title: Development of a practical NF1 genetic testing method through the pilot analysis of five Japanese families with neurofibromatosis type 1.
abstract: OBJECTIVE
  Mutation analysis of NF1, the responsible gene for neurofibromatosis type 1 (NF1), is still difficult due to its large size, lack of mutational hotspots, the presence of many pseudogenes, and its wide spectrum of mutations. To develop a simple and inexpensive NF1 genetic testing for clinical use, we analyzed five Japanese families with NF1 as a pilot study.
  
  
  METHODS
  Our original method, CEL endonuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) was optimized for NF1 mutation screening, and reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the effect of transcription. Also, we employed DNA microarray analysis to evaluate the break points of the large deletion.
  
  
  RESULTS
  A new nonsense mutation, p.Gln209(∗), was detected in family 1 and the splicing donor site mutation, c.2850+1G>T, was detected in family 2. In family 3, c.4402A>G was detected in exon 34 and the p.Ser1468Gly missense mutation was predicted. However mRNA analysis revealed that this substitution created an aberrant splicing acceptor site, thereby causing the p.Phe1457(∗) nonsense mutation. In the other two families, type-1 and unique NF1 microdeletions were detected by DNA microarray analysis.
  
  
  CONCLUSIONS
  Our results show that the combination of CHIPS and RT-PCR effectively screen and characterize NF1 point mutations, and both DNA and RNA level analysis are required to understand the nature of the NF1 mutation. Our results also suggest the possibility of a higher incidence and unique profile of NF1 large deletions in the Japanese population as compared to previous studies performed in Europe.
authors.affiliation: Department of Pediatrics, Kanazawa University Graduate School of Medical Science, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Okumura
authors.affiliation: Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, Ishikawa, Japan; Center for Medical Genetics, Kanazawa Medical University Hospital, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Ozaki
authors.affiliation: Division of Genomic Medicine, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, Ishikawa, Japan; Center for Medical Genetics, Kanazawa Medical University Hospital, Japan. Electronic address: niida@kanazawa-med.ac.jp.
authors.forename: Yo
authors.initials: Y
authors.lastname: Niida
mesh_terms: D000293:Adolescent; D000328:Adult; D044466:Asian People; D002648:Child; D002675:Child, Preschool; D018389:Codon, Nonsense; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007564:Japan; D008297:Male; D009154:Mutation; D020125:Mutation, Missense; D009456:Neurofibromatosis 1 / Q000175:diagnosis* / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010865:Pilot Projects; D017354:Point Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016622:Silver Staining / Q000379:methods; D055815:Young Adult
chemical_list: D018389:Codon, Nonsense
keywords: CEL nuclease; DNA microarray; Enzyme mismatch cleavage; Genetic testing; NF1; Neurofibromatosis type 1