Functional Evaluation of Paraplegin Mutations by a Yeast Complementation Assay
Functional Evaluation of Paraplegin Mutations by a Yeast Complementation Assay
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DOI：
10.1002/humu.21226
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发表时间：
2010-05-01
期刊：
影响因子：
3.9
通讯作者：
Mannan, Ashraf U.
中科院分区：
文献类型：
--
作者：
Bonn, Florian;Pantakani, Krishna;Mannan, Ashraf U.
遗传性痉挛性截瘫（AR-HSP）的常染色体隐性形式主要由SPG 7基因中的突变引起，SPG 7基因编码paraplegin，paraplegin是线粒体中异源寡聚体m-AAA蛋白酶的亚基。在本研究中，25个不相关的HSP个体/家族的基因组DNA中的SPG 7基因的测序导致在SPG 7基因的编码序列中鉴定出两个具有复合杂合突变（p.G349S/p.W583C和p.A510V/p.N739KfsX741）的HSP患者。我们使用酵母互补试验来评估在HSP患者中检测到的新型SPG 7序列变体的功能后果。我们评估了由paraplegin变体和AFG 3L 2的蛋白水解失活形式（AFG 3L 2（E575 Q）或AFG 3L 2（K354 A））组成的异源寡聚m-AAA蛋白酶在m-AAA蛋白酶缺陷型酵母细胞中表达时的蛋白水解活性。我们证明，新鉴定的paraplegin变体干扰异源寡聚体m-AAA蛋白酶的蛋白水解功能。此外，在我们的HSP队列中，常见的沉默多态性如p.T503A和p.R688Q可以与突变（p.G349S，p.W583C，p.A510V和p.N739KfsX744）区分开来。因此，酵母互补测定可以作为一个可靠的系统来区分致病性突变与任何新的SPG 7序列变体的沉默多态性，这将有助于解释SPG 7的遗传数据。Mutat 31：617-621，2010. (C)2010 Wiley-Liss，Inc.
An autosomal recessive form of hereditary spastic paraplegia (AR-HSP) is primarily caused by mutations in the SPG7 gene, which codes for paraplegin, a subunit of the hetero-oligomeric m-AAA protease in mitochondria. In the current study, sequencing of the SPG7 gene in the genomic DNA of 25 unrelated HSP individuals/families led to the identification of two HSP patients with compound heterozygous mutations (p.G349S/p.W583C and p.A510V/p.N739KfsX741) in the coding sequence of the SPG7 gene. We used a yeast complementation assay to evaluate the functional consequence of novel SPG7 sequence variants detected in the HSP patients. We assessed the proteolytic activity of hetero-oligomeric m-AAA proteases composed of paraplegin variant(s) and proteolytically inactive forms of AFG3L2 (AFG3L2(E575Q) or AFG3L2(K354A)) upon expression in m-AAA protease-deficient yeast cells. We demonstrate that the newly identified paraplegin variants perturb the proteolytic function of hetero-oligomeric m-AAA protease. Moreover, commonly occurring silent polymorphisms such as p.T503A and p.R688Q could be distinguished from mutations (p.G349S, p.W583C, p.A510V, and p.N739KfsX744) in our HSP cohort. The yeast complementation assay thus can serve as a reliable system to distinguish a pathogenic mutation from a silent polymorphism for any novel SPG7 sequence variant, which will facilitate the interpretation of genetic data for SPG7. Hum Mutat 31:617-621, 2010. (C) 2010 Wiley-Liss, Inc.