Balkan Journal of Medical Genetics

NEXT GENERATION SEQUENCING IDENTIFIED A NOVEL MULTI EXON DELETION OF THE NF1 GENE IN A CHINESE PEDIGREE WITH NEUROFIBROMATOSIS TYPE 1
Yang J, An J-X, Liu X-L, Wang Z-Q, Xie G-M, Yang X-L, Xu S-J, Feng F, Ni Y -- Jie Yang and Jinxia An contributed equally to this study.
*Corresponding Author: Dr. Yali Ni, Gansu Provincial Maternity and Child-Care Hospital, 143 Qilihe North Street, LanZhou, Gansu Province, 730050, People’s Republic of China. Tel.: +86-0931-2231241. E-mail: niyali@126.com
page: 45

CASE REPORTS

Two individuals in a Chinese family were clinically diagnosed with NF1, based on the consensus criteria of the National Institutes of Health (NIH) [2], 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 NF1 genes that are associated with NF1. 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 [3]. 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 NF1 Gene. Next generation sequencing identified a novel multi exon c.(4661+1_4662-1)_(5748 +1_5749-1)del; EX36_39DEL deletion on the NF1 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 Quantitative Real-Time Polymerase Chain Reaction (qPCR). We performed qPCR to validate the multi exon deletion on the NF1 gene identified by NGS. The qPCR was performed using an ABI PRISM® 7900HT realtime 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.

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