AR-Arg840Cys突变在雄性激素不敏感综合征（AIS）患者中功能机制的分析
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摘要
雄性激素不敏感综合征(AIS)是雄性激素受体(AR)缺陷引起的雄性性发育失常，是一种X染色体连锁疾病。患者通常具有正常的46，XY核型，其表型变化范围相当广泛，从完全的女性表型到不同程度的男性化不足。
本实验室曾报道了一个雄性激素不敏感综合征中国患者大家系，遗传分析表明，雄性激素受体的Arg840Cys突变是引起本家系疾病的原因。家系中的患者个体呈现高度变异的表型特征。本文利用了具有显著代表性的三个患者个体，利用已建立的这些个体的生殖器皮肤成纤维细胞系(GSF)，在细胞水平上研究突变受体的功能改变与表型差异的关系。
研究表明，在激素受体-配体结合实验中，和正常对照相比，患者的受体-配体最大结合能力没有明显的差异，但是患者的受体－配体结合亲和力明显降低。在受体-配体解离实验中，患者的解离速度明显快于正常个体，且三位患者中病症严重程度与受体-配体解离速度呈正相关。此外，患者中的两位不育个体在受体-配体热稳定性实验中表现为热不稳定性，而可育患者则具有热稳定性。共聚焦显微镜观察发现家系中三位患者的AR-Arg840Cys转运能力也随着男性化不足病症的加重而递减。利用荧光素酶报告基因分别研究了野生型AR和突变型AR在每株细胞内转激活能力的差异后发现，和正常个体相比突变携带者的AR转激活能力明显受损，且受损的程度与患者病症有着一定的相关性；转激活能力越低，男性化不足程度越严重。当野生型AR转入后，其转激活能力得到明显的恢复，尤其在症状最轻的患者中这一现象相当明显。推测在该患者细胞中，可能存在一些特殊的辅因子与AR作用，形成一种有效的机制，使得由突变所致的AR转激活信号传输的受损得到最大程度的恢复。本研究基于患者的GSFs，代表着患者自身的基因背景。正是由于患者间基因背景的差异，导致该家系患者显著的表型变化。为此，已利用蛋白质双向电泳及抑制消减杂交的方法，在这些个体中寻找差异表达的相关辅调控基因，以期进一步阐明AR-Arg840Cys突变一因多效性的机制。
Androgen Insensitivity Syndrome (AIS) is an X-linked disorder of male sexual differentiation, caused by an absent or dysfunctional androgen receptor (AR). Mutations in the AR gene result in a wide range of AIS phenotypes. The phenotypic spectrum ranges from a complete female phenotype to diverse male phenotype deficiency.
We have recently reported an AR-Arg840Cys from a large Chinese family affected with AIS. Genetic analysis showed that the disease trait was completely linked to an Arg840Cys mutant of AR and that all patients shared an identical Arg840Cys mutant but displayed large variation in disease phenotypes. Some of the affected males were infertile and had gynecomastia and/or hypospadias but some had fathered children normally. With an attempt to unveil the molecular mechanisms of the pleiotropic phenomenon, we have created genital skin fibroblast (GSF) cell lines directly from three patients in a Chinese family affected with androgen insensitivity syndrome (AIS). The donors of the GSF samples represent a typical pattern of the disease phenotypic variation in the affected members in the family. By making use of these primary cell lines, we find that this mutant AR has not influenced a normal androgen-binding capacity at 37℃ but has a reduced affinity for androgens and is thermolabile in the two cell lines whose donors suffer more severe disease symptom. The confocal analysis reveals that the impaired unclear trafficking of the androgen-receptor in the cell lines is highly correlated with severity of the donors' disease phenotype. Comparison in transcriptional activation between the normal and the mutant AR shows that the transactivity is significantly weakened in the mutant cell lines and the extent of the reduced transactivity depends on the donors' disease symptom. Moreover, the impaired transactivity is recovered, when induced by the plasmids carrying normal androgen receptors, to a normal level in one of the three cell lines whose donor has the mildest AIS. It suggested that there would be an effective mechanism that acts a role in regulating and compensating the defective transmission of the activation signal
Our data reveals that although etiology of AIS is monogenic, the disease phenotype varies because the main biological functions of the AR such as binding
affinity, trafficking and transcriptional activation are altered to different degrees. The study also reveals existence of the other factors that act very likely interactive roles with the AR gene in contributing to the disease phenotypic variation. As an initial attempt to explore the interaction between the AR and its co-regulators, we carried out the 2D-PAGE and suppression subtractive hybridization (SSH) experiments to hunt for these co-regulators.
