associated with CDKN2A/p16 inactivation. Am J Pathol 155:1879–1884

    CAS  PubMed  Google Scholar 

  21. Mawrin C, Kirches E, Boltze C, Dietzmann K, Roessner A, Schneider-Stock R (2002) Immunohistochemical and molecular analysis of p53, RB, and PTEN in malignant peripheral nerve sheath tumors. Virchows Arch 440:610–615

    Article  CAS  PubMed  Google Scholar 

  22. Mantripragada KK, Spurlock G, Kluwe L, Chuzhanova N, Ferner RE, Frayling IM, Dumanski JP, Guha A, Mautner V, Upadhyaya M (2008) High-resolution DNA copy number profiling of malignant peripheral nerve sheath tumors using targeted microarray-based comparative genomic hybridization. Clin Cancer Res 14:1015–1024

    Article  CAS  PubMed  Google Scholar 

  23. Upadhyaya M, Spurlock G, Monem B, Thomas N, Friedrich RE, Kluwe L, Mautner V (2008) Germline and somatic NF1 gene mutations in plexiform neurofibromas. Human Mutation, Mutation in Brief 29:E112–E122

    Google Scholar 

  24. Stewart H, Bowker C, Edees S, Smalley S, Crocker M, Mechan D, Forrester N, Spurlock G, Upadhyaya M (2008) Congenital disseminated neurofibromatosis type 1: a clinical and molecular case report. 2008. Am J Med Genet 146A(11):1444–1452

    Article  CAS  PubMed  Google Scholar 

  25. Cichowski K, Shih TS, Schmitt E, Santiago S, Reilly K, McLaughlin ME, Bronson RT, Jacks T (1999) Mouse models of tumor development in neurofibromatosis type 1. Science 286:2172–2176

    Article  CAS  PubMed  Google Scholar 

  26. Vogel KS, Klesse LJ, Velasco-Miguel S, Meyers K, Rushing EJ, Parada LF (1999) Mouse tumor model for neurofibromatosis type 1. Science 286:2176–2179

    Article  CAS  PubMed  Google Scholar 

  27. Parada LF (2000) Minireview neurofibromatosis type 1. Biochim Biophys Acta 1471:M13–M19

    CAS  PubMed  Google Scholar 

  28. Le LQ, Shipman T, Burns DK, Parada LF (2009) Cell of origin and microenvironment contribution for NF1-associated dermal neurofibromas. Cell Stem Cell 8;4(5):453–463

    Article  Google Scholar 

  29. Parrinello S, Lloyd AC (2009) Neurofibroma development in NF1—insights into tumor initiation. Trends Cell Biol 9(8):395–403

    Article  Google Scholar 

  30. 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 Mutation 23(2):134–146

    Article  CAS  Google Scholar 

  31. Sutter C, Gebert J, Bischoff P, Herfarth C, von Knebel Doeberitz M (1999) Molecular screening of potential HNPCC patients using a multiplex microsatellite PCR system. Mol Cell Probes 13:157–165

    Article  CAS  PubMed  Google Scholar 

  32. Kozlowski P, Jasinska AJ, Kwiatkowski DJ (2008) New applications and developments in the use of multiplex ligation-dependent probe amplification. Electrophoresis 29(23):4627–4636

    Article  CAS  PubMed  Google Scholar 

  33. Legius E, Herman D, Wu R, Hall BK, Marynen P, Cassiman J (1994) TP53 mutations are frequent in malignant NF1 tumors. Genes Chromosomes Cancer 10:250–255

    Article  CAS  PubMed  Google Scholar 

  34. Futreal PA, Barrett JC, Wiseman RW (1991) An Alu polymorphism intragenic to the TP53 gene. Nucleic Acids Res 19:6977

    Article  CAS  PubMed  Google Scholar 

  35. Storey A, Thomas M, Kalita A, Harwood C, Gardiol D, Mantovani F, Breuer J, Leigh IM, Matlashewski G, Banks L (1998) Role of a p53 polymorphism in the development of human papillomavirus-associated cancer. Nature 21;393(6682):229–234

    Google Scholar 

  36. McDaniel T, Carbone D, Takahashi T, Chumakov P, Chang EH, Pirollo KF, Yin J, Huang Y, Meltzer SJ (1991) The MspI polymorphism in intron 6 of p53 (TP53) detected by digestion of PCR products. Nucleic Acids Res 11;19(17):4796

    Article  Google Scholar 

  37. Belchis DA, Meece CA, Benko FA, Rogan PK, Williams RA, Gocke CD (1996) Loss of heterozygosity and microsatellite instability at the retinoblastoma locus in osteosarcomas. Diagn Mol Pathol 5(3):214–219

    Article  CAS  PubMed  Google Scholar 

  38. Yandell DW, Dryja TP (1989) Detection of DNA sequence polymorphisms by enzymatic amplification and direct genomic sequencing. Am J Hum Genet 45(4):547–555

    CAS  PubMed  Google Scholar 

  39. Hartmann A, Rosner U, Schlake G, Dietmaier W, Zaak D, Hofstaedter F et al (2000) Clonality and genetic divergence in multifocal low-grade superficial urothelial carcinoma as determined by chromosome 9 and p53 deletion analysis. Lab Invest 80(709):2000

