#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Molecular Diagnostics of NF1 in Slovakia Using cDNA and MLPA Analysis


Authors: M. Némethová 1,2;  A. Bolčeková 3;  S. Požgayová 2;  D. Ilenčíková 3;  A. Hlavatá 3;  Ľ. Kádaši 1,4;  L. Kovácz 3;  A. Zaťková 1
Authors place of work: Laboratórium genetiky, ÚMFG SAV, Bratislava 1;  Laboratórium klinickej a molekulárnej genetiky, II. detská klinika LF UK a DFNsP Bratislava 2;  II. detská klinika LF UK a DFNsP Bratislava 3;  Katedra molekulárnej biológie, PriF UK, Bratislava 4
Published in the journal: Cesk Slov Neurol N 2014; 77/110(6): 721-733
Category: Original Paper

Summary

Aim:
The aim of our study was to identify causative germline mutation in suspected NF1 patients, in order to help differential diagnostics, as well as to collect as large as possible group of patients for a possible genotype-phenotype correlations.

Material and methods:
Our set of patients consisted of 107 Slovak patients, recruited between 2008 and 2013, who were considered for a diagnosis NF1 by clinical genetics. In order to identify mutations, we employed NF1 cDNA sequencing that enabled us to also detect splicing mutations, as well as MLPA analysis that enables identification of larger deletions.

Results:
By employing the selected methods in our set of 94 unrelated Slovak patients who fulfilled the basic NF1 diagnostic criteria, we uncovered germline mutations in the NF1gene in 83 of them (88.3%). We observed a high proportion of mutations identified in Slovak population only so far (41/83, 49.4%), and we confirmed de novo mutation in 27/42 tested families (64.3%). Genotype-phenotype correlations revealed an increased incidence of optic pathway glioma in patients with a mutation in the 5´end of the NF1 gene.

Conclusion:
Combination of cDNA analysis and MLPA provides an effective method for identification of mutations in the NF1 gene. By employing these methods, we were able to also identify frequently incorrectly classified atypical splicing mutations. Clinical geneticists used our results in the differential diagnosis of the disease, especially in contested cases.

Key words:
NF1 gene – causative germline mutation – RNA splicing – genotype-phenotype correlation – de novo mutations –differential diagnostics

The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.

The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers.


Zdroje

1. Boyd KP, Korf BR, Theos A. Neurofibromatosis type 1. J Am Acad Dermatol 2009; 61(1): 1-14. doi: 10.1016/ j.jaad.2008.12.051.

2. DeBella K, Szudek J, Friedman JM. Use of the national institutes of health criteria for diagnosis of neurofibromatosis 1 in children. Pediatrics 2000; 105(3 Pt 1):608-614.

3. Neurofibromatosis. Conference statement. National Institutes of Health Consensus Development Conference. Arch Neurol 1988; 45(5): 575-578.

4. Evans DG, Baser ME, McGaughran J, Sharif S, Howard E, Moran A. Malignant peripheral nerve sheath tumours in neurofibromatosis 1. J Med Genet 2002; 39(5): 311-314.

5. Cichowski K, Jacks T. NF1 tumor suppressor gene function: narrowing the GAP. Cell 2001; 104(4): 593-604.

6. Brannan CI, Perkins AS, Vogel KS, Ratner N, Nordlund ML, Reid SW et al. Targeted disruption of the neurofibromatosis type  1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues. Genes Dev 1994; 8(9): 1019-1029.

7. Viskochil D, Buchberg AM, Xu G, Cawthon RM, Stevens J, Wolff RK et al. Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell 1990; 62(1): 187-192.

8. Cawthon RM, O‘Connell P, Buchberg AM, Viskochil D, Weiss RB, Culver M et al. 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(4): 555-565.

9. Wallace MR, Marchuk DA, Andersen LB, Letcher R,Odeh HM, Saulino AM et al. Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients. Science 1990; 249(4965): 181-186.

10. Li Y, O‘Connell P, Breidenbach HH, Cawthon R, Stevens J, Xu G et al. Genomic organization of the neurofibromatosis 1 gene (NF1). Genomics 1995; 25(1): 9-18.

