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Table of Contents
Year : 2020  |  Volume : 38  |  Issue : 4  |  Page : 217-220

Clinical manifestations and neurofibromatosis type 1 gene mutations of 25 patients with neurofibromatosis type 1 from 10 Chinese pedigrees

Department of Dermatology, Xuanwu Hospital, Capital Medical University, Changchun, Xicheng, Beijing, China

Date of Submission03-May-2019
Date of Decision11-Dec-2019
Date of Acceptance26-Dec-2019
Date of Web Publication16-Dec-2020

Correspondence Address:
Prof. Wei Zhu
Department of Dermatology, Xuanwu Hospital, Capital Medical University, No.45, Changchun St, Xicheng District, Beijing 100053
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ds.ds_49_19

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This study enrolled 25 patients with neurofibromatosis type 1 (NF1) from 10 Chinese pedigrees. Sanger sequencing analysis and multiplex ligation-dependent probe amplification analysis were used to detect mutations and large fragment losses of the NF1 gene. This study identified 10 NF1 mutations, which comprised six novel and four recurrent mutations. Majority of the mutations can lead to termination codon production, which results in the synthesis of the truncated gene product neurofibromin.

Keywords: Chinese pedigrees, clinical manifestations, neurofibromatosis type 1, neurofibromatosis type 1 gene mutations

How to cite this article:
Chen H, Lin X, Lian S, Zhu W. Clinical manifestations and neurofibromatosis type 1 gene mutations of 25 patients with neurofibromatosis type 1 from 10 Chinese pedigrees. Dermatol Sin 2020;38:217-20

How to cite this URL:
Chen H, Lin X, Lian S, Zhu W. Clinical manifestations and neurofibromatosis type 1 gene mutations of 25 patients with neurofibromatosis type 1 from 10 Chinese pedigrees. Dermatol Sin [serial online] 2020 [cited 2022 Nov 26];38:217-20. Available from: https://www.dermsinica.org/text.asp?2020/38/4/217/303705

  Introduction Top

Neurofibromatosis type 1 (NF1; OMIM # 162200) is one of the most common autosomal dominant disorders and is typically characterized by café-au-lait macules, skin neurofibromas, axilla, or abdominal freckles. Other associated features are hypophrenia, central nervous system neurofibromas, Lisch nodules, optic pathway gliomas, short stature, and increased risk for specific malignancies. NF1 is caused by mutations in the NF1 gene, which is one of the largest human genes located at 17q11.2.[1] NF1 encodes neurofibromin, which is a 2818 amino acid polypeptide that is expressed in nearly all tissues but is most highly expressed in the brain, spinal cord, and peripheral nervous system. Neurofibromin is known as a negative regulator of the Ras/Raf/MEK/ERK pathway[2] (i.e., MAPK signal pathway); however, its function is not fully understood. The NF1 gene is extremely large in terms of gene size due to the presence of pseudogenes and a lack of clear mutation hotspot. NF1 has a wide mutation spectrum from single-nucleotide substitution to large deletions. In this study, Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) technology were used to screen NF1 sequences in 25 patients with NF1 and their relatives.

  Materials and Methods Top

General clinical data

This study enrolled 25 patients that fulfilled the clinical diagnostic criteria for NF1 and were members of 10 Chinese pedigrees (i.e., 1 four generation, 2 three generation, and 7 two generation). Among these patients, 10 and 15 were male and female, respectively, with ages ranging from 3 years to 77 years.

Neurofibromatosis type 1 mutation screening

All patients with NF1 and their relatives signed the informed consent form before the start of the experiment. Subsequently, blood samples of the pedigree members were collected, and peripheral blood DNA was extracted. This procedure was approved by the Ethical Committee of Xuanwu Hospital, Capital Medical University (approval letter obtained on Feb. 13th, 2018). Polymerase chain reaction (PCR) was used to amplify all exons and exon/intron boundaries of the NF1 gene. Sanger sequencing analysis was directly conducted on the amplification products of PCR. Primers for PCR analysis were used as described by Zhang J. et al.[3] Mutations were identified after a sequence was compared with the reference complementary deoxyribonucleic acid (accession number NM_000267.3 in GenBank). A total of 100 normal blood samples without a genetic relationship to the patients with NF1 were used as controls to exclude the possibility of gene polymorphism. MLPA was sensitive to the relative quantification of nucleic acid sequences. Each MLPA probe consisted of two fluorescent-labeled oligonucleotide fragments, and each fragment contained a primer sequence and a specific sequence. During the MLPA reaction, both the probes were hybridized with the target sequence. After hybridization, the two oligonucleotides were ligated, denatured from the target sequence, and amplified by dye-labeled PCR with universal primers. The relative quantity of each PCR product was proportional to the number of copies of the target sequence.[4] MLPA was performed on patients without abnormalities in Sanger sequencing to detect possible large fragment losses in the NF1 gene.

