雄激素受体基因新突变引起的完全型雄激素不敏感综合征

目的 对1例完全型雄激素不敏感综合征(complete androgen insensitivity syndrom,CAIS)的雄激素受体(androgen receptor,AR)基因进行分析,寻找致病突变.方法 提取外周血全基因组DNA,扩增位于X染色体上的AR基因所有8个外显子及邻近外显子与内含子之间的剪切位点DNA序列,对扩增片段直接进行DNA序列测定,与基因库中的序列进行比对.结果 该病例AR基因第1外显子的441位密码子发生无义突变(GAA→TAA),由编码谷氨酸(Glu)变为终止密码,导致雄激素受体蛋白翻译至441位时终止,产生没有功能的氨基酸多肽残段.结论 Glu441stop(GAA→TAA)是一种导致CAIS的AR基因新的突变方式,之前在世界范围内均未见报道,丰富了AR基因突变谱,增加对CAIS发病机制的了解.     

雄激素受体基因新突变引起的完全型雄激素不敏感综合征

目的 对1例完全型雄激素不敏感综合征(complete androgen insensitivity syndrom,CAIS)的雄激素受体(androgen receptor,AR)基因进行分析,寻找致病突变.方法 提取外周血全基因组DNA,扩增位于X染色体上的AR基因所有8个外显子及邻近外显子与内含子之间的剪切位点DNA序列,对扩增片段直接进行DNA序列测定,与基因库中的序列进行比对.结果 该病例AR基因第1外显子的441位密码子发生无义突变(GAA→TAA),由编码谷氨酸(Glu)变为终止密码,导致雄激素受体蛋白翻译至441位时终止,产生没有功能的氨基酸多肽残段.结论 Glu441stop(GAA→TAA)是一种导致CAIS的AR基因新的突变方式,之前在世界范围内均未见报道,丰富了AR基因突变谱,增加对CAIS发病机制的了解.     

雄激素受体基因新突变引起的完全型雄激素不敏感综合征

目的 对1例完全型雄激素不敏感综合征(complete androgen insensitivity syndrom,CAIS)的雄激素受体(androgen receptor,AR)基因进行分析,寻找致病突变.方法 提取外周血全基因组DNA,扩增位于X染色体上的AR基因所有8个外显子及邻近外显子与内含子之间的剪切位点DNA序列,对扩增片段直接进行DNA序列测定,与基因库中的序列进行比对.结果 该病例AR基因第1外显子的441位密码子发生无义突变(GAA→TAA),由编码谷氨酸(Glu)变为终止密码,导致雄激素受体蛋白翻译至441位时终止,产生没有功能的氨基酸多肽残段.结论 Glu441stop(GAA→TAA)是一种导致CAIS的AR基因新的突变方式,之前在世界范围内均未见报道,丰富了AR基因突变谱,增加对CAIS发病机制的了解.

Abstract：

Objective To identify the mutation of human androgen receptor gene(AR) in a patient with complete androgen insensitivity syndrome (CAIS). Methods DNA sequences of 8 exons and their exon/intron boundaries of the AR gene in the patient were amplified by PCR and directly sequenced. Results DNA sequencing revealed a nonsense mutation in exon 1, resulting in a change of codon 441 GAA (glutamic acid) to a stop codon (TAA). Conclusion A novel mutation Glu441stop (GAA to TAA) of the androgen receptor gene leading to complete androgen insensitivity syndrome was identified in this study in a Chinese patient. It may help us further understanding the pathogenesis of CAIS.     

一种导致完全性雄激素不敏感综合征的AR基因移码突变的鉴定

目的 对1个男性假两性畸形完全性雄激素不敏感综合征的家系雄激素受体(androgen receptor,AR)基因进行突变检测,并分析其致病原因.方法 用PCR扩增及DNA测序等技术分析男性假两性畸形先证者候选基因AR的外显子及外显子内含子接头序列,根据检测到的突变位点情况,检测患者及其家系其他成员的相应DNA区段的碱基序列.结果 先证者及其家庭成员共3例患者均为AR基因1910delA的移码突变.其母亲为AR基因突变杂合子,是此疾病的携带者.该突变导致AR基因的N637I(AAU→AUC)、L638*(CTG→TGA)改变,导致AR蛋白283个氨基酸的截短.正常人群未发现该移码突变,该突变尚未见文献报道.结论 基因水平确定了该家系为AR基因突变引起的完全性雄激素不敏感综合征男性假两性畸形家系,同时发现了1种AR基因病理性新突变.     

