Tusie-Luna M T, Speiser P W, Dumic M, New M I, White P C
Division of Pediatric Endocrinology, Cornell University Medical College, New York 10021.
Mol Endocrinol. 1991 May;5(5):685-92. doi: 10.1210/mend-5-5-685.
The mild nonclassic form of steroid 21-hydroxylase deficiency is one of the most common autosomal recessive disorders in humans, occurring in almost 1% of caucasians and about 3% of Ashkenazi Jews. Many patients with this disorder carry a Val-281----Leu missense mutation in the CYP21 gene. This and most other mutations causing 21-hydroxylase deficiency are normally present in the CYP21P pseudogene and have presumably been transferred to CYP21 by gene conversion. To identify other potential nonclassic alleles, we used recombinant vaccinia virus to express two mutant enzymes carrying the mutations Pro-30----Leu (normally present in CYP21P) and Ser-268----Thr (considered a normal polymorphism of CYP21). Whereas the activity of the protein carrying the Ser----Thr mutation was indeed indistinguishable from the wild type, the enzyme with the Pro----Leu substitution had 60% of wild-type activity for 17-hydroxyprogesterone and about 30% of normal activity for progesterone when assayed in intact cells. When kinetic analysis of the latter mutant enzyme was performed in cellular lysates, the first order rate constants (maximum velocity/dissociation constant) for both substrates were reduced 10- to 20-fold compared with those for the wild-type enzyme. Pro-30 is conserved in many microsomal P450 enzymes and may be important for proper orientation of the enzyme with respect to the aminoterminal transmembrane segment. The Pro----Leu mutation was present in 5 of 18 patients with nonclassic 21-hydroxylase deficiency, suggesting that this mutation indeed acts as a nonclassic deficiency allele.
类固醇21-羟化酶缺乏症的轻度非经典形式是人类最常见的常染色体隐性疾病之一，在近1%的白种人和约3%的德系犹太人中出现。许多患有这种疾病的患者在CYP21基因中携带Val-281----Leu错义突变。导致21-羟化酶缺乏的这种突变和大多数其他突变通常存在于CYP21P假基因中，并且据推测已通过基因转换转移到CYP21。为了鉴定其他潜在的非经典等位基因，我们使用重组痘苗病毒来表达两种携带突变Pro-30----Leu（通常存在于CYP21P中）和Ser-268----Thr（被认为是CYP21的正常多态性）的突变酶。携带Ser----Thr突变的蛋白质的活性确实与野生型无法区分，而在完整细胞中进行检测时，具有Pro----Leu替代的酶对17-羟孕酮的活性为野生型活性的60%，对孕酮的活性约为正常活性的30%。当在细胞裂解物中对后一种突变酶进行动力学分析时，与野生型酶相比，两种底物的一级速率常数（最大速度/解离常数）降低了10至20倍。Pro-30在许多微粒体P450酶中是保守的，并且可能对于酶相对于氨基末端跨膜区段的正确取向很重要。Pro----Leu突变存在于18例非经典21-羟化酶缺乏症患者中的5例中，表明该突变确实作为一种非经典缺乏等位基因起作用。
