RMND5A基因在遗传性泛发性色素异常症中的致病机理研究

Dyschromatosis universalis hereditaria (DUH) is an autosomal inherence genodermatosis, characterised by hyperpigmented and hypopigmented macules of the whole body. We have recruited 3 families and 8 sporadic DUH and sent them for mutational analysis in ABCB6 gene. Only one sporadic patient was found carrying ABCB6 muation. We selected one definitely diagnosed DUH family (DUH1) with no ABCB6 mutation for exome genotyping and whole-exome sequencing, one missense mutation in RMND5A gene was identified. By mutational analysis in other patients, one additional missense mutation in RMND5A was found in DUH2 family. The results suggest that RMND5A is a causative gene for DUH. Based on these achievements, the project focuses on undercovering the pathogenic role of RMND5A in DUH. By transfecting RMND5A coding and siRNA into melanocyte, we detected the related gene transcription and functional protein expression in melanocyte maturation and melanin synthesis by QRT-PCR, Western-blot and co-immunoprecipitation technologies. By constructing the transgenic zebrafish with RMND5A-PSGH2 and RMND5A siRNA injection into embryonic cells, we detect functional difference in melanocyte in vivo. By transfecting miR-138 mimics and RMND5A siRNA into human Melanoma A375 cells, we investigate the mechnism of miR-138-RMND5A-Exportin-5 in abnormal function of melanoma cell. This project will improve the cognition of DUH. Meanwhile, we will recruit more DUH cases for the RMND5A gene sequencing and improve the mutation spectrum, which attends to achieve more comprehensive genetic diagnosis.

遗传性泛发性色素异常症是一种常染色体显性遗传病，皮损表现为泛发全身的色素沉着和色素减退斑。本研究前期收集3例DUH家系和8例散发病例，在1例未携带ABCB6基因突变的DUH家系进行外显子组芯片扫描以及外显子组测序，发现了致病基因RMND5A，在另一例DUH家系中检测到了RMND5A基因的第二种突变。在前期基础上，本研究拟对人培养黑素细胞进行基因转染和siRNA干扰，构建RMND5A-PSGH2重组质粒转染斑马鱼胚胎细胞，通过QRT-PCR、Western-blot及酪氨酸酶活性测定等技术检测成鱼活体内黑素细胞功能变化。利用miR-138 mimics，RMND5A siRNA对人黑色瘤A375细胞进行转染，探讨miR-138-RMND5-Exportin-5通路在黑素细胞功能异常中的作用机制。丰富DUH发病机理认识。同时完善RMND5A基因突变谱，为日后基因诊断打好基础。

遗传性泛发性色素异常症（Dyschromatosis universalis hereditaria, DUH）是一类存在遗传异质性的以色素异常为特征表现的遗传性皮肤病。本课题在前期收集的一例包含4代48例个体的DUH家系中未检测到既往基因突变。在该家系内使用外显子组测序在既往报道的6q24.2-q25.2定位区域内发现致病突变SASH1：c.1761C>G (p.Ser587Arg)。构建定点突变质粒并转染细胞，发现该突变使得SASH1表达下调76.8%。使用人类表达谱芯片分析筛查出该突变导致的SASH1表达改变可能通过THBS1影响TGF-β1通路介导的黑色素细胞迁移功能改变。GeneChip primeview human arrays对SASH1突变型及野生型转染细胞进行分析，IPA分析发现突变组内SASH1表达改变可能通过THBS1影响TGF-β1通路介导的黑色素细胞迁移功能改变，进一步的功能研究证实了SASH1下调促进细胞增殖、迁移，提高细胞侵袭能力，诱导细胞EMT，并抑制细胞的黑色素生成。反之，SASH1过表达抑制细胞增殖、迁移、细胞侵袭以及EMT，增强细胞的黑色素生成。下调SASH1，使得TGF-β1的表达显著升高，外源TGF-β1刺激抑制SASH1表达。THBS1过表达显著减弱SASH1沉默诱导的TGF-β1表达升高。活体实验SASH1下调显著促进A375细胞在裸鼠体内的成瘤能力，TGF-β1抗体可以逆转SASH1下调对裸鼠体内成瘤能力的促进作用。