完全型雄激素不敏感综合征雄激素受体基因突变分析

目的 对完全型雄激素不敏感综合征一家系雄激素受体(androgen receptor,AR)基因进行突变检测;并对发现突变的基因进行分析.方法 应用PCR扩增、DNA序列测定等技术分析所有AR基因外显子及其邻近DNA序列片段;应用核苷酸内切酶诊断方法观察其是否存在于正常人群;应用跨物种比对方法探讨突变所在位置的保守性.结果 3例患者AR基因第4外显子均发生E681D(GAG→GAT)错义突变,患者母亲为此突变杂合子携带者;患者父亲未见异常;正常人群未发现AR基因E681D突变;681位谷氨酸在不同物种间高度保守.结论 AR基因E681D(GAG→GAT)突变可能是导致完全型雄激素不敏感综合征新的突变方式.     

一个遗传性出血性毛细血管扩张症家系患者的ACVRL1基因突变分析

目的 确定一个遗传性出血性毛细血管扩张症家系患者ACVRL1基因突变位置及分析临床表型.方法 对该家系先证者ACVRL1基因常见突变位置第3、7、8外显子进行PCR和变性高效液相色谱方法筛查,随后DNA测序证实,确定突变位点后,扩增其家系另5名成员的相应区域筛查,并行DNA序列分析.结果 先证者胃黏膜见出血和毛细血管扩张,彩超显示右肾小动静脉瘘;4例患者ACVRL1基因第3外显子145delG,形成移码突变,导致第53密码子为UAG终止密码子,另2名无临床表现者未见此突变.结论 ACVRL1基因145delG突变是这个家系致病遗传基础.     

男性不育症患者X染色体雄激素受体基因外显子1、3、4状态筛选研究

目的研究雄激素受体（androgen receptor,AR）基因与特发无精症之间的关系。方法运用分子生物学方法分别检测35名无精症患者和20名对照组正常男性雄激素受体基因的3个功能区的DNA片段,结果实验组患者和正常男性AR外显子1中（CAG）n重复数在11~27之间,两组无显著差别,35例无精症患者用PCR扩增出雄激素受体外显子3（exon3）片段35例（100%）和外显子4（exon4）片段34例（97.15%）,20名正常已生育男性扩增出AR外显子3（exon3）片段20例（100%）和外显子4（exon4）片段20例（100%）。结论（CAG）n基因多态性与男性精子生成障碍无相关性,exon4缺失通过影响靶细胞核的类固醇激素-受体结合物数量在精子发生中的作用需进一步探讨。     

雄激素受体基因剪接受点突变G3346→T引起一例完全型雄激素不敏感综合征

目的－探讨完全型雄素不敏感综合征（complete androgen insensitivity syndrome,cAIS)发病的分子机理,并研究雄激素受体（androgen receptor,AR)结构,功能及其与临床表现的关系。方法 用银染聚合酶链反应－单链构象多态性（polymerase chain reaction-single strand conformation polymorphism,PCR-SSCP)及PCR产物直接测序的方法,对1例完全型AIS患者AR基因外显子B－H分别进行了研究。结果 外显子F片段在SSCP分析时有泳动变位,经测序,突变发生在内含子5与外显子F交界处G^3346→T,结论 为首例发现的AR基因剪接受点突变,GU－AG的高度保守对于维持AR的正常功能非常重要。     

45例人前列腺癌雄激素受体基因B～H外显子的突变研究

目的和方法:为探讨雄激素受体(AR)与前列腺癌(PC)发生发展及内分泌治疗不敏感的关系,我们首先建立了用PC的穿刺和石蜡切片组织检测AR基因突变的PCR-SSCP(双链构象多态性)-双链DNA循环测序法.并用上述方法检测了45例国人前列腺癌组织(6例穿刺组织,39例石蜡切片)AR基因B～H外显子的基因突变.结果:在6例患者的癌组织中证实有7个SSCP泳动变位片段(其中一患者有两个外显子异常),这6例患者中4例为低分化腺癌,其中2例已有远处转移,而序列分析证实这2例转移患者SSCP异常的外显子H片段各存在一错义突变(Glu 872 Gln、Met 886 Ile).这两种AR的基因突变国内外均未见报道,为在PC中新发现的突变.结论:实验发现AR突变均发生在前列腺癌的中晚期,提示AR基因突变可能与PC的进展和转移有关.     

多发性骨骺发育不良患者软骨寡聚物基质蛋白基因新突变

目的 对1例多发性骨骺发育不良女性患者的软骨寡聚物基质蛋白(cartilage oligomeric matrix protein,COMP)基因进行突变分析,为遗传咨询和产前分子诊断提供指导.方法 采集患者血样,提取基因组DNA,用外显子序列捕获+第二代测序技术对COMP基因进行基因突变的检测并用聚合酶链反应结合Sanger法核苷酸序列测定进行突变位点验证.结果 该患者COMP基因第9外显子存在c.956A＞T错义突变,其COMP基因第319位密码子由原来编码天冬氨酸的密码子GAC突变为编码缬氨酸的密码子GTC(p.Asp319Val).SIFT功能预测该错义突变将影响蛋白结构.结论 该患者发病是由COMP基因c.956A＞T突变导致,该突变国内外未见报道.     

