Murono K, Mendonca B B, Arnhold I J, Rigon A C, Migeon C J, Brown T R
Hospital das Clinicas, Faculdade de Medicina da Universidade de Sao Paolo, Brazil.
Hum Mutat. 1995;6(2):152-62. doi: 10.1002/humu.1380060208.
Mutations of the human androgen receptor gene were identified in five subjects from four families with androgen insensitivity syndrome. Individual exons of the androgen receptor gene were amplified by the polymerase chain reaction from genomic DNA and screened for sequence-dependent differences in their melting characteristics by denaturing gradient gel electrophoresis. DNA fragments from exons with altered mobility were sequenced. Four different single nucleotide base substitutions were found within exons 5, 6, and 7 encoding the steroid-binding domain of the androgen receptor. In one subject with ambiguous genitalia, amino acid residue 763 was changed from tyrosine to cysteine (TAC-->TGC; Y763C). Four subjects, including two siblings, had complete androgen insensitivity. In one subject, residue 779 was changed from arginine to tryptophan (CGC-->TGG; R779W), another subject (M807V) had a substitution of valine (GTG) for methionine (ATG) residue at position 807, and the two siblings (R855C) had a mutation in residue 855 changing arginine (CGC) to cysteine (TGC). Binding of the synthetic androgen ligand, methyltrienolone (R1881), by the mutant receptor Y763C was decreased by 54% compared to the normal receptor. Transcriptional activation of a mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene by AR mutant Y763C was negligible at 0.1 nM R1881 and only 55% at 10 nM R1881 when compared to the maximal response with the normal AR, as assessed by CAT activity. Mutant M807V retained only 22% of normal R1881 binding and mutant R855C was unable to bind the steroid. In accordance with the steroid binding, transcriptional activation of MMTV-CAT by M807V rose to only 26% of control in the presence of 10 nM R1881, a concentration at which R855C remained functionally inactive. In summary, missense mutations within the exons of the androgen receptor gene encoding the steroid-binding domain of the receptor are common causes of both partial and complete forms of androgen insensitivity syndrome.
在来自四个家族的五名雄激素不敏感综合征患者中鉴定出了人类雄激素受体基因的突变。通过聚合酶链反应从基因组DNA中扩增雄激素受体基因的各个外显子，并通过变性梯度凝胶电泳筛选其熔解特性的序列依赖性差异。对迁移率改变的外显子的DNA片段进行测序。在编码雄激素受体类固醇结合结构域的外显子5、6和7中发现了四种不同的单核苷酸碱基替换。在一名生殖器模糊的患者中，氨基酸残基763从酪氨酸变为半胱氨酸（TAC→TGC；Y763C）。四名患者，包括两名兄弟姐妹，患有完全雄激素不敏感。在一名患者中，残基779从精氨酸变为色氨酸（CGC→TGG；R779W），另一名患者（M807V）在第807位的甲硫氨酸（ATG）残基被缬氨酸（GTG）取代，两名兄弟姐妹（R855C）在残基855处发生突变，精氨酸（CGC）变为半胱氨酸（TGC）。与正常受体相比，突变受体Y763C对合成雄激素配体甲基三烯olone（R1881）的结合减少了54%。通过AR突变体Y763C对小鼠乳腺肿瘤病毒-氯霉素乙酰转移酶（MMTV-CAT）报告基因的转录激活，在0.1 nM R1881时可忽略不计，与正常AR的最大反应相比，在10 nM R1881时仅为55%，通过CAT活性评估。突变体M807V仅保留了正常R1881结合的22%，突变体R855C无法结合类固醇。与类固醇结合情况一致，在存在10 nM R1881的情况下，M807V对MMTV-CAT的转录激活仅升至对照的26%，在该浓度下R855C仍然功能失活。总之，编码受体类固醇结合结构域的雄激素受体基因外显子内的错义突变是雄激素不敏感综合征部分和完全形式的常见原因。
