Clinical characteristics and mutation Spectrum of NF1 in 12 Chinese families with orbital/periorbital plexiform Neurofibromatosis type 1

A retrospective review was performed of consecutive patients diagnosed with OPPNs at Ninth People’s Hospital of Shanghai between January 2013 and June 2016. NF1 diagnosis was based on clinical features conforming to at least two of the following National Institutes of Health criteria [13]: six or more café au lait spots, axillary or inguinal freckling, two or more cutaneous neurofibromatosis, one plexiform NF, characteristic bony defects, optic glioma, two or more iris Lisch nodules, or a first-degree relative with NF1. Moreover, the probands of all families presented with plexiform NF in the orbital or periorbital region. The exclusion criteria were as follows: (1) a follow-up period < 6 months; and (2) incomplete data collection. We contacted all patients and their relatives who met the inclusion criteria and explained the purpose of the study; the participants were voluntary without any compensation. A total of 36 OPPN patients were included in this study. The OPPNs include 32 hereditary OPPNs (14 families) and 2 sporadic OPPNs. Of the 34 patients from 14 families who met the inclusion criteria, 4 were not reached, and 2 declined to participate for other reasons, such as time or geographic limitations. Of the 28 patients who agreed to return for the follow-up visit, 2 were excluded for incomplete data, leaving a final sample size of 26 patients. All 26 patients had familial OPPN.

Informed consent was obtained from all patients or their guardians at the follow-up visit. This study adhered to the tenets of the Declaration of Helsinki and was approved by the Shanghai Jiaotong University research ethics committee. The medical records were reviewed. Data collected included patient demographics, clinical characteristics, treatments and final outcomes at follow-up. The demographics consisted of age and sex. The clinical characteristics included the presence of amblyopia, motility disturbances, corneal changes, optic nerve disorders, ptosis, proptosis, canthal abnormalities, cafe au lait spots, facial descent, cheek deformities, bony orbital expansion and soft tissue expansion of eyelids. If the patients’ relative had received treatment elsewhere, his/her prior clinical details and pathological sections were retrieved for review. Regarding outcome measures, the duration from the initial diagnosis to the first recurrence was documented.

Genomic DNA was isolated from the peripheral blood leukocytes of the participants. We collected genomic DNA samples from at least two affected patients of each family (24 patients in 12 families and 2 unaffected volunteers were chosen as controls). The affected patients shared the same NF1 mutation site, which was wild-type in the unaffected patients. These mutations were not present in 100 normal subjects or in the dbSNP database (http://​www.​ncbi.​nlm.​nih.​gov/​SNP), predicted to be not tolerated by SIFT (http://​sift.​bii.​a-star.​edu.​sg/​index.​html) and to be a disease-causing variant by Mutation Taster (http://​www.​mutationtaster.​org/​). A panel of NF1 gene exons, splicing sites and promoter regions was designed and sequenced following the instructions of Ion AmpliSeq™ Library Kit 2.0 (Thermo Fisher, USA). A targeted next-generation sequencing (NGS) approach, bioinformatics analyses, and Sanger sequencing were utilized. Library preparation, qualification, and NGS were conducted using the Illumina Hiseq2000 platform (Illumina, Inc., San Diego, CA, USA). Bioinformatics analyses, including read alignments and calculations of coverage and depth, were also carried out according to a previously described protocol [14]. The following 5 databases were used for annotation of all identified variants, including dbSNP137 (http://​hgdownload.​cse.​ucsc.​edu/​goldenPath/​hg19/​database/​snp137.​txt.​gz.), HapMap Project (ftp://​ftp.​ncbi.​nlm.​nih.​gov/​hapmap), 1000 Genomes Project (ftp://​ftp.​1000genomes.​ebi.​ac.​uk/​vol1/​ftp), YH (http://​yh.​genomics.​org.​cn/​), and Exome Variant Server (http://​evs.​gs. washington.edu/EVS/). NF1 microarray data are listed in Additional file 1: Table S1.

Genomic DNA was extracted from the patients’ peripheral blood leukocytes (51,206; QIAGEN, Hilden, Germany). The region of the mutated NF1 genomic fragments was amplified by polymerase chain reaction (PCR), which was performed with 100 ng genomic DNA, 25 μL 2× GC-rich buffer, 8 μL of a dNTP mixture (2.5 mmol/L), 1 U LA Taq (Takara Biotechnology (Dalian) Co., Ltd), 2 μL (10 μmol/L) of each primer, and ddH₂O to a final volume of 50 μL. The PCR amplification protocol was as follows: 95 °C for 5 min; 40 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final elongation step at 72 °C for 3 min. The PCR products were then Sanger sequenced to produce a chromatogram. These primers used are listed in Additional file 2: Table S2.

A total of 26 eyes from 26 patients were recruited for this study. Among them, 9 patients (35%) were male and 17 (65%) female. The mean age was 26.9 ± 15.3 years, ranging from 6.0 years to 50.0 years. The mean follow-up period was 35.1 ± 19.6 months. Various clinical characteristics of Chinese patients with OPPN are displayed in Fig. 1. The most commonly involved area is the upper eyelid (Fig. 2a). Histologically, the nodules consisted of abundant collagen fibre bundles, nerve sheaths and Schwann cells (Fig. 2b). Moreover, these OPPN patients often exhibited orbital expansion (Fig. 2c), facial osteoclasia (Fig. 2d), intracranial invasion (Fig. 2e), corneal ulcer and symblepharon (Fig. 2f). Systematic manifestations, such as café au lait spots (Fig. 2g) and coexisting extraocular NFs nodules (Fig. 2 h, i), were also found.

Demographic and clinical characteristics were compared between the patients and their previous generation, and the results are summarized in Table 1. Most patients exhibited OPPN in the same laterality as their parents and/or grandparents. Of note, significantly earlier onset (p = 0.001) and more severe symptoms were observed in successive generations. Additionally, compared to their ancestors, a larger proportion of patients displayed amblyopia (p = 0.034), motility disorders (p = 0.009) and bony orbital expansion (p = 0.019). However, no statistical significance was found regarding the following: sex; the presence of ptosis; proptosis; cafe au lait spots; facial descent; cheek deformities; soft tissue expansion of the eyelids or corneal or optic nerve; or canthal abnormalities. Among the 12 patients who underwent reconstruction surgery at our hospital, 10 (83.3%) experienced recurrence. Based on Kaplan-Meier survival estimates, the 1-year recurrence rate was 83.3%, and the median duration to the initial recurrence was 7.0 months (95% confidence interval: 5.30–8.70 months).

Mutation analysis revealed a wide spectrum of NF1 mutations in this cohort (Table 2), with fourteen different mutations identified. Eleven (91.7%) OPPN families presented NF1 mutations, and 6 identified mutations were novel. Nine (64.3%) of the fourteen identified mutations were distributed in exons, three (21.4%) in splicing sites and one (7%) in an intron. Among exon mutations, three (33.3%) nonsense mutations causing premature termination of neurofibromin were detected; three frameshift mutations and three missense mutations were also discovered. These mutations were evenly distributed from exon 12 to intron 49 of the gene. Additionally, no common mutations were detected across the families, suggesting the absence of mutational hot spots. The regions of NF1 genomic DNA mutation sited were amplified by PCR (Fig. 3a), and a Sanger chromatogram confirmed novel mutations (Fig. 3b).