A new diagnostic procedure to detect unknown transthyretin (TTR) mutations in familial amyloidotic polyneuropathy (FAP)
A new diagnostic procedure to detect unknown transthyretin (TTR) mutations in familial amyloidotic polyneuropathy (FAP)
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DOI：
10.1016/s0022-510x(99)00326-3
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发表时间：
2000-02-15
影响因子：
4.4
通讯作者：
Ando, M
中科院分区：
文献类型：
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作者：
Yamashita, T;Ando, Y;Ando, M
对两例甲状腺素运载蛋白（TTR）所致的淀粉样变性患者进行了免疫组织病理学、质谱和分子遗传学研究。在用抗TTR多克隆抗体证实淀粉样沉积物中TTR的免疫反应性后，采用离心浓缩-电喷雾质谱（ESI-MS）检测血清中TTR的变异体。分析仅需50 μ l血清和30 μ l抗TTR抗体。与抗体孵育后，将样品通过1000 kDa截留离心浓缩器以保留抗体，此后，通过ESI-MS分析滤液。在血清样品中检测到几种形式的正常和变异TTR：未缀合的TTR、半胱氨酸和半胱氨酸-甘氨酸缀合的TTR。在患者中，检测到TTR的变异形式，其分子量比正常TTR高26.0 Da。单链构象多态性（SSCP）和直接序列分析证实存在一个碱基取代位于密码子50 AGT（Ser）ATT（Ile）在这两名患者，这对应于增加的分子量为26.0。目前的诊断程序表明，ESI-MS和SSCP的有用性，以快速筛选TTR相关的淀粉样变性。此外，从SSCP中显示异常电泳迁移模式的条带获得的DNA样品有助于直接序列分析以检测未知突变，并且在ESI-MS中观察到的变体PTR分子量的变化证实了碱基取代。(C)2000 Elsevier Science B. V.保留所有权利。
Two patients with amyloidosis caused by transthyretin (TTR) were investigated by immunohistopathologic, mass spectrometric, and molecular genetic methods. After confirming the immunoreactivity of TTR in the amyloid deposits using anti-TTR polyclonal antibody, a new method: centrifugal concentration and electrospray ionization mass spectrometry (ESI-MS) was employed to detect the variant TTR in the serum. Only 50 mu l of the serum and 30 mu l of the anti-TTR antibody were needed for the analysis. After incubation with the antibody, the samples were passed through a 1000 kDa cut off centrifugal concentrator to retain the antibody, thereafter, the filtrate was analyzed by ESI-MS. Several forms of normal and variant TTR were detected in the serum samples: unconjugated TTR, cysteine and cysteine-glycine conjugated TTR. In the patients, a variant form of TTR was detected with a 26.0 Da higher molecular weight than that of normal TTR. Single-strand conformation polymorphism (SSCP) and direct sequence analysis confirmed the presence of a one-base substitution situated at the codon 50 from AGT (Ser) to ATT (Ile) in both patients, that corresponded to the increased molecular weight of 26.0. The present diagnostic procedure demonstrates the usefulness of both ESI-MS and SSCP to screen for TTR related amyloidosis rapidly. Moreover, the DNA samples obtained from the band showing abnormal electrophoretic migration pattern in SSCP, facilitate the direct sequence analysis to detect the unknown mutation, and the observed shift in molecular weight of the variant PTR in ESI-MS confirms the base substitution. (C) 2000 Elsevier Science B.V. All rights reserved.