11. Skuse GR, Cappione AJ. RNA processing and clinical variability in neurofibromatosis type I (NF1). Hum Mol Genet 1997; 6(10): 1707-1712.

12. Stocker KM, Baizer L, Coston T, Sherman L, Ciment G. Regulated expression of neurofibromin in migrating neural crest cells of avian embryos. J Neurobiol 1995; 27(4): 535-552.

13. Williams VC, Lucas J, Babcock MA, Gutmann DH,Korf B, Maria BL. Neurofibromatosis type 1 revisited. Pediatrics 2009; 123(1): 124-133. doi: 10.1542/ peds.2007-3204.

14. Martin GA, Viskochil D, Bollag G, McCabe PC, Crosier WJ, Haubruck H et al. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell 1990; 63(4): 843-849.

15. Xu GF, O‘Connell P, Viskochil D, Cawthon R, Robertson M, Culver M et al. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell 1990; 62(3): 599-608.

16. Izawa I, Tamaki N, Saya H. Phosphorylation of neurofibromatosis type 1 gene product (neurofibromin) by cAMP-dependent protein kinase. FEBS Lett 1996; 382(1-2): 53-59.

17. Mangoura D, Sun Y, Li C, Singh D, Gutmann DH,Flores A et al. Phosphorylation of neurofibromin by PKC is a possible molecular switch in EGF receptor signaling in neural cells. Oncogene 2006; 25(5): 735-745.

18. Korf BR, Rubenstein AE. Neurofibromatosis: a handbook for patients, families, and health care professionals. 2nd ed. New York: NY Thieme Medical Publishers 2005.

19. Viskochil D. Genetics of neurofibromatosis 1 and the NF1 gene. J Child Neurol 2002; 17(8): 562-570.

20. Yunoue S, Tokuo H, Fukunaga K, Feng L, Ozawa T,Nishi T et al. Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GTPase-activating protein function toward Ras. J Biol Chem 2003; 278(29): 26958-26969.

21. Radtke HB, Sebold CD, Allison C, Haidle JL, Schneider G. Neurofibromatosis type 1 in genetic counseling practice: recommendations of the National Society of Genetic Counselors. J Genet Couns 2007; 16(4): 387-407.

22. Kern SE, Winter JM. Elegance, silence and nonsense in the mutations literature for solid tumors. Cancer Biol Ther 2006; 5(4): 349-359.

23. Kluwe L, Siebert R, Gesk S, Friedrich RE, Tinschert S, Kehrer-Sawatzki H et al. Screening 500 unselected neurofibromatosis 1 patients for deletions of the NF1 gene. Hum Mutat 2004; 23(2): 111-116.

24. Messiaen L, Wimmer K. Mutation analysis of the NF1 gene by cDNA-based sequencing of the coding region. In: Cunha KSG, Geller M (eds). Advances in neurofibromatosis research. New York: Nova Science 2012: 89-108.

25. Pasmant E, Sabbagh A, Spurlock G, Laurendeau I, Grillo E, Hamel MJ et al. NF1 microdeletions in neurofibromatosis type 1: from genotype to phenotype. Hum Mutat 2010; 31(6): E1506-E1518. doi: 10.1002/ humu.21271.

26. Chang YF, Imam JS, Wilkinson MF. The nonsense-mediated decay RNA surveillance pathway. Annu Rev Biochem 2007; 76: 51-74.

27. Den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 2000; 15(1): 7-12.

28. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P et al. A method and server for predicting damaging missense mutations. Nat Methods 2010; 7(4): 248-249. doi: 10.1038/ nmeth0410-248.

29. Bromberg Y, Rost B. SNAP: predict effect of non-synonymous polymorphisms on function. Nucleic Acids Res 2007; 35(11): 3823-3835.

30. Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PloS One 2012; 7(10): e46688. doi: 10.1371/ journal.pone.0046688.

31. Choi Y. A fast computation of pairwise sequence alignment scores between a protein and a set of single-locus variants of another protein. In: Proceedings of the ACM Conference on bioinformatics, computational biology and biomedicine (BCB‚12). ACM, New York, NY, USA 2012: 414-417.

32. Desmet FO, Hamroun D, Lalande M, Collod-Beroud G, Claustres M et al. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 2009; 37(9): e67. doi: 10.1093/ nar/ gkp215.