  Results Top

Skin manifestations

All patients with NF1 (25/25) manifested café-au-lait macules. Among these patients, 19 displayed Crowe's sign, 24 had skin neurofibromas, and 4 had plexiform neurofibroma [Table 1].
Table 1: Clinical manifestations of neurofibromatosis Type 1 patients

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Manifestations beyond the skin

Further manifestations were as follows: three had intracranial neoplasm, 1 had mediastinal neurofibroma, 1 had multiple pulmonary neoplasms, and 1 had neurofibroma in the spinal cord. Three patients had difficulty in learning, and three had Lisch nodules [Table 1].

Neurofibromatosis type 1 mutation screening

This study identified 10 NF1 mutations, which comprised six novel and four recurrent mutations [Table 2]. No mutation was found in the control group (N = 100).
Table 2: Spectrum of neurofibromatosis Type 1 gene mutations identified in the present study

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  Discussion Top

Clinical manifestations of patients with neurofibromatosis type 1

NF1 mutations weaken the tumor-inhibiting function of neurofibromin, and other tumors are present in patients with NF1 at an increased incidence. Age dependence was observed in patients with NF1. Among the confirmed cases, the rates of diagnosis at 1, 8, and 20 year old were 54%, 97%, and 100%, respectively. Thus, the final diagnosis requires long-term clinical observation and follow-up.[11]

Types of mutations in the neurofibromatosis type 1 gene

The NF1 gene is located at 17q11.2. This gene is a large fragment that spans 350 kb and includes approximately 57 structural exons, 3 splicing exons (i.e., 9a, 23a, and 48a), and 60 introns. Neurofibromin, which is encoded by the NF1 gene, exists in all cells and has the highest expression in Schwann cells, neurons, and neurogliocytes. The guanosine triphosphatase-activating protein-related domain (GRD), which is encoded by exon 20a to exon 27a, is a highly conserved key functional area. Neurofibromin can activate the Guanosine triphosphate (GTP) enzyme, catalyze Ras-GTP hydrolysis into Ras-GDP (Guanosine diphosphate, GDP), and inactivate the Ras signaling pathway of proto-oncogenes. Neurofibromin can function as a tumor inhibitor by negatively regulating the RAS/MAPK and PI3K/AKT/mTOR signaling pathways.[12]

More than 3000 mutations in the NF1 gene, such as single-nucleotide substitution, oligonucleotide deletion/insertion, and large fragment deletion, have been reported. However, clear mutation hotspots have not been observed. Ars et al.[13] found that 41% of mutations are located in eight exons/flanking introns (i.e., 4b, 7, 10b, 13, 15, 20, 29, and 37), which represents 16% of the entire coding region. Yao et al.[3] proposed that the mutation frequency of exon 15 is higher than those of other exons after weighing the exon size. A 1-bp nucleotide deletion is typical in NF1 mutations. The majority of NF1 mutations can lead to the production of termination codons, which results in the synthesis of truncated neurofibromin.[14] Only 10% of NF1 mutations produce amino acid variants.

The 10 NF1 mutations identified in the present study were four nonsense mutations (i.e., c. 6686G>A, c. 2088G>A, c. 3826C>T, and c. 1318C>T), three deletion mutations (i.e., c. 7096_7101delAACTTT, c. 8077delT, and c. 4840delT), two suspected splicing mutations (i.e., IVS22 + 5G>A and IVS10 + 5G>C), and one duplication mutation (i.e., c. 3236_3240 dup TTCTA). These mutations were distributed across eight exons and two introns. Seven of eight amino acid variants produced truncated proteins, and one produced an amino acid deletion (i.e., p. 2366_2367delNF). Eukaryotic cells can identify and degrade mRNA with premature termination codon, thereby preventing the toxicity of the truncated protein on the cells.[15] Although the termination codon produced by mutation is located at the NF1 gene terminal, the extraordinary degradation of abnormal mRNA also causes significant clinical symptoms in patients with NF1.

