【作者】 李端琢；

【导师】 刘木根；

【作者基本信息】 华中科技大学 ， 遗传学， 2013， 博士

【摘要】 遗传性泛发型色素异常症（DUH）是一种遗传性皮肤色素异常，以全身泛发性色素沉着斑和色素减退斑相互夹杂存在为典型特征。该病与1933年被报道，但发病的病理机制尚不清楚。遗传学上已发现两个DUH致病位点，分别定位于6q24.2-q25.2和12q21-q23，但迄今为止其致病基因尚未被克隆。本论文中，我们研究了一个湖北枣阳五代汉族DUH家系，首先通过微卫星连锁分析将致病基因定位于2q33.3-2q36.1，其中微卫星标记D2S2382的LOD值为3.49。将家系中一个正常和两个病人的DNA样本进行外显子测序，结果发现了29个变异。这29个变异中位于2q33.3-2q36.1的仅有位于ABCB6基因内的c.1067T>C（p.Leu356Pro），家系中的其他成员的DNA测序，发现家系中所有的病人都含有这个突变，而所有的正常人都不携带此突变。对500个湖北正常人进行ABCB6的c.1067T>C突变验证，在所有正常人对照中均未检测到此突变。在6个散发的DUH病人中，进行ABCB6基因突变分析，发现其中的两个病人分别具有两个不同的ABCB6错义突变，分别为c.508A>G(p.Ser170Gly)和c.1736G>A (p.Gly579Glu)，而对400个正常人进行DNA测序验证，均未发现这两个突变。ABCB6是ABC转运蛋白(ATP-binding cassette transporter)家族成员。为从功能上了解释ABCB6与DUH发病之间的关系，我们首先检测了ABCB6在皮肤组织和细胞中的表达。取正常人表皮进行免疫组化检查，发现ABCB6在表皮基底层中高表达，并在黑素细胞和角质化细胞中都呈现胞质定位。人黑素瘤细胞A375和人角质化细胞系HaCaT中的Western蛋白印迹实验，证实了ABCB6在这两种表皮细胞中均有表达。进一步构建ABCB6野生型及三个突变型的EGFP融合蛋白，在鼠黑素瘤细胞B16-F1中观察ABCB6蛋白的亚细胞定位，发现ABCB6在细胞质中呈现类似黑素小体的分布，并且可在突触中聚集，而导致DUH的突变则破坏了ABCB6蛋白的正常定位，突变型ABCB6蛋白不能呈现在细胞质中的正常分布，主要滞留在细胞核周围的高尔基体中。本论文鉴定了第一个遗传性泛发型色素异常的致病基因，并对ABCB6突变引起DUH的病理机制进行初步研究。我们的发现为进一步研究ABCB6功能，揭示DUH这一疾病的分子病理机制打下了良好的基础。人类皮肤色素形成是一个复杂而有趣的生理过程，受到内外环境因子的精确调控，发现ABCB6作为一个新的色素调控因子，也为色素代谢的研究开拓了一个新的方向。更多还原

【Abstract】 Dyschromatosis universalis hereditaria (DUH) is a pigmentary genodermatosischaracterized by a mixture of hyper-and hypo-pigmented macules distributed randomlyover the body. It was initially described in1933, but the pathogenic mechanism is remainsunknown. Two chromosome loci have been reported to be associated with DUH, one ismapped to chromosome6q24.2-q25.2, and the other is on12q21-q23. However, nocausative gene(s) for DUH has been reported to date.In this study, we investigated a large five-generation Han nationality familyassociated with DUH from Hubei province. After excluding the two known loci, weperformed genome-wide linkage analysis and identified a novel DUH locus onchromosome2q33.3-q36.1with a maximum LOD score of3.49with marker D2S2382.Exome sequencing identified a c.1067T>C（p.Leu356Pro）mutation in exon3of ABCB6inthe DUH family. Direct DNA sequencing and co-segregation analysis showed that thec.1067T>C mutation occurred in all affected members, but not in any of the unaffectedindividuals of the family. DNA sequence analysis with a panel of500unaffected controlindividuals matched for the geographical location did not detect this mutation.Furthermore, additional missense mutations, c.508A>G (p.Ser170Gly) in exon1, andc.1736G>A (p.Gly579Glu) in exon12of ABCB6were found in two out of six patients bymutational screening using sporadic DUH patients. Neither of the ABCB6mutations wasfound in400control individuals.ABCB6belongs to the large family of ATP-binding cassette transporters. Toinvestigate the mechanism by which mutations of ABCB6causing DUH, we performedimmunohistologic examination in normal human skin. We found that ABCB6is expressedin the epidermis and had a diffuse cytoplasmic distribution. More over, western blotresults showed that ABCB6express in HaCaT (human keratinocyte line) and A375cells (ahuman malignant melanoma cell line). In addition, the wild-type and three mutant formsABCB6were tagged with EGFP fusion proteins and then transfected into mouse B16cells,respectively. The localization of the various fusion proteins was determined by confocalmicroscopy. Subcellular localization examination of wild-type ABCB6revealed that it localized to the endosome-like compartment, and its accumulation in the dendrites couldbe clearly seen. In comparison, disease-causing mutations of ABCB6resulted in itsretention in the Golgi apparatus.In conclusion, we identified the first causative gene for DUH. Linked theATP-binding cassette transporter6to Dyschromatosis universalis hereditaria, may be greathelp to understand the molecular basis of this disease. The metabolism of skin pigment iscomplicated and delicated. Identification of ABCB6as an pathogenic gene for pigmentarygenodermatosis, will open a novel avenue to understanding of the mechanism ofpigmentogenesis.更多还原