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/jc.76.6.1560
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发表时间：
1993-06-01
影响因子：
5.8
通讯作者：
LEVINE, MA
中科院分区：
文献类型：
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作者：
MIRIC, A;VECHIO, JD;LEVINE, MA
奥尔布赖特遗传性骨营养不良（AHO）是一种遗传性疾病，与鸟嘌呤核苷酸结合调节蛋白（G（s）α）的α亚基活性不足有关，该蛋白将受体与腺苷酸环化酶偶联。为了鉴定导致G（s）α缺陷的突变，我们从AHO患者中分离基因组DNA，并使用聚合酶链反应扩增G（s）α基因的外显子。使用G（s）α基因外显子侧翼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-夹合成来自每个引物对的一个寡核苷酸。通过变性梯度凝胶电泳分析扩增的G（s）α基因片段，以检测改变双链DNA片段熔点的突变。使用这种技术，我们已经确定和特点的三个突变和一个中性多态性。该多态性位于外显子5，由保留密码子131处异亮氨酸残基的T -> C取代（ATT -> ATC）组成。两个突变是错义突变，在一个家族中由外显子4中的核苷酸取代（T -> C）组成，其导致G（s）α分子的密码子99处的Pro取代Leu。第二个家族中的受影响受试者在外显子6中有一个单碱基（C -> T）突变，导致密码子165处的Arg被Cys取代。在第三个家族中每个受影响的受试者的一个G（s）α等位基因中鉴定出外显子8 +214位的4个碱基对缺失（GTGG）。该突变导致Gln 213密码子后的移码，导致缺失后提前终止密码子81个碱基对。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，在所有受影响的受试者中，免疫活性G（s）α蛋白的水平降低。我们的结论是编码G（s）α的基因中的异质性突变，包括缺失和单个氨基酸取代，是AHO中G（s）α缺乏的原因。
Albright hereditary osteodystrophy (AHO) is an inherited disorder associated with deficient activity of the alpha-subunit of the guanine nucleotide-binding regulatory protein (G(s)alpha) that couples receptors to adenylyl cyclase. To identify mutations that lead to G(s)alpha deficiency, we isolated genomic DNA from patients with AHO and used the polymerase chain reaction to amplify exons of the G(s)alpha genes. DNA was amplified using intron-specific oligonucleotide primers flanking exons of the G(s)alpha gene. To optimize our ability to detect mutations, one oligonucleotide from each primer pair was synthesized with a 5' GC-clamp. Amplified G(s)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 G(s)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 G(s)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 G(s)alpha protein were decreased in all affected subjects. We conclude that heterogeneous mutations in the gene encoding G(s)alpha, including deletions and single amino acid substitutions, are responsible for G(s)alpha deficiency in AHO.