Neurofibromatosis type 1 (NF1) [MIM# 162200] is a rare, autosomal dominant neurocutaneous disease. Neuro-fibromatosis type 1 manifests with neurofibroma (fibromatous skin tumors), café-au-lait spots and skin fold freckling . Neurofibromatosis type 1 patients usually present with extreme inter- and intra-familial phenotypic variability with an incidence of 1/3500 live births . Germline mutations in the NF 1 gene causes NF 1. The NF 1 gene encodes neurofibromin consisting of 2818 amino acids, highly expressed in neurons and different types of neuronal cells. In addition, neurofibromin activates protein by down-regulating the function of the renin-angiotensin system (RAS) .
Two individuals in a Chinese family were clinically diagnosed with NF1, based on the consensus criteria of the National Institutes of Health (NIH) , at the Gansu Provincial Maternity and Child-Care Hospital, LanZhou, Gansu Province, People’s Republic of China (PRC) (Figure 1). In our hospital, detailed physical examination, laboratory investigations were done. The proband (II-1) was a 31-year-old Chinese male with neurofibroma and café-au-lait macules on his chest and back [Figure 2(A), 2(B)]. In the proband, café-au-lait macules are gradually increasing in size as well as developing new café-au-lait macules. Several tender masses were identified on the chest and back of the proband. Physical examination of these tender masses were consistent with neurofibroma.
The proband’s mother (I-2) was 65 years old and presented with multiple subcutaneous neurofibromas and café-au-lait spots [Figure 2(C), 2(D) and 2(E)]. The proband’s father (I-1) was normal. Physical growth and cognitive ability was normal and no other abnormality was identified in either the proband or his mother.
The Ethics Committee of the Gansu Provincial Maternity and Child-Care Hospital approved the present study. Next generation sequencing (NGS) and Sanger sequencing was performed to identify the candidate gene and mutation in the proband. The proband’s blood sample was collected and genomic DNA was extracted.
Next Generation Sequencing. DNA samples obtained from the proband (II-1) were sequenced using target exome-based NGS. A sequence capture human array (Roche NimbleGen Inc., Madison, WI, USA) was designed to capture the targeted sequence, covering all 57 exons and flanking sequences (including the 100 bp introns) of the NF 1 genes that are associated with NF 1. On average, there are 6,857,687 reads per sample, with 87.48% of mapping to the targeted regions. The average sequencing depth of the target area was 428.14-times with 99.86% coverage.
The procedure for preparation of libraries was consistent with standard operating protocols published previously . According to the standard protocol, we simultaneously sequenced 30 samples on Illumina HiSeq 2500 Analyzers (Illumina, San Diego, CA, USA) for each pooling batch of 90 cycles (specially designed rare disease screening). We applied Illumina Pipeline software (version 1.3.4) to generate the raw data that was later used for bioinformatic analysis. We extracted the clean reads from the raw reads by using already established filtering criteria. We then selectively used at least 90 bp long clean reads for aligning to the human reference genome (Build 37) of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/grc) database using Burrows Wheeler aligner (BWA). The BWA is a multi-vision software package (http://bio-bwa.sourceforge.net/index.shtml) generating the output file in BAM format. After that, we analyzed the target region coverage, sequencing depth, SNP/InDel/CNV detection by using the bam data. Later, SOAPsnp software (www.soap/genomics.org.cn), Sam tools pileup software (www.htslib.org) and Bioinformatic computational framework (this explains the pipeline made by the software), were established for identifying single nucleotide polymorphisms (SNPs), insertions/deletions (InsDels) and copy number variations (CNVs). Filtering criteria for a SNP or InDel has been set with at least 10 reads with >20.0% of total reads. The SNPs are filtered out if it’s frequency is <0.05 in dbSNP (www.ncbi.nlm.nih.gov/projects/SNP/), Hapmap (https://www.genome.gov), 1000 Genomes Project (http://www.1000genomes.org), the 200 healthy reference samples sequenced in this study, it would consider as a disease-causing mutation.
Identification of a Novel Heterozygous Multi Exon Deletion in the NF 1 Gene. Next generation sequencing identified a novel multi exon c.(4661+1_4662-1)_(5748+1_5749-1)del; EX36_39DEL deletion on the NF 1 gene in the proband and in his mother. This multi-exon deletion leads to the formation of a truncated neurofibromin instead of the wild type neurofibromin. This mutation has been validated in proband and his parents by q-PCR (Figure 3). This mutation has not been identified among the 100 normal healthy control subjects.
