【作者】 王志红；

【导师】 兰风华；

【作者基本信息】 福建医科大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 X-连锁肾上腺-脑白质营养不良(adrenoleukodystrophy, X-ALD)是最常见的过氧化物酶体脂质代谢病,由ABCD1基因突变所致。该疾病基因定位于染色体Xq28,编码一个含745个氨基酸残基的蛋白质,称为肾上腺-脑白质营养不良蛋白(adrenoleukodystrophy protein, ALDP)。由ABCD1基因突变引起的ALDP结构改变或功能缺陷,使患者体内的极长链饱和脂肪酸(saturated very long chain fatty acid, VLCFA)不能进入过氧化物酶体进行β-氧化,从而在血浆和组织中蓄积,最终引起神经系统脱髓鞘和肾上腺皮质功能减退等病理改变。本研究小组在对30多个X-ALD家系、100多名家系成员进行ABCD1基因突变分析时,检测出4名尚未出现临床症状的X-ALD患者;在国内首次报道ABCD1基因新生突变和散发性X-ALD病例;对1个X-ALD家系中的2名女性杂合子患者进行了X染色体失活模式检测,并分析了突变型与野生型ABCD1基因表达量之比,结果提示:由X染色体非随机失活导致的野生型ABCD1基因表达量降低,可能是女性杂合子患者发病的原因。为了研究ABCD1基因突变对ALDP结构和功能的影响,本研究利用公认的生物信息学软件,构建了ALDP立体结构的计算机模型,对ALDP的结构功能域进行了预测,并在此基础上,对本研究小组鉴定的多个错义突变进行独立的建模,籍此为各特定突变对蛋白质稳定性的影响做出预测和评估。综合不同算法的预测结果,上述突变均位于进化保守区域,大多突变会造成蛋白质稳定性降低,易被蛋白酶体水解,从而无法行使正常的生物学功能,为致病性突变。为进一步验证以上生物信息学预测的准确性,选择R280L和L576P两个突变进行功能研究。利用分子克隆技术,构建野生型ALDP的真核重组载体:pEGFP-N2-ABCD1,并且通过人工诱导突变获得了2个突变体的真核重组载体:pEGFP-N2-ABCD1-R280L和pEGFP-N2-ABCD1-L576P。重组质粒转染HeLa细胞后,荧光显微镜和免疫印迹均可检测到野生型ALDP-GFP融合蛋白表达,但未检测到突变型ALDP表达,提示R280L和L576P突变可能造成相应的ALDP突变体稳定性下降,易被蛋白酶体水解。该结果与生物信息学预测一致。本研究可为设计更好的X-ALD诊断、预防和治疗方法提供实验依据,并深化对单基因遗传病发病机制的认识。更多还原

【Abstract】 X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder, which is characterized pathologically by progressive demyelination of the central nervous system and the adrenal dysfunction. ABCD1 is the gene responsible for X-ALD, which lies on chromosome Xq28 and encodes a peroxisomal membrane protein of 745 amino acid residues called adrenoleukodystrophy protein (ALDP). Defect in the ABCD1 gene impairs the peroxisomal capacity to metabolize very long chain saturated fatty acids (VLCFAs) viaβ-oxidation, leading to accumulation of VLCFAs in all parts of the body, including the brain and adrenal glands.From 2001 to 2010, the ABCD1 gene from more than 30 unrelated Chinese X-ALD families were analyzed in our laboratory. Here, we report the identification of 4 asymptomatic males with X-ALD from 3 pedigrees, 2 de novo mutations from 2 pedigrees and 2 heterozygous female patients from 1 pedigree, and the results of the X chromosome inactivation analysis showed that skewed X chromosome inactivation in favor of the mutant allele would be associated with the manifestation of heterozygous symptoms.To study the effects of the ABCD1 mutations on the structure and function of ALDP, different computational algorithms were used. The results showed that all of the missense mutations identified in our laboratory were at conserved codons, and the mutant ALDP might be degraded by proteasomes because of the abnormal folding, suggesting that all the mutations would be classified as pathogenic.To evaluate the accuracy of the bioinformatic predictions, functional studies on two variants (R280L and L576P) were carried out. The wild type and mutant recombinant eukaryotic expression vectors were constructed, named pEGFP-N2-ABCD1, pEGFP-N2-ABCD1-R280L and pEGFP-N2-ABCD1-L576P. Immunofluorescence study and western blotting analysis showed the expression of wild type ALDP-GFP in HeLa cells, while the mutant ALDP-GFP could not be detected, which might be due to rapid degradation. Therefore, the results of functional experiments were consistent with the computational predictions. The present study provided experimental data for the improvement of diagnosis, prevention and treatment of X-ALD, and furthers our understanding on the pathogenic mechanism of monogene disorders.更多还原