    Google Scholar 

  40. Cairns P, Polascik TJ, Eby Y, Tokino K, Califano J, Merlo A, Mao L, Herath J, Jenkins R, Westra W et al (1995) Frequency of homozygous deletion at p16/ CDKN2 in primary human tumors. Nature (Genet) 11:210–212

    Article  CAS  Google Scholar 

  41. Pollock PM, Welch J, Hayward NK (2001) Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development. Cancer Res 1;61(3):1154–1161

    Google Scholar 

  42. Gusev VD, Nemytikova LA, Chuzhanova NA (1999) On the complexity measures of genetic sequences. Bioinformatics 15:994–999

    Article  CAS  PubMed  Google Scholar 

  43. Chuzhanova NA, Anassis EJ, Ball E, Krawczak M, Cooper DN (2003) Meta-analysis of indels causing human genetic disease: mechanisms of mutagenesis and the role of local DNA sequence complexity. Hum Mutat 21:28–44

    Article  CAS  PubMed  Google Scholar 

  44. Ball EV, Stenson PD, Krawczak M, Cooper DN, Chuzhanova NA (2005) Micro-deletions and micro-insertions causing human genetic disease: common mechanisms of mutagenesis and the role of local DNA sequence complexity. Hum Mut 26:205–213

    Article  CAS  PubMed  Google Scholar 

  45. Abeysinghe SS, Chuzhanova N, Krawczak M, Ball EV, Cooper DN (2003) Translocation and gross deletion breakpoints in human inherited disease and cancer I: nucleotide composition and recombination-associated motifs. Hum Mut 22:229–244

    Article  CAS  PubMed  Google Scholar 

  46. Bacolla A, Jaworski A, Larson JE, Jakupciak JP, Chuzhanova N, Abeysinghe SS, O'Connell CD, Cooper DN, Wells RD (2004) Breakpoints of gross deletions coincide with non-B DNA conformations. Proc Natl Acad Sci U S A 101:14162–14167

    Article  CAS  PubMed  Google Scholar 

  47. Kondrashov AS, Rogozin IB (2004) Context of deletions and insertions in human coding sequences. Hum Mutat 23:177–185

    Article  CAS  PubMed  Google Scholar 

  48. Maertens O, Brems H, Vandesompele J, De Raedt T, Heyns I, Rosenbaum T, De Schepper S, De Paepe A, Mortier G, Janssens S, Speleman F, Legius E, Messiaen L (2006) Comprehensive NF1 screening on cultured Schwann cells from neurofibromas. Hum Mutat 27(10):1030–1040

    Google Scholar 

  49. Edwards RA (2007) Laser capture microdissection of mammalian tissue. J Vis Exp 8:309

    PubMed  Google Scholar 

  50. 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 tumors. Hum Genet 107(1):33–39

    Article  CAS  PubMed  Google Scholar 

  51. Luijten M, Redeker S, van Noesel MM, Troost D, Westerveld A, Hulsebos TJ (2000) Microsatellite instability and promoter methylation as possible causes of NF1 gene inactivation in neurofibromas. Eur J Hum Genet 8(12):939–945

    Article  CAS  PubMed  Google Scholar 

  52. 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

    Article  CAS  PubMed  Google Scholar 

  53. Däschner K, Assum G, Eisenbarth I, Krone W, Hoffmeyer S, Wortmann S, Heymer B, Kehrer-Sawatzki H (1997) Clonal origin of tumor cells in a plexiform neurofibroma with LOH in NF1 intron 38 and in dermal neurofibromas without LOH of the NF1 gene. Biochem Biophys Res Commun 19;234(2):346–350

    Google Scholar 

  54. John AM, Ruggieri M, Ferner R, Upadhyaya M (2000) A search for evidence of somatic mutations in the NF1 gene. J Med Genet 37(1):44–49

    Google Scholar 

  55. Serra E, Rosenbaum T, Nadal M, Winner U, Ars E, Estivill X, Lázaro C (2001) Mitotic recombination effects homozygosity for NF1 germline mutations in neurofibromas. Nat Genet 28(3):294–296

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We are grateful to Cancer Research UK and Cancer IRG, Cardiff University for financial support.

The authors declare that they have no conflict of interest.

We declare that the experiments comply with the current laws of the United Kingdom.

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Correspondence to Meena Upadhyaya.

Electronic supplementary materials

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Supplementary Table 1

Somatic and germline mutations in 89 tumors and paired blood DNA samples from three NF1 patients. Shading of consecutive tumor samples represents tumors found located in close proximity to each other (DOC 42 kb)

Supplementary Table 2

Summary of 158 published NF1 somatic mutations in neurofibromas (DOC 42 kb)

Supplementary Table 3

Summary of TP53 LOH analysis (DOC 26 kb)

Supplementary Table 4

Summary of RB1 LOH analysis (DOC 26 kb)

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Thomas, L., Kluwe, L., Chuzhanova, N. et al. Analysis of NF1 somatic mutations in cutaneous neurofibromas from patients with high tumor burden. Neurogenetics 11, 391–400 (2010). https://doi.org/10.1007/s10048-010-0240-y

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  • DOI: https://doi.org/10.1007/s10048-010-0240-y

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