Dermatol Sinica, June 2006 190

Form the Department of Dermatology, ChiaLi General Hospital,1 and College of Medicine, National Cheng Kung University,2 TaiwanAccepted for publication: December 20, 2005Reprint requests: Dr. Sheau-Chiou Chao, Department of Dermatology, National Cheng-Kung University Hospital, 138, Sheng-LiRoad, Tainan, TaiwanTEL: 886-6-2353535-5417 FAX: 886-6-2766180 E-mail: joly@mail.ncku.edu.tw

NF1 Gene

A Novel Deletion Mutation in the NF1 Gene in aTaiwanese Patient with Neurofibromatosis Type 1

Yi-Chen Liao1 Mei-Hui Yang2 Sheau-Chiou Chao2

Neurofibromatosis type 1 (NF1) or von Recklinghausen neurofibromatosis is one of the most com-mon autosomal dominant disorders in humans, primarily affecting cells of neural crest origin and result-ing in developmental, pigmentary, and neoplastic abnormalities. NF1 affecting 1 in 3500 individualsand fully penetrant. Mutation detection is complex due to the large size of NF1gene, the presence ofpseudogenes and the great variety of lesions. Here we presented a 20-year-old female patient who hadNF1 manifestation in school age and gene analysis revealed a novel deletion mutation (263delA).(Dermatol Sinica 24: 190-193, 2006)

Key words: Neurofibromatosis 1 (NF1), von Recklinghausen neurofibromatosis, NF1 gene, Cafe-au-laitmacules

von Recklinghausen neurofibromatosis

NF1

1/3500 NF1

20

NF1 (263delA) (

24: 190-193, 2006)

191 Dermatol Sinica, Sep 2006

INTRODUCTIONThe neurofibromatosis (NF) is a heteroge-

nous set of genetic disorder having clinicalmanifestations that involve the skin, the nervoussystem or both. At least two distinct autosomaldominant disorders have been definitely recog-nized. The most common type of NF is neurofi-bromatosis type 1 (NF1; MIM# 162200) (85%patients), which should be distinguished fromneurofibromatosis type 2 (NF2) (10% patients).

Neurofibromatosis type 1 (NF1), formerlyknown as Von Recklinghausen neurofibromato-sis, is a common genetic disorder affectingapproximately 1 in 3500 people. The conditionappears to be fully penetrant but has a highlyvariable expression, even within families.1

Diagnosis is based on the clinical criteria rec-ommended by an NIH Consensus Conference,2

which include multiple cafe-au-lait spots, cuta-neous or subcutaneous neurofibromas, plexi-form neurofibromas, axillary or inguinal freck-ling, optic gliomas, and iris Lisch nodules.Complications occur in some patients andinclude learning difficulties or mental retarda-tion, focal neurological deficits, dysplasticskeletal lesions, hypertension, and, rarely,malignancy.1 The disease is fully penetrant andexhibits a mutation rate some 10-fold higherthan that reported for most other disease genes.3

As a consequence, a high number of sporadiccases (up to 50%) are observed.4 Most diseasefeatures are present in more than 90% ofpatients at puberty.5

The disease is caused by mutations in theNF1 gene, one of the largest human genes, com-posed of 60 exons and spanning more than 300kb of genomic DNA. Due to the large numberof coding exons and the considerable mutationalheterogeneity, the determination of the NF1mutational spectrum has been complex. In thepresent study we performed mutation analysisof NF1 gene in a Taiwanese family

HISTORYThis 20-year-old female patient was quite

well in the past. Multiple cafe-au-lait maculesand neurofibromas, axillary freckles were

developed during school age. (Fig. 1)Examination of the skin revealed areas withpigmentary alterations consisting of numerous1-5 mm sized light-brown macules in a speck-led distribution, as well as several larger cafe-au-lait macules. In her family, only patient'smother has the similar skin lesions. (Fig. 2)

GENE ANALYSIS AND RESULTThe NF1 genenmapes to chromosome

17q11.2. It spans a region of about 350kb ofgenomic DNA and contains 60 exons.6-8

Phenotype expression of individual patients inthe same family can be uniquely different.Therefore gene mutation analysis was per-

Fig. 1 (A)Freckles and cafe-au-lait macules were noted over theelbow (B) Neurofibroma over the trunk.

Fig. 2 Only patient and her mother have NF1 skin manifestations.

Dermatol Sinica, Sep 2006 192

NF1 Gene

formed with PCR amplification and autosomat-ed sequencing.

Genomic DNA was extracted from periph-eral blood of the patient and family memberswith informed consent. DNA samples were thensubjected to mutation screening by amplifica-tion of segments of NF1 gene spanning all thegene using primers synthesized on the basis ofintronic sequences (GenBank accession no.M82814.1 and NT010799.1). The first base(position +1) of the initiator methionine is takenas the start of the cDNA.

For PCR amplification, approximately 200ng of genomic DNA, 12.8 pmol of each primer,10 mole dNTP and 1.25 U of AmpliTaq Gold(Perkin Elmer, Roche Molecular Systems, Inc.,Branchburg, New Jersey, USA) were used in atotal volume of 50 l . PCR and sequencingwere performed over all exons. Only one dele-tion mutation was found over exon 3.