Quantitative Real-Time Polymerase Chain Reaction (qPCR). We performed qPCR to validate the multi exon deletion on the NF 1 gene identified by NGS. The qPCR was performed using an ABI PRISM® 7900HT real-time PCR system (Life Technologies, Carlsbad, CA, USA) and hot-start qPCR Master Mix. The primer sequences and PCR conditions will be provided by the corresponding author upon request. Data were analyzed using the comparative threshold cycle (2–ΔΔCt) method.
Neurofibromatosis type 1 is a rare neurocutaneous inherited genetic disease caused by germline mutations in the NF 1 gene. In the human genome, NF 1 is one of the largest genes with a very high mutation rate with extreme mutational heterogeneity. Over 1000 germline (missense, deletion, insertion, nonsense, splice site mutations) mutations of the NF 1 gene have been reported so far . In this study, the novel heterozygous multi exon deletion causes the formation of a truncated neurofibromin which affects the normal function of the NF 1 gene. Neurofibromatosis type 1 patients usually presented with two major clinical symptoms; i.e., neurofibromata and the café-au-lait spots, which increase in number and gradually developed into larger spots with increasing age. Hence, a clear gradient or degree of phenotypic variation is common in affected individuals of the same family . Recently, in their two studies, Banerjee et al. [1,6] reported that clinical manifestations for NF1 patients are extremely heterogenous, even associated with astrocytoma, tibial pseudarthrosis and anemia. In this study, we showed that the proband and his mother presented with severe and mild phenotypes, respectively, although they carry the same mutation. Moreover, in addition of the genetic factors, there are also epigenetic factors that influence the phenotypic heterogeneity in NF1 patients .
In conclusion, we identified a Chinese family with NF1. Next generation sequencing and qPCR identified a novel heterozygous multi exon deletion c.(4661+1_4662-1)_(5748+1_5749-1)del; (EX36_39DEL) on the NF 1 gene causing NF1 in this family. We have added a heterozygous novel NF1 mutation associated with an NF1 phenotype as well as understanding the signification of the application of NGS for rapid and accurate screening of the specific disease-associated genes that provide the correct clinical diagnosis for NF1.
We wish to thank all the members of this family for participating in our study. We also thank Dr. Santasree Banerjee (Beijing Genomics Institute-Qingdao, Beijing Genomics Institute-Shenzen, Qingdao, Shandong Province, PRC) for her careful reading and editing of the manuscript.
This project was supported by Grant from The Gansu Natural Science Foundation of China (Grant Number: 17JR5RA027).
Banerjee S, Dai Y, Liang S, Chen H, Wang Y, Tang L, et al. A novel mutation in NF1 is associated with diverse intra-familial phenotypic variation and astrocytoma in a Chinese family. J Clin Neurosci. 2016; 31(9): 182-184.
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Yang L, Banerjee S, Cao J, Bai X, Peng Z, Chen H, et al. Compound heterozygous variants in the coiled-coil domain containing 40 gene in a Chinese family with primary ciliary dyskinesia cause extreme phenotypic diversity in cilia ultrastructure. Front Genet. 2018; 9(2): 23. doi: 10.3389/fgene.2018.00023. eCollection 2018.
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Yang L, Banerjee S, Cao J, Bai X, Peng Z, Chen H, et al. Compound heterozygous variants in the coiled-coil domain containing 40 gene in a Chinese family with primary ciliary dyskinesia cause extreme phenotypic diversity in cilia ultrastructure. Front Genet. 2018; 9(2): 23. doi: 10.3389/fgene.2018.00023. eCollection 2018.)| false 10.3389/fgene.2018.00023
Pasmant E, Parfait B, Luscan A, Goussard P, Briand-Suleau A, Laaaaurendeau I, et al. Neurofibromatosis type 1 molecular diagnosis: What can NGS do for you when you have a large gene with loss of function mutations? Eur J Hum Genet 2015; 23(5): 596-601.
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Pasmant E, Parfait B, Luscan A, Goussard P, Briand-Suleau A, Laaaaurendeau I, et al. Neurofibromatosis type 1 molecular diagnosis: What can NGS do for you when you have a large gene with loss of function mutations? Eur J Hum Genet 2015; 23(5): 596-601.)| false 10.1038/ejhg.2014.145
Banerjee S, Lei D, Liang S, Yang L, Liu S, Wei Z, et al. Novel phenotypes of NF1 patients from unrelated Chinese families with tibial pseudarthrosis and anemia. Oncotarget. 2017; 8(24): 39695-39702.