摘要
肾上腺脑白质营养不良(ALD)为X-连锁隐性遗传病，生化上以超长链饱和脂肪酸蓄积为特征。该疾病临床表现多样且诊断困难、死亡率高，如何早期诊断和治疗十分重要，但长期以来不被重视。近几年国外相继开展了ALD的研究，明确了其诊断方法。在分子生物学方面发现ALD基因定位于X染色体长臂2区8带(Xq28)，由10个外显子构成，每个外显子均可发生突变，且突变类型多样。至今国内对ALD病例很少报道，且尚未见到关于ALD基因的研究。
目的：探讨中国人ALD的临床诊断方法及分子生物学发病机制，为ALD在临床及基因水平上的诊断提供方法和实验依据。
方法：(1)依据临床表现、神经影像学及生化检测诊断12例ALD患者(2)应用聚合酶链反应(PCR)结合DNA测序技术，对收集的12例患者中抽取4例患者和3例患者母亲ALD基因外显子1-10及其侧翼进行突变检测。
结果：(1)根据患者临床表现，结合颅脑MRI显示特征性改变和超长链脂肪酸(VLCFA)检测结果诊断ALD。(2)通过对ALD基因研究发现一例患者ALD基因外显子5内含子5交界处发生了1875+G→A。这种突变可能导致ALD基因5’端剪切信号改变，使外显子5和外显子6剪接异常，合成异常不稳定ALD编码蛋白(ALDP)。(3)一例患者在ALD基因外显子6发生了Val517Ile(G→A)的碱基错义突变。另一例患者在ALD基因外显子7发生了Gln556Arg(A→G)的碱基错义突变。ALD基因编码的745个氨基酸组成的ALDP位于过氧化酶体膜上，由两个部分组成，一个为跨膜区，一个为ATP结合区。外显了6，7，8编码ATP结合区。外显子6、7突变，合成异常不稳定的ALDP，不能将二十四酸酰COA合成酶转运至过氧化物体内发挥正常功能，使VLCFA在脑白质、肾上腺及血浆聚集增多，导致疾病发生。
结论：(1)ALD为X-连锁隐性遗传病，由该基因突变而发病。通过临床表现和特异实验室检测诊断ALD。(2)ALD基因内含子5的5'剪切点突变，导致外显子5
肾上腺脑白质营养不良临床及分子生物学研究
和外显子6剪接异常，外显子6的Va1517lle（G—A）碱基错义突变和外显子7的
Gin556Arg（A—G）的碱基错义突变为卜连锁隐性遗传ALD发病原因之一。
Adrenoleukodystrophy is an X-linked disease , The characteristic biochemical abnormality of ALD is the accumulation of very-long-chain fatty acides(VLCFA). The disease have different phenotypes and high mortality rate. It is important for us to diagnose the disease and treat it in early stage. Resently,the other country's workers have been researching into the causes of ALD and have found the method of diagnosing the disease. In the aspect of molecular biology, ALD gene has been mapped to Xq28 and contains ten exons. Many kinds of mutations have been found in ALD gene. Now , we have less reports concerning the disease and have not research into the ALD gene in china.
Objective To explore the method of diagnosis and molecular mechanism of X-linked adrenoleukodystrophy,and to provide the method and the experimental evidence of diagnosis and treatment of ALD. Methods (1) Provide the method of diagnosis with physical examination, coronal MRI and biochemical measure. (2) Direct DNA sequencing and polymerase chain reaction in exons and their flanking sequences of ALD gene were performed in four patients and their mothers . Result (1) We can diagnose the disease with clinic presentation, brain MRI and increased levels of VLCFA. (2) A splice mutation were identified in interface of exon 5 and intron 5(1875+G?A). The splice mutation due to the change of splice signal on 5 end of ALD gene and leads to abnormal splice in exonS and exon6. This mutation causes unstable and abnormal ALD protein. (3) A missense mutation were identified in exon6(Va!517Tle G -A). The other missense mutation were identified in exon7(Gln556Arg A
G).The ALD gene is predicted to encode a protein of 745 amino acide consisting of six-membrane-spanning segments and a ATP-binding domain on the membrane of peroxisome. The exon 6, 7, 8 encode ATP-binging domain of ALD gene. The mutations in exon6 and exon7 lead to abnormal ALDP. The lignoceroyl COA ligase can not transport into peroxisome and can not develop his normal function. Defective 3 -oxidation of VLCFA in peroxisome has been shown to lead to an accumulation of VLCFA in the central nerve systems, adrenal gland and blood.
Conclusion (1) Adrenoleukodystrophy is an X^linked disease and caused by the gene mutation. We can diagnose the disease with clinic presentation and laboratory data. (2) The splice mutation in 5 end of intron 5 leading to abnormal splice in exonS and exonG , two missense mutation in exon6( Val517Ile G-A) and in exon7(Gln556Arg A桮) appear to be causes of X-linked recessive adrenoleukodystrophy.
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