The NF1 primer sets used for exon 3 were5'- CTG GGA GGT AAA ATG GAA GA and5'- CTC TTG GTC CAC ATC TGT AC -3'. Theamplification conditions were 94 for 5 min,followed by 35 cycles of 94 for 45 second,annealing temperature 55 for 45 second and72 for 45 second, and extension at 72 for10 min. The PCR product (289 bp) was exam-

ined on 2% agarose gel. The PCR product wassubjected to direct automated sequencing (377ABI Advanced Biotechnologies, Columbia,MD.).

Direct sequencing revealed 263delA inexon 3, which resulting frameshift and prema-ture termination of codon (PTC + 15 aa) (Fig.3).

DISCUSSIONNF1 is an autosomal dominant disease

with variable clinical manifestations. Phenotypeexpression of individual patients in the samefamily can be uniquely different. The highlyvariable clinical features of NF1 are attributedto the face that NF1 mutations involve differentfunctional domains of the protein and that othermodifier genes could also affect the diseasemanifestation.

Mutation detection in NF1 has been madedifficult by the large size of the gene, the exis-tence of a number of homologous pseudogenesequences spread throughout the genome, andthe lack of defined mutational hotspots.9 Themost common transcript codes for a polypeptideof 2818 amino acids called neurofibromin.9-12

Analysis of the protein sequence of neurofi-bromin revealed striking similarity with the cat-alytic domain of proteins that function as nega-tive regulators of Ras guanosine triphosphate(GTP)ase proteins. Loss of NF1 gene expres-sion results in absent neurofibromin Ras GAPfunction, which leads to increased Ras activityand results in increased cell proliferation andtumor formation. To date, more than 709 NF1gene mutation had been reported in the literature(http://archive.uwcm.ac.uk/uwcm/mg/search/120231.html ). Frameshift and nonsense mutationsthat would be expected to truncate the readingframe constituted about 50% mutations detect-ed. In this case, we detected a novel deletionmutation (263delA) in exon 3 which resulted inpremature termination of codon (PTC+15aa).Mutation analysis in families with NF1 allowsfor genetic counseling and prenatal diagnosis.

Fig. 3 (A) Automated DNA sequencing of the NF1 gene showed263delA in the exon 3 which resulting frameshift and pre-mature termination of codon (PTC+8aa) (B) normalsequence from the father.

193 Dermatol Sinica, Sep 2006

REFERENCES1. Huson SM, Hughes R: The neurofibromatoses: a

clinical and pathogenetic overview. London:Chapman and Hall. 1994.

2. Stumpf DA, Alksne JF, Annegers JF, et al.:Neurofibromatosis: conference statement. ArchNeurol 45: 575-578, 1988.

3. Huson SM, Compston DA, Clark P, et al.: Agenetic study of von Recklinghausen neurofibro-matosis in south east Wales. I. Prevalence, fitness,mutation rate, and effect of parental transmissionon severity. J Med Genet 26: 704-711, 1989.

4. Upadhyaya M, Cooper DN: The mutational spec-trum in neurofibromatosis 1 and its underlyingmechanisms. In: Upadhyaya M, Cooper DN (edi-tors), 1998.

5. Cawthorn RM, Weiss R, Xu G, et al.: A majorsegment of the neurofibromatosis type 1 gene:cDNA sequence, genomic structure and pointmutations. Cell 62: 193-201, 1990.

6. Viskochil D, Buchberg AM, Xu G, et al.:Deletions and a translocation interrupt a clonedgene at the neurofibromatosis type 1 locus. Cell62: 187-192, 1990.

7. Wallace MR, Marchuk DA, Anderson LB, et al.:

Type 1 neurofibromatosis gene: identification of alarge transcript disrupted in three NF1 patients.Science 249: 181-186, 1990.

8. De Raedt T, Brems H, Wolkenstein P, et al.:Elevated risk for MPNST in NF1 microdeletionpatients. Am J Hum Genet 72: 1288-1292, 2003.

9. Mattocks C, Baralle D, Tarpey P, et al.:Automated comparative sequence analysis identi-fies mutations in 89% of NF1 patients and con-firms a mutation cluster in exons 11-17 distinctfrom the GAP related domain. J Med Genet 41:e48, 2004.

10. Marchuk DA, Saulino AM, Tavakkol RL, et al.:cDNA cloning of the type 1 neurofibromatosisgene: complete sequence of the NF1 gene productGenomics 11: 931-940, 1991.

11. Danglot G, Regnier V, Fauvet D, et al.:Neurofibromatosis 1 (NF1) mRNAs expressed inthe central nervous system are differentiallyspliced in the 5' part of the gene. Hum Mol Genet 4:915-920, 1995.

12. Li Y, O'Connell P, Briedenbach HH, et al.:Genomic organization of the Neurofibromatosis 1gene (NF1). Genomics 25: 9-18, 1995.