Partial androgen resistance due to a distinctive qualitative defect of the androgen receptor

Using whole genital skin fibroblasts, we have characterized a novel androgen receptor mutation in a family with partial androgen resistance. The proposita was born with bilateral labioscrotal folds and a single perineal urogenital orifice. Her similarly affected maternal aunt was raised as a female with the support of gonadectomy and vaginoplasty. The mutant androgen receptor has a normal maximum binding capacity (Bmax), but an increased apparent equilibrium dissociation constant (Kd) with 5 alpha-dihydrotestosterone (DHT) and 2 synthetic androgens, methyltrienolone (MT) and mibolerone (MB). Preformed mutant DHT-receptor complexes dissociate (k) at a near-normal rate, but their MT and MB counterparts dissociate twice as quickly as normal. The native free mutant receptor is not more thermolabile than normal, but its recently dissociated counterpart is. Prolonged incubation of the cells with each of the 3 androgens causes the mutant receptor to acquire a normal increment of increased androgen-receptor activity. This androgen-sensitive pattern of misbehavior of the present mutant receptor distinguishes it from those responsible for 3 other families with partial androgen resistance studied previously. These differences will help to identify structure-function domains on the androgen receptor protein, particularly in conjunction with the use of DNA probes to analyze mutations at the X-linked androgen receptor locus.     

Decrease in androgen binding and effect of androgen treatment in a case of X-linked bulbospinal neuronopathy

X-linked recessive bulbospinal neuronopathy is a motoneuron disorder to be distinguished from amyotrophic lateral sclerosis. Effective treatment is not known. Patients with X-linked recessive bulbospinal neuronopathy may show gynecomastia and testicular atrophy, and a mutation in the androgen receptor gene has been found associated with the disease. Intermediate steps leading from the androgen receptor abnormality to the clinical syndrome have not yet been elucidated. Therefore, binding of androgen ([³H]dihydrotestosterone) to its specific receptor by genital skin fibroblasts cultured from a patient with X-linked recessive bulbospinal neuronopathy and confirmed androgen receptor mutation was studied. Markedly decreased binding capacity was found. We treated the patient for 6 months with nandrolone-decanoate. No effect on his neuromuscular status was observed during 2 years of follow-up.Abbreviations AR androgen receptor - BSN X-linked recessive bulbospinal neuronopathy     

Amber mutation creates a diagnostic MaeI site in the androgen receptor gene of a family with complete androgen insensitivity

We have discovered in the X-linked androgen receptor gene a single nucleotide substitution that is the putative cause of complete androgen insensitivity (resistance) in a family with affected individuals in 2 generations. Earlier studies on the family indicated cosegregation of mutant phenotype and the RFLPs at the loci DXS1 and DXYS1. The mutation is an adenine-to-thymine transversion in exon 8 that changes the sense of codon 882 from lysine to an amber (UAG) translation termination signal. The substitution creates a recognition sequence for the restriction endonuclease MaeI: this permits ready recognition of hemizygotes and heterozygotes after amplification of genomic exon 8 by the polymerase chain reaction. The mutation predicts the synthesis of a truncated receptor that lacks 36 amino acids at the carboxy terminus of its 252-amino acids androgen-binding domain. The cultured genital skin fibroblasts of the one affected patient examined have normal levels of androgen receptor mRNA, but negligible androgen-receptor binding activity. These results accord with a variety of data from spontaneous and artificial mutations indicating that all portions of the steroid binding domain contribute to normal steroid binding by a steroid receptor.     

Amber mutation creates a diagnostic MaeI site in the androgen receptor gene of a family with complete androgen insensitivity.

We have discovered in the X-linked androgen receptor gene a single nucleotide substitution that is the putative cause of complete androgen insensitivity (resistance) in a family with affected individuals in 2 generations. Earlier studies on the family indicated co-segregation of mutant phenotype and the RFLPs at the loci DXS1 and DXYS1. The mutation is an adenine-to-thymine transversion in exon 8 that changes the sense of codon 882 from lysine to an amber (UAG) translation termination signal. The substitution creates a recognition sequence for the restriction endonuclease MaeI: this permits ready recognition of hemizygotes and heterozygotes after amplification of genomic exon 8 by the polymerase chain reaction. The mutation predicts the synthesis of a truncated receptor that lacks 36 amino acids at the carboxy terminus of its 252-amino acid androgen-binding domain. The cultured genital skin fibroblasts of the one affected patient examined have normal levels of androgen receptor mRNA, but negligible androgen-receptor binding activity. These results accord with a variety of data from spontaneous and artificial mutations indicating that all portions of the steroid binding domain contribute to normal steroid binding by a steroid receptor.     