33. Reese MG, Eeckman FH, Kulp D, Haussler D. Improved splice site detection in Genie. J Comput Biol 1997; 4(3): 311-323.

34. Nemethova M, Bolcekova A, Ilencikova D, Durovcikova D, Hlinkova K, Hlavata A et al. Thirty-nine novel neurofibromatosis 1 (NF1) gene mutations identified in Slovak patients. Ann Hum Genet 2013; 77(5): 364-379. doi: 10.1111/ ahg.12026.

35. Sharif S, Upadhyaya M, Ferner R, Majounie E, Shenton A, Baser M et al. A molecular analysis of individuals with neurofibromatosis type 1 (NF1) and optic pathway gliomas (OPGs), and an assessment of genotype-phenotype correlations. J Med Genet 2011; 48(4): 256-260. doi: 10.1136/ jmg.2010.081760.

36. Alkindy A, Chuzhanova N, Kini U, Cooper DN, Upadhyaya M. Genotype-phenotype associations in neurofibromatosis type 1 (NF1): an increased risk of tumor complications in patients with NF1 splice-site mutations? Hum Genomics 2012; 6: 12. doi: 10.1186/ 1479-7364-6-12.

37. Robinson PN, Buske A, Neumann R, Tinschert S, Nurnberg P. Recurrent 2-bp deletion in exon 10c of the NF1 gene in two cases of von Recklinghausen neurofibromatosis. Hum Mutat 1996; 7(1): 85-88.

38. Park VM, Pivnick EK. Neurofibromatosis type 1(NF1): a protein truncation assay yielding identification of mutations in 73% of patients. J Med Genet 1998; 35(10): 813-820.

39. Fahsold R, Hoffmeyer S, Mischung C, Gille C, Ehlers C, Kücükceylan N et al. 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(3): 790-818.

40. Viskochil D, Carey JC. Alternate and related forms of the neurofibromatoses In: Huson SM, Hughes RA (eds). The Neurofibromatoses a clinical and pathogenetic overview. London: Chapman Hall 1994: 445-474.

41. De Readt T, Brems H, Wolkenstein P, Vidaud D, Pilotti S, Perrone F et al. Elevated risk for MPNST in NF1 microdeletion patients. Am J Hum Genet 2003; 72(5): 1288-1292.

42. Rodriguez-Viciana P, Tetsu O, Tidyman WE, Estep AL, Conger BA, Cruz MS et al. Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 2006; 311(5765): 1287-1290.

43. Stevenson DA, Swensen JJ, Viskochil DH. Neurofibromatosis type 1 and other syndromes of the Ras Pathway. In: Kaufman D et al (eds). Neurofibromatoses. Basel: Karger 2008: 32-45.

44. Tinschert S, Naumann I, Stegmann E, Buske A, Kaufmann D, Thiel G et al. Segmental neurofibromatosis is caused by somatic mutation of the neurofibromatosis type 1 (NF1) gene. Eur J Hum Genet 2000; 8(6): 455-459.

45. Lee MJ, Su YN, You HL, Chiou SC, Lin LC, Yang CC et al. Identification of forty-five novel and twenty-three known NF1 mutations in Chinese patients with neurofibromatosis type 1. Hum Mutat 2006; 27(8): 832.

46. Abernathy CR, Colman SD, Kousseff BG, Wallace MR. Two NF1 mutations: frameshift in the GAP-related domain, and loss of two codons toward the 3‘ end of the gene. Hum Mutat 1994; 3(4): 347-352.

47. Osborn MJ, Upadhyaya M. Evaluation of the protein truncation test and mutation detection in the NF1 gene: mutational analysis of 15 known and 40 unknown mutations. Hum Genet 1999; 105(4): 327-332.

48. Wimmer K, Yao S, Claes K, Kehrer-Sawatzki H, Tinschert S, De Raedt T et al. Spectrum of single- and multiexon NF1 copy number changes in a cohort of 1,100 unselected NF1 patients. Genes Chromosomes. Cancer 2006; 45(3): 265-276.

