一个新FPH致病基因的鉴定与功能分析

Familial progressive hyperpigmentation(FPH) is an autosomal dominant hereditary disorder characterized by patches of hyperpigmentation in the skin which were present at birth or in early infancy and increase in size and number with age. It is likely to be a heterogenous disease. To date, a Chinese family with FPH has been demonstrated to be caused by the KITLG N36S mutation, which has a gain-of-function effect on the melanin synthesis. We have collected six FPH pedigrees so far and excluded the possibility that they link to KITLG by Sanger sequencing and linkage analysis of microsatellite markers next to KITLG. At the same time, we have screened 3 key individuals from a five-generation Chinese family with FPH (Pedigree 1）by exome sequencing, and found 34 variants candidate for disease related pathogenic mutation. In this project，these variants will be first validated by using direct DNA sequence in other members of the FPH pedigree 1, a disease-associated variant among of them will be identified for its cosegregation perfectly with affected, but not with unaffected, members of the pedigree. After excluded the possibility of Single Nucleotide Polymorphisms （SNPs） in a panel of 500 unaffected control individuals matched for the geographical location, the candidate causative gene will be further confirmed by sequencing its exons in other families with FPH and sporadic cases, then a novel causative gene responsible for FPH will be verified. After a comparative analysis for its origin is performed in database online, its functions will be analyzed with over-expression gene technology or RNA interference technology. These results will clarify the pathologic mechanisms of FPH, and help us understand skin pigmentation.

家族性进行性色素过度沉着症(FPH)是一种常染色体显性遗传病，具有遗传异质性。迄今仅有一个中国FPH家系发现KITLG基因突变（N36S）与发病有关。近年来，本课题组通过对KITLG基因测序分析和与之紧密连锁微卫星标记的连锁分析，排除了已收集的六个FPH家系与KITLG基因位点连锁的可能性；同时，采用全基因组外显子测序技术对一个五代FPH家系（家系1）中3个关键成员进行了测序分析，筛选得到34个候选突变位点。本项目拟首先应用DNA测序技术对候选致病基因在该家系与收集的其它家系成员，以及500名正常对照者中进行验证，鉴定一个新FPH致病基因；经Internet数据库同源性分析后，利用基因过表达技术和RNA干扰技术进行该基因的功能分析，阐明该基因的突变如何引起皮肤色素过度沉着的病理生理机制。

家族性进行性色素过度沉着症(FPH)是一种常染色体显性遗传性皮肤病。为了精确定位我们收集的一个五代FPH家系1的致病基因，首先采用SNPs芯片对该家系进行基因分型与连锁分析(Human Zhonghua-8 Bead–Chips)，发现在第15号染色体rs1026369～rs11857925区域间LOD值介于3.386-3.806，表明一个新FPH致病基因紧密连锁于该区域。结合前期全基因组外显子测序结果，综合分析发现了一个候选致病基因---ADAM10。采用DNA测序法对该FPH家系1所有成员进行ADAM10突变筛查，发现该基因第12号外显子剪接位点突变c.1511+1G>A与疾病表型共分离。在相同地区人群中招募正常对照者300人，采用DNA测序法分析该位点碱基状态，未发现相同突变，排除其为SNPs的可能性。采用ADAM10单抗对FPH家系1先证者的色素沉着斑和正常皮肤进行免疫组化染色分析，结果显示：ADAM10在这些皮肤中均有表达。同时运用DNA测序法分析了另一个两代FPH家系成员的ADAM10所有外显子，发现该基因第9号外显子存在错义突变 c.1172C>T (p.Ser391Phe)，且与疾病表型共分离。SNPs排除实验未发现相同突变。经UCSC网站查询，该位点在多种物种进化过程中均保持高度保守性，提示ADAM10错义突变 c.1172C>T (p.Ser391Phe)具有致病性。以上结果证实ADAM10致病突变导致了FPH发病。. 利用常规分子生物学技术构建野生型质粒pEZ-M98-ADAM10，并测序验证正确。采用QuikChange定点突变试剂盒进行突变型pEZ-M98-ADAM10质粒构建，经测序验证而得到突变型ADAM10。采用Lipofectamine 2000试剂盒将这些表达质粒分别转染A375细胞，采用NaOH裂解法测定各组细胞黑素含量，发现野生型与突变型质粒转染组的黑素含量均较对照组升高 (P<0.05)。采用Real-time PCR和Western blot法分别检测各组细胞黑素合成酶TYR和TYRP1的mRNA和蛋白表达水平，均未发现统计学差异。提示ADAM10调控黑素代谢可能存在其它分子机制，需要进一步深入研究。.