Identification of sterol 27 hydroxylase gene mutations in CTX patients

脑腱硬化症(CTX)是一种常染色体隐性遗传性脂肪储存疾病，由固醇27-羟基酶基因(CyP27)突变引起。对一名24岁的日本女性CTX患者及其父母进行了细胞色素P27基因突变的研究。患者来源的成纤维细胞中的甾醇27-羟基酶活性检测不到，而其母亲和父亲的成纤维细胞中的活性分别为正常水平的54%和41%。直接测序分析显示，患者的CYP27基因第362位密码子(CGT 362Arg至CAT 362His)存在A替换G的错义突变，患者为纯合子模式，父母为杂合型模式。该突变消除了cDNA1195位的正常Hga I酶切位点，位于该基因的肾上腺素结合区，很可能是导致这个日本CTX家族中固醇27-羟基酶活性降低的原因。来自两个无关家族的另外三名日本CTX患者也被进行了遗传学研究。通过DNA序列分析，在其中一个CTX家族中发现了一个新的氨基酸372位A替代G突变(CGG372Arg到CAG372Gln)。另一家系的两名患者也发现了该突变，其第441位氨基酸(CGG441Arg至CAG441Gln)为A替代G的复合杂合型模式。后一种突变与本课题组以前报道的相同(J.Lipid Res.1994，35：1031-1039)。由于这两个突变改变了限制性内切酶的位点，因此发展了检测携带者的快速筛查方法。将这两个突变的cDNA导入COS细胞后，其甾醇27-羟基酶活性明显降低。这些结果表明，这三个突变是导致这些患者的甾醇27-羟基酶活性不足的原因。

Cerebrotendinous xathomatosis (CTX), an autosomal recessive lipid-storage hereditary disorder, is caused by mutations in the sterol 27-hydroxylase gene (CYP27). A 24-year-old female Japanese CTX patient and her parents were studied for a CYP27 mutation. Sterol 27-hydroxylase activity in fibroblasts derived from the patient was undetectable, while the activities in fibroblasts from her mother and father were 54% and 41% of the normal level, respectively. Direct sequence analysis showed a missense mutation of A for G substitution in the CYP27 gene at codon 362 (CGT 362Arg to CAT 362His) with a homozygous pattern in the patient, and a heterozygous pattern in the parents. The mutation, which eliminates a normal Hga I endonuclease site at position 1195 of the cDNA and is located at the adrenodoxin binding region of the gene, is most probably responsible for the decreased sterol 27-hydroxylase activity in this Japanese CTX family.Other three Japanese CTX patients from two unrelated families were also studied genetically. By DNA sequence analysis a novel mutation of A for G substitution at amino acid position 372 (CGG 372Arg to CAG 372Gln) was identified in one of the CTX families. The mutation was also found in two patients from the other family, with a compound heterozygous pattern of A for G substitution at amino acid position 441 (CGG 441Arg to CAG441Gln). The latter mutation was the same as previously reported by our group (J.Lipid Res. 1994,35 : 1031-1039). As the two mutations changed the restriction enzyme sites, rapid screening methods were developed for the detection of the carriers. Transfection of the two mutant cDNAs into COS cells resulted in markedly reduced sterol 27-hydroxylase activity. These results indicate that the three mutations are responsible for the deficiency of the sterol 27-hydroxylase activity in these patients.