Two forms of suspected splice mutations, namely IVS22 + 5G>A and IVS10 + 5G>C, were found in two NF1 pedigrees but not in unaffected relatives and healthy controls. Splice mutations might involve the GT-AG site or regulation sequence, which leads to incorrect NF1 splicing.[16],[17] Potential splice site variants were analyzed using Alamut Visual 2.10 (Interactive Biosoftware, Rouen, France) to verify the presence of a consistent predicted splice effect across the majority of tools. As to the Alamut Visual splicing predictions, the intronic variants IVS22 + 5G>A and IVS10 + 5G>C might interfere with the recognition of natural acceptor splice sites.

Missense mutations are common in the GRD coding region.[18] Lee et al.[14] predicted a protein defect in Leu1390Pro and Thr1787Met in conserved GRD sequences among Taiwanese patients with NF1. Thr1787Met does not affect the recognition of the myristyl anchor site. By contrast, Leu1390Pro destroys the recognition of the Ras-GTP enzyme site and may lead to the occurrence of NF1.[14] Although the GRD region is an important domain of neurofibromin, the segment that encodes the GRD region accounts for only 10% of the entire NF1 gene. Moreover, known NF1 gene mutations are not concentrated in the GRD coding region. Among the 10 mutations found in the current study, only one mutation was located in the GRD region (i.e., exon25). The other nine mutations were scattered in the other region of the NF1 gene. This finding indicated the presence of other functional regions outside GRD.

Relationship between genotypes and phenotypes

NF1 expression is highly variable and unpredictable, and its phenotypic variability may be affected by several factors, such as a second hit, modifier gene, and environmental factors.[3]

No clear correlations have been observed between a specific NF1 mutation and a particular clinical feature. The gross deletion of NF1 was observed in approximately 5% of the patients with NF1; this phenomenon may be associated with severe clinical phenotypes, such as cognitive defects, facial or body dysmorphism, and early-onset cutaneous neurofibroma.[19] However, the symptoms caused by a 3-bp in-frame deletion (i.e., c. 2970_2972delAAT) in exon 17 were mild. These patients showed typical café-au-lait macules and Lisch nodules but without neurofibromas.[13] When the missense mutation of the NF1 gene affects p. Arg1809, the patients may manifest café-au-lait macules, Lisch nodules, and Noonan syndrome-related symptoms, such as short stature, pulmonic stenosis, and less neurofibromas.[20]

Patients without detectable NF1 gene mutations may be chimeric due to postzygotic mutations. The somatic NF1 mutation of the affected tissues should be analyzed for these patients. Other NF1 gene alterations might be undetectable using the current techniques, that is, several gross NF1 gene rearrangements or alterations in introns and regulatory elements might affect transcription, RNA processing, and translation.


This study identified 10 NF1 mutations, which comprised six novel and four recurrent mutations. Majority of the mutations can lead to termination codon production, which results in the synthesis of the truncated gene product neurofibromin. The findings regarding NF1 mutations are limited by the small number of patients with NF1. Thus, we recommend that future work should explore the relationship between the genotypes and phenotypes of NF1.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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Barron VA, Lou H. Alternative splicing of the neurofibromatosis type I pre-mRNA. Biosci Rep 2012;32:131-8.  Back to cited text no. 2
Zhang J, Tong H, Fu X, Zhang Y, Liu J, Cheng R, et al. Molecular characterization of NF1 and neurofibromatosis type 1 genotype-phenotype correlations in a Chinese population. Sci Rep 2015;5:11291.  Back to cited text no. 3
Sørensen KM, Andersen PS, Larsen LA, Schwartz M, Schouten JP, Nygren AO. Multiplex ligation-dependent probe amplification technique for copy number analysis on small amounts of DNA material. Anal Chem 2008;80:9363-8.  Back to cited text no. 4
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Nasim Z, Fahim M, Ahn JH. Possible role of MADS AFFECTING FLOWERING 3and B-BOX DOMAIN PROTEIN 19 in flowering time regulation of arabidopsis mutants with defects in nonsense-mediated mRNA decay. Front Plant Sci 2017;8:191.  Back to cited text no. 15
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  [Table 1], [Table 2]


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