Heterogeneous mutations in the gene encoding the alpha-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy.
Heterogeneous mutations in the gene encoding the alpha-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy.
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DOI：
10.1210/jcem.76.6.8388883
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发表时间：
1993-06
期刊：
影响因子：
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通讯作者：
Alexander Miric;J. D. Vechio;Michal A. Levine
中科院分区：
文献类型：
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作者：
Alexander Miric;J. D. Vechio;Michal A. Levine
奥尔布赖特遗传性骨营养不良症(Aho)是一种遗传性疾病，与鸟嘌呤核苷酸结合调节蛋白(Gsα)的α亚基活性缺陷有关，Gsα是将受体偶联到腺苷环化酶的受体。为了确定导致Gsα缺陷的突变，我们从AHO患者中提取基因组DNA，并使用聚合酶链式反应来扩增Gsα基因的外显子。使用Gsα基因外显子两侧的内含子特异性寡核苷酸引物扩增DNA。为了优化我们检测突变的能力，用5‘GC-钳从每对引物中合成了一个寡核苷酸。用变性梯度凝胶电泳法分析扩增的Gsα基因片段，以检测改变双链DNA片段熔点的突变。利用这项技术，我们已经鉴定和表征了三个突变和一个中性多态。该多态位于第5外显子，由保守第131密码子异亮氨酸残基(ATT->；ATC)的T->；C替代组成。两个突变是错义突变，其中一个家族包括外显子4的核苷酸替换(T->；C)，导致Gsα分子密码子99处的亮氨酸被Pro取代。在第二个家系中，受影响的受试者在第6外显子有一个单碱基(C->；T)突变，导致第165位密码子上的Arg被Cys取代。在第三个家系中每个受试者的一个Gsα等位基因中发现了外显子8+214位的4碱基对缺失(GTGG)。这种突变会导致Gln213密码子之后的移码，从而在缺失后导致过早终止密码子81碱基对。从培养的成纤维细胞或红细胞制备的质膜的免疫印迹分析表明，所有受影响的受试者免疫活性Gsα蛋白水平都降低。我们的结论是，编码Gsα基因的异质性突变，包括缺失和单一氨基酸替换，是AHO中Gsα缺陷的原因。
Albright hereditary osteodystrophy (AHO) is an inherited disorder associated with deficient activity of the alpha-subunit of the guanine nucleotide-binding regulatory protein (Gs alpha) that couples receptors to adenylyl cyclase. To identify mutations that lead to Gs alpha deficiency, we isolated genomic DNA from patients with AHO and used the polymerase chain reaction to amplify exons of the Gs alpha genes. DNA was amplified using intron-specific oligonucleotide primers flanking exons of the Gs alpha gene. To optimize our ability to detect mutations, one oligonucleotide from each primer pair was synthesized with a 5' GC-clamp. Amplified Gs alpha gene fragments were analyzed by denaturing gradient gel electrophoresis in order to detect mutations that alter the melting point of the double-stranded DNA fragment. Using this technique, we have identified and characterized three mutations and one neutral polymorphism. The polymorphism, located in exon 5, consisted of a T-->C substitution that conserves the isoleucine residue at codon 131 (ATT-->ATC). Two mutations were missense mutations, which in one family consisted of a nucleotide substitution (T-->C) in exon 4 that results in replacement of Leu by Pro at codon 99 of the Gs alpha molecule. Affected subjects in a second family had a single base (C-->T) mutation in exon 6 that resulted in replacement of Arg by Cys at codon 165. A 4-base pair deletion (GTGG) in exon 8 at position +214 was identified in one Gs alpha allele from each affected subject in the third family. This mutation causes a frameshift after the codon for Gln213 that results in a premature stop codon 81 base pair after the deletion. Immunoblot analysis of plasma membranes prepared from cultured fibroblasts or erythrocytes indicated that levels of immunoactive Gs alpha protein were decreased in all affected subjects. We conclude that heterogeneous mutations in the gene encoding Gs alpha, including deletions and single amino acid substitutions, are responsible for Gs alpha deficiency in AHO.