Supplementary Material for: Childhood-onset Refractory Hypertension Results from Neurofibromatosis type 1 Caused by a Splicing NF1 mutation

Background: Neurofibromatosis type 1 (NF-1) is caused by mutations in the NF1 gene that encodes neurofibromin, a negative regulator of RAS proto-oncogene. Approximately one-third of the reported pathogenic mutations in NF1 are splicing mutations, but most consequences are unclear. The objective of this study was to identify the pathogenicity of splicing mutation in a Chinese family with NF-1 and determine the effects of the pre-mRNA splicing mutation by in vitro functional analysis. Methods: Next-generation sequencing was used to screen candidate mutations. We performed a minigene splicing assay to determine the effect of the splicing mutation on NF1 expression and three-dimensional structure models of neurofibromin were generated using SWISS-MODEL and PROCHECK method, respectively. Results: A pathogenic splicing mutation c.479+1G>C in NF1 was found in the proband characterized by childhood-onset refractory hypertension. In vitro analysis demonstrated that c.479+1G>C mutation caused skipping of exon 4, leading to a Glutamine to Valine substitution at position 97 in neurofibromin and an open reading frame shift terminating at codon 108. Protein modelling showed that several major domains were missing in the truncated neurofibromin protein. Conclusion: The splicing mutation c.479+1G>C identified in a Chinese patient with NF-1 and childhood-onset refractory hypertension caused skipping of exon 4 and a truncated protein. Our findings offered new evidence for the molecular diagnosis of NF-1.

背景：神经纤维瘤病1型（Neurofibromatosis type 1, NF-1）由NF1基因突变所致，该基因编码的神经纤维蛋白（neurofibromin）是RAS原癌基因（RAS proto-oncogene）的负调控因子。目前已报道的NF1致病性突变中，约三分之一为剪接突变，但多数此类突变的致病效应尚不明确。本研究旨在明确一个中国NF-1家系中剪接突变的致病性，并通过体外功能分析阐明该前mRNA（pre-mRNA）剪接突变的分子效应。
方法：采用下一代测序（Next-generation sequencing）技术筛选候选突变。通过迷你基因剪接实验（minigene splicing assay）验证该剪接突变对NF1基因表达的影响，并分别借助SWISS-MODEL与PROCHECK工具构建神经纤维蛋白的三维结构模型。
结果：先证者表现为儿童起病的难治性高血压，其携带NF1基因致病性剪接突变c.479+1G>C。体外实验证实，c.479+1G>C突变可导致第4号外显子（exon 4）跳读，进而使神经纤维蛋白第97位的谷氨酰胺（Glutamine）突变为缬氨酸（Valine），并引发开放阅读框（open reading frame）移码，最终在第108位密码子（codon）处提前终止翻译。蛋白质结构建模显示，截短后的神经纤维蛋白缺失了多个关键结构域。
结论：本研究在1例合并儿童起病难治性高血压的中国NF-1患者中鉴定出剪接突变c.479+1G>C，该突变可导致第4号外显子跳读并产生截短型蛋白。本研究结果为NF-1的分子诊断提供了新的实验依据。