本实验室曾报道了一个雄性激素不敏感综合征中国患者大家系，遗传分析表明，雄性激素受体的Arg840Cys突变是引起本家系疾病的原因。家系中的患者个体呈现高度变异的表型特征。本文利用了具有显著代表性的三个患者个体，利用已建立的这些个体的生殖器皮肤成纤维细胞系(GSF)，在细胞水平上研究突变受体的功能改变与表型差异的关系。
研究表明，在激素受体-配体结合实验中，和正常对照相比，患者的受体-配体最大结合能力没有明显的差异，但是患者的受体－配体结合亲和力明显降低。在受体-配体解离实验中，患者的解离速度明显快于正常个体，且三位患者中病症严重程度与受体-配体解离速度呈正相关。此外，患者中的两位不育个体在受体-配体热稳定性实验中表现为热不稳定性，而可育患者则具有热稳定性。共聚焦显微镜观察发现家系中三位患者的AR-Arg840Cys转运能力也随着男性化不足病症的加重而递减。利用荧光素酶报告基因分别研究了野生型AR和突变型AR在每株细胞内转激活能力的差异后发现，和正常个体相比突变携带者的AR转激活能力明显受损，且受损的程度与患者病症有着一定的相关性；转激活能力越低，男性化不足程度越严重。当野生型AR转入后，其转激活能力得到明显的恢复，尤其在症状最轻的患者中这一现象相当明显。推测在该患者细胞中，可能存在一些特殊的辅因子与AR作用，形成一种有效的机制，使得由突变所致的AR转激活信号传输的受损得到最大程度的恢复。本研究基于患者的GSFs，代表着患者自身的基因背景。正是由于患者间基因背景的差异，导致该家系患者显著的表型变化。为此，已利用蛋白质双向电泳及抑制消减杂交的方法，在这些个体中寻找差异表达的相关辅调控基因，以期进一步阐明AR-Arg840Cys突变一因多效性的机制。
Androgen Insensitivity Syndrome (AIS) is an X-linked disorder of male sexual differentiation, caused by an absent or dysfunctional androgen receptor (AR). Mutations in the AR gene result in a wide range of AIS phenotypes. The phenotypic spectrum ranges from a complete female phenotype to diverse male phenotype deficiency.
We have recently reported an AR-Arg840Cys from a large Chinese family affected with AIS. Genetic analysis showed that the disease trait was completely linked to an Arg840Cys mutant of AR and that all patients shared an identical Arg840Cys mutant but displayed large variation in disease phenotypes. Some of the affected males were infertile and had gynecomastia and/or hypospadias but some had fathered children normally. With an attempt to unveil the molecular mechanisms of the pleiotropic phenomenon, we have created genital skin fibroblast (GSF) cell lines directly from three patients in a Chinese family affected with androgen insensitivity syndrome (AIS). The donors of the GSF samples represent a typical pattern of the disease phenotypic variation in the affected members in the family. By making use of these primary cell lines, we find that this mutant AR has not influenced a normal androgen-binding capacity at 37℃ but has a reduced affinity for androgens and is thermolabile in the two cell lines whose donors suffer more severe disease symptom. The confocal analysis reveals that the impaired unclear trafficking of the androgen-receptor in the cell lines is highly correlated with severity of the donors' disease phenotype. Comparison in transcriptional activation between the normal and the mutant AR shows that the transactivity is significantly weakened in the mutant cell lines and the extent of the reduced transactivity depends on the donors' disease symptom. Moreover, the impaired transactivity is recovered, when induced by the plasmids carrying normal androgen receptors, to a normal level in one of the three cell lines whose donor has the mildest AIS. It suggested that there would be an effective mechanism that acts a role in regulating and compensating the defective transmission of the activation signal
Our data reveals that although etiology of AIS is monogenic, the disease phenotype varies because the main biological functions of the AR such as binding
affinity, trafficking and transcriptional activation are altered to different degrees. The study also reveals existence of the other factors that act very likely interactive roles with the AR gene in contributing to the disease phenotypic variation. As an initial attempt to explore the interaction between the AR and its co-regulators, we carried out the 2D-PAGE and suppression subtractive hybridization (SSH) experiments to hunt for these co-regulators.
引文
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