49. Upadhyaya M, Spurlock G, Monem B, Thomas N, Friedrich RE, Kluwe L et al. Germline and somatic NF1 gene mutations in plexiform neurofibromas. Hum Mutat 2008; 29(8): E103-E111. doi: 10.1002/ humu.20793.

50. Ars E, Kruyer H, Morell M, Pros E, Serra E, Ravella Aet al. Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients. J Med Genet 2003; 40(6): e82.

51. Heim RA, Kam-Morgan LN, Binnie CG, Corns DD,Cayouette MC, Farber RA et al. Distribution of 13 truncating mutations in the neurofibromatosis 1 gene. Hum Mol Genet 1995; 4(6): 975-981.

52. Griffiths S, Thompson P, Frayling I, Upadhyaya M.Molecular diagnosis of neurofibromatosis type 1: 2 years experience. Fam Cancer 2007; 6(1): 21-34.

53. Wimmer K, Roca X, Beiglbock H, Callens T, Etzler J,Rao AR et al. Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5‘ splice-site disruption. Hum Mutat 2007; 28(6): 599-612.

54. General information. LOVD Gene homepage [on-line]. Available from URL: https:/ / grenada.lumc.nl/ LOVD2/ mendelian_genes/ home.php?select_db=NF1.

55. Origone P, Bellini C, Sambarino D, Banelli B, Morcaldi G, La RC et al. Neurofibromatosis type 1 (NF1): Identification of eight unreported mutations in NF1 gene in Italian patients. Hum Mutat 2003; 22(2): 179-180.

56. Han SS, Cooper DN, Upadhyaya MN. Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 (NF1) gene. Hum Genet 2001; 109(5): 487-497.

57. Bottillo I, Ahlquist T, Brekke H, Danielsen SA, van den Berg E, Mertens F et al. Germline and somatic NF1 mutations in sporadic and NF1-associated malignant peripheral nerve sheath tumours. J Pathol 2009; 217(5): 693-701. doi: 10.1002/ path.2494.

58. Mattocks C, Baralle D, Tarpey P, French-Constant C,Bobrow M, Whittaker J. 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 2004; 41(4): e48.

59. Bolcekova A, Nemethova M, Zatkova A, Hlinkova K, Pozgayova S, Hlavata A et al. Clustering of mutations in the 5‘ tertile of the NF1 gene in Slovakia patients with optic pathway glioma. Neoplasma 2013; 60(6): 655-665. doi: 10.4149/ neo_2013_084.

60. Messiaen LM, Wimmer K. NF1 mutational spectrum. In: Kaufman D et al (eds). Neurofibromatoses. Basel: Karger 2008: 63-77.

61. Bendova S, Krepelova A, Petrak B, Kinstova L, Musova Z, Rausova E et al. Novel mutations in the NF1 gene in Czech patients with neurofibromatosis type 1. J Mol Neurosci 2007; 31(3): 273-279.

62. Cnossen MH, van der Est MN, Breuning MH, van Asperen CJ, Breslau-Siderius EJ, van der Ploeg AT et al. Deletions spanning the neurofibromatosis type 1 gene: implications for genotype-phenotype correlations in neurofibromatosis type 1? Hum Mutat 1997; 9(5): 458-464.

63. Lázaro C, Gaona A, Ainsworth P, Tenconi R, Vidaud D, Kruyer H et al. Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients. Hum Genet 1996; 98(6): 696-699.

64. Messiaen LM, Callens T, Mortier G, Beysen D, Vandenbroucke I, Van RN et al. 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(6): 541-555.

65. Rasmussen SA, Colman SD, Ho VT, Abernathy CR,Arn PH, Weiss L et al. Constitutional and mosaic large NF1 gene deletions in neurofibromatosis type 1. J Med Genet 1998; 35(6): 468-471.

66. Valero MC, Martin Y, Hernandez-Imaz E, Marina Hernandez A, Melean G, Valero AM et al. A highly sensitive genetic protocol to detect NF1 mutations. J Mol Diagn 2011; 13(2): 113-122. doi: 10.1016/ j.jmoldx.2010.09.002.

Štítky
Paediatric neurology Neurosurgery Neurology

Článok vyšiel v časopise

Czech and Slovak Neurology and Neurosurgery

Číslo 6

2014 Číslo 6
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#