基于综合嵌合突变分析技术的节段型1型神经纤维瘤病的诊断策略及局部神经纤维瘤发生规律的探究

Segmental neurofibromatosis type 1 (NF1) is caused by mosaic NF1 mutation. The patients with segmental NF1 have distinct prognosis. Some only present with solitary café au-lait macule (CALM), others have neurofibromas that dramatically affect the patients' quality of life. But in clinical settings, it is hard to differentiate at an early stage. It has been known that the earlier embryo phase a mosaic mutation was derived from, the more systems of a patient would be affected. Comprehensive mosaic mutation analysis can resolve this problem. In our previous studies, we built a big NF1 genotype-phenotype database in Chinese population, and successfully identified segmental NF1 only with CALM by isolate and culture cells. We found that there existed biallelic mutations only in melanocytes, and the involved range of CALMs had a positive correlation with percentage of NF1 mutation in blood samples from segmental NF1 patients, which is suggestive of a relation with phenotypic heterogeneity. However, currently, research regarding mosaic mutation analysis and genotype-phenotype correlation analysis of segmental NF1 was lacking. In this study, we aim to establish a diagnostic algorithm based on mosaic mutation analysis, and try to take the lead in establishing a diagnosis platform for segmental NF1. Based on measurement of the mutation type and NF1 protein expression levels in cultured melanocytes and quantification of the mutation frequency of different tissues, we try to investigate the correlations with the presence of neurofibroma, which can help to predict the prognosis and lay a foundation for precision medicine.

节段型1型神经纤维瘤病（NF1）由NF1基因嵌合突变导致，不同患者间预后差异大，可只表现为咖啡斑，也可合并神经纤维瘤严重影响患者生活质量，但临床上早期难以鉴别。已知嵌合突变来源于越早的胚胎发育阶段常造成多系统累及，因此明确诊断和判断预后需综合的嵌合突变分析。我们前期构建了中国人群大样本的NF1基因型表型数据库；并成功分离培养细胞鉴定了只表现为咖啡斑的节段型NF1，发现咖啡斑皮损中仅黑素细胞存在双等位基因突变，且患者咖啡斑受累范围与血液中突变比例呈正相关，提示其与临床表型异质性相关。但目前国际上对节段型NF1的嵌合突变鉴定和基因型表型相关性研究缺乏。因此本研究旨在1）通过综合的嵌合突变分析，建立节段型NF1的基因诊断平台；2）通过分离培养咖啡斑中黑素细胞后鉴定NF1基因突变类型及蛋白表达水平，以及测定不同胚层来源组织的突变比例，探讨神经纤维瘤发生的规律，从而判断患者预后及为精准医疗奠定基础。

背景：节段型1型神经纤维瘤病(NF1, 又称为镶嵌型NF1)的特点是节段性分布咖啡斑和(或)神经纤维瘤。然而,只有节段型咖啡斑表现的镶嵌型NF1往往漏诊。节段型咖啡斑可以是非综合征性咖啡斑, 也可能与节段型NF1和其并发症密切相关。因此,一个正确的诊断对临床咨询、产前诊断和精准医学在有重要意义。.主要方法：在这项研究中, 纳入了50例伴节段型咖啡斑表现，怀疑为节段型NF1的患儿。我们从咖啡斑和正常皮肤的活检中分离培养不同细胞（黑素细胞，成纤维细胞和角质形成细胞）并进行体细胞突变分析,此外,我们在不同组织样本中进行深度测序确定镶嵌突变频率。并且研究不同节段型 NF1 患者血液中突变比例是否与节段型咖啡斑的受累范围相关..重要结果：最终测到42人有致病突变，35人明确诊断为节段型NF1，6人诊断为斑痣（为国内首次检测斑痣中的嵌合突变），1人诊断为节段型Legius综合征，另8人未测到咖啡斑相关综合征的基因突变，明确诊断为非综合征性咖啡斑（单纯性咖啡斑）。研究中发现不同节段型 NF1 患者血液中存在不同程度的突变比例， 并且与节段型咖啡斑的受累范围呈正相关..科学意义：首次在中国人群中收集了节段型NF1的临床表型与突变的数据. 通过测定血液中的突变比例， 通过评估咖啡斑的受累范围，可以判断神经纤维瘤的发生。证实了使用设计的基因测序包进行靶向深度测序是发现镶嵌型NF1的有效方法。