A de novo G to T transversion in a pro-alpha 1 (I) collagen gene for a moderate case of osteogenesis imperfecta. Substitution of cysteine for glycine 178 in the triple helical domain.
A de novo G to T transversion in a pro-alpha 1 (I) collagen gene for a moderate case of osteogenesis imperfecta. Substitution of cysteine for glycine 178 in the triple helical domain.
复制标题
中度成骨不全病例的 pro-alpha 1 (I) 胶原蛋白基因从头 G 到 T 颠换。
DOI：
10.1016/s0021-9258(18)52374-7
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
P. Pignatti
中科院分区：
文献类型：
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作者：
M. Valli;M. Mottes;R. Tenni;A. Sangalli;M. G. Lira;Antonio Rossi;Franco Antoniazzi;Giuseppe Cetta;P. Pignatti
从患有中度成骨不全症的患者身上培养的成纤维细胞在 I 型胶原分子的合成方面存在缺陷；大约一半的 α 1(I) 链在三螺旋结构域中含有半胱氨酸残基，并且当两条突变的 α 1(I) 链掺入 I 型胶原异源三聚体时形成二硫键。先证者的父母临床和生化均正常。使用 2-硝基-5-硫氰基苯甲酸的化学方法将半胱氨酸定位在三螺旋结构域残基 170 和 200 之间的肽 α 1(I)CB8 内（Tenni, R.、Rossi, A.、Valli, M.、Mottes, M.、Pignatti, P. F. 和 Cetta, G. (1990) Matrix 10, 20-26)。含有一个或两个突变链的 I 型前胶原异三聚体表现出 (i) 细胞分泌的轻微异常； (ii) 低程度的翻译后过度修饰； (iii) 热稳定性相同，但低于正常值。从先证者的真皮成纤维细胞培养物中分离总RNA，并使用总RNA制备pro-α1(I)的cDNA。通过聚合酶链式反应扩增了编码包含α1(I)三螺旋结构域残基119-193的区域的cDNA的一部分。通过 DNA.DNA 异源双链体的化学裂解鉴定出单碱基对错配，表明编码甘氨酸 178 或 181 的三联体中的鸟嘌呤可能被取代。通过化学裂解，在患者的 pro-alpha 1(I) mRNA 和正常 cDNA 探针之间形成的异源双链体中大约一半的分子中检测到相同的独特错配。对扩增产物进行克隆和测序，确认患者的杂合性并证明错义突变的存在和位置；在编码 α1(I) 三螺旋结构域残基 178 的三联体的第一个碱基中发现了单个 T 取代 G，导致半胱氨酸取代甘氨酸。等位基因特异性寡核苷酸与扩增的 DNA 杂交证实了先证者基因组中的从头点突变。该患者的研究结果与表型梯度模型一致，该模型将结构缺陷的定位与成骨不全的临床结果相关联。该突变蛋白具有一些与甘氨酸 175 的半胱氨酸取代所引起的特性不同的特性，表明邻近氨基酸对突变效果的直接影响。
Cultured fibroblasts from a patient affected with a moderate form of osteogenesis imperfecta were defective for the synthesis of type I collagen molecules; about half of the alpha 1(I) chains contained a cysteine residue in the triple helical domain and a disulfide link formed when two mutant alpha 1(I) chains were incorporated into a type I collagen heterotrimer. The proband's parents were clinically and biochemically normal. The cysteine was localized within peptide alpha 1(I)CB8 between residues 170 and 200 of the triple helical domain using a chemical procedure with 2-nitro-5-thiocyanobenzoic acid (Tenni, R., Rossi, A., Valli, M., Mottes, M., Pignatti, P. F., and Cetta, G. (1990) Matrix 10, 20-26). Type I procollagen heterotrimers containing either one or two mutant chains showed (i) a slight abnormality in secretion from cells; (ii) a low degree of post-translational overmodifications; (iii) the same, but lower than normal, thermal stability. Total RNA was isolated from the proband's dermal fibroblast cultures, and cDNAs for pro-alpha 1(I) were prepared d using total RNA. A portion of cDNA, coding for the region encompassing residues 119-193 of alpha 1(I) triple helical domain, was amplified by polymerase chain reaction. A single base pair mismatch was identified by chemical cleavage of DNA.DNA heteroduplexes, indicating a possible substitution of a guanine in the triplet coding for glycine 178 or 181. The same unique mismatch was detected by chemical cleavage in about one-half of the molecules in heteroduplexes formed between patient's pro-alpha 1(I) mRNAs and a normal cDNA probe. The amplified products were cloned and sequenced, confirming the heterozygous nature of the patient and demonstrating the presence and the location of a missense mutation; a single T for G substitution was found in the first base of the triplet coding for residue 178 of alpha 1(I) triple helical domain, leading to a cysteine for glycine substitution. Allele-specific oligonucleotide hybridization to amplified DNA confirmed a de novo point mutation in the proband's genome. The findings in this patient are in accord with the phenotypic gradient model, which correlates the localization of the structural defect with the clinical outcome of osteogenesis imperfecta. The mutant protein has some properties that differ from the caused by the cysteine for glycine 175 substitution, suggesting a direct influence of the neighboring amino acids on the effects of the mutation.