Leu-676-Pro mutation of the androgen receptor causes complete androgen insensitivity syndrome in a large Hutterite kindred

A large Manitoba Hutterite kindred with X-linked receptor negative complete androgen insensitivity syndrome (CAIS) was studied. In attempts to identify all carriers of the syndrome in this kindred, using the androgen receptor (AR) cDNA, we have found a novel diagnostic Mspl polymorphic pattern, which cosegregates with the disease. This polymorphism was not detected in 79 unrelated X-chromosomes of which 22 were from Hutterite controls. We were able to localize the polymorphism to exon 4, which is known to encode part of the androgen receptor hormone binding domain. A single base substitution (T→C) was detected, which creates a new Mspl site. This novel transition mutation replaces Leu-676 with Pro at a site which is conserved in numerous members of the steroid receptor gene family. Sequencing all 8 exons of the AR revealed the Leu-676→Pro mutation as the only change in the primary structure of the receptor. Transfection of COS-l cells with an expression vector of the mutant AR demonstrates that this point mutation of nucleotide 2558 abolishes receptor binding activity. The mutation can easily be detected by MspI digestion of the polymerase chain reaction (PCR) amplified exon 4 product.© 1995 wiley-Liss, Inc.     

DNA linkage analysis and studies of the androgen receptor gene in a large kindred with complete androgen insensitivity

DNA linkage analysis of the X chromosome and studies with cDNA probes specific for the androgen receptor gene were performed on the largest known kindred with the syndrome of complete androgen insensitivity. The affected subjects (XY) have absent binding of dihydrotestosterone to the androgen receptor (the receptor negative form of androgen insensitivity). In this kindred there was maternal transmission of the gene, with all affected males expressing complete genital feminization. Linkage analysis studies were conducted with two DNA probes, DXS1 and PGK1, localized to the Xq11-Xq13 region of the long arm of the X chromosome near the centromere. The results demonstrate linkage to the markers in the order of DXS1-(AR; PGK1), thus localizing the AR gene to an area between Xq11 and Xq13. Three cDNA probes that span various parts of the androgen receptor gene, including the DNA and steroid binding domain, were used to evaluate the androgen receptor gene in normal individuals, carrier mothers, and affected subjects. Identical restriction fragment patterns were found in all three groups studied. Thus the androgen receptor gene was present in affected subjects without detectable DNA polymorphism at the androgen binding domain. Therefore, despite complete absence of binding to the androgen receptor, the defect in the androgen receptor gene in this kindred is not the result of a gene deletion. The results point to a mutation or a small insertion/deletion as the probable cause of the syndrome.     

Fish granulocytes express a constitutively active androgen receptor variant

In humans, alternative splicing of androgen receptor (AR) is usually involved in some diseases. However, our knowledge about the presence of AR variants in other species and its importance for immunity is scant. Here, we report the identification of a constitutively active AR variant lacking the ligand-binding domain (LBD), ARΔLBD, in the fish gilthead seabream. ARΔLBD is expressed in the testis and the head-kidney (HK), and its expression varies with the reproductive stage and is correlated with plasma testosterone (T). In addition, ARΔLBD is expressed in acidophilic granulocytes (AGs), which are the functional equivalent of mammalian neutrophils, but not in macrophages, and its expression is modulated by both T and immune stimuli. Notably, AR and ARΔLBD were able to interact, being the activity of AR dominant at all concentrations tested of the ligand. These results reveal a new mechanism for the regulation of neutrophil biology in vertebrates and explain the conflicting results that suggest that androgens are less important than AR in human and mouse neutrophil homeostasis.     

雄激素对正常男性及一睾丸女性化综合征患者外阴部皮肤成纤维细胞雄激素受体的调节

本文研究了人工合成的雄激素－Ｒ１８８１对三例正常男性及一例睾丸女性化（ｔｅｓｔｉｃｕｌａｒｆｅｍｉｎｉｚａｔｉｏｎ，ｔｆｍ）综合征患者外阴部皮肤成纤维细胞（ｇｅｎｉｔａｌｓｋｉｎｆｉｂｒｏｂｌａｓｔｓ，ＧＳＦ）系雄激素受体（ＡＲ）的影响。实验表明，Ｒ１８８１均可增加上述细胞系ＡＲ的水平。当正常ＧＳＦ和患者ＧＳＦ（ＧＳＦｔｆｍ３）与５ｎｍｏｌ／Ｌ的Ｒ１８８１预温育２４ｈ后，其ＡＲ水平分别增至１．９６倍（为三个正常ＧＳＦ系的平均值）及１．８倍。这种增加具有时间、浓度依赖性及可逆性，表明雄激素可正向调节这些细胞系ＡＲ的水平。