Oxygen consumption rate for evaluation of COQ2 variants associated with multiple system atrophy
Oxygen consumption rate for evaluation of COQ2 variants associated with multiple system atrophy
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用于评估与多系统萎缩相关的 COQ2 变体的耗氧率
DOI：
10.1007/s10048-018-0563-7
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发表时间：
2019
期刊：
影响因子：
2.2
通讯作者：
Tsuji Shoji
中科院分区：
文献类型：
--
作者：
Yasuda Tsutomu;Matsukawa Takashi;Mitsui Jun;Tsuji Shoji
致编辑：我们在患有多系统萎缩症（MSA）的多重家族中鉴定了COQ 2的纯合或复合杂合突变，并发现功能受损的COQ 2变体与散发性MSA相关[1]。特别是，最近的荟萃分析证实了东亚人中V393 A变异与散发性MSA风险的相关性[2]。COQ 2编码对羟基苯甲酸-聚戊烯基转移酶，该转移酶对于线粒体中充当电子载体的辅酶Q10（CoQ 10）的生物合成至关重要。尽管具有杂合V393 A的淋巴母细胞样细胞系（LCL）的COQ 2活性轻微但显著降低，但携带V393 A COQ 2 cDNA的酵母菌株的生长速率没有[1]。由于LCL的可用性有限，因此COQ 2变体的灵敏测定方法很重要。在这里，我们测量的基础耗氧率（OCR）的酵母coq 2空株窝藏野生型或突变体的人COQ 2（hCOQ 2）cDNA，以评估COQ 2的功能变化。用RT-PCR方法从人胎盘RNA中扩增出hCOQ 2 cDNA。克隆的cDNA跨越1266 bp，包括第一个ATG密码子（NM_015697.（七）.通过定点诱变，随后亚克隆到pAUR 123（Takara Bio，滋贺，日本）中，产生突变hCOQ 2 cDNA（V393 A [1-3]、S146 N [4]和M128 V [1，3]）。用携带野生型或突变型hCOQ 2 cDNA的pAUR 123转化BY 4741 Δ coq 2菌株。根据制造商的说明书，使用XFe 24细胞外通量分析仪（Seahorse Bioscience，North Billerica，MA）在30 ℃下测量基础OCR。将酵母细胞重悬于600 μL不可发酵甘油培养基中，并接种到预包被聚-L赖氨酸的XF 24细胞培养微孔板上（和子，大坂，日本），然后以50× g离心1 min。通过测量不同数量酵母细胞的基础OCR，评价基础OCR测量的线性（0.5× 105至4.0× 105个细胞）用野生型hCOQ 2转化。接下来，使用Bonferroni校正的双尾t检验比较1.0× 105个具有野生型和突变型hCOQ 2的酵母的平均基础OCR。认为P值小于0.05表示具有统计学显著性。在0.5× 105 - 4.0× 105个细胞/孔范围内确认了OCR测量的线性（R2= 0.9998;图1 a）。测定野生型、V393 A、S146 N和M128 V的平均基础OCR分别为148.7±12.6、103.4±17.6、75.7±14.6和98.7±24.7 pmol/min（图1 B;平均值±SD）。
To the editor: We identified homozygous or compound heterozygous mutations in COQ2 in multiplex families with multiple system atrophy (MSA) and found that functionally impaired COQ2 variants are associated with sporadic MSA [1]. In particular, association of V393A variant with risk of sporadic MSA has been confirmed in East Asians by a recent meta-analysis [2]. COQ2 encodes parahydroxybenzoate-polyprenyl transferase, which is essential for the biosynthesis of coenzyme Q10 (CoQ10) that serves as an electron carrier in the mitochondria. Although the COQ2 activities of lymphoblastoid cell lines (LCLs) with heterozygous V393A were mildly but significantly decreased, the growth rate of the yeast strain harboring V393A COQ2 cDNA was not [1]. Since the availability of LCLs is limited, sensitive assay methods for COQ2 variants are important. Here, we measured the basal oxygen consumption rate (OCR) of the yeast coq2 null strain harboring wildtype or mutant human COQ2 (hCOQ2) cDNAs to evaluate the functional changes of COQ2. hCOQ2 cDNA was prepared from human placenta RNA by RT-PCR. The cloned cDNA spans 1266 bp including the first ATG codon (NM_015697. 7). Mutant hCOQ2 cDNAs (V393A [1–3], S146N [4], and M128V [1, 3]) were generated by site-directed mutagenesis followed by subcloning into pAUR123 (Takara Bio, Shiga, Japan). The BY4741Δcoq2 strain was transformed with pAUR123 carrying wild-type or mutant hCOQ2 cDNAs. Basal OCR was measured at 30 C using an XFe24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA) according to the manufacturer’s instructions. Yeast cells were resuspended in 600 μL of nonfermentable glycerol medium and seeded onto the XF24 Cell Culture Microplates precoated with Poly-L lysine (Wako, Osaka, Japan) followed by centrifugation at 50× g for 1 min. The linearity of the basal OCR measurement was evaluated by measuring the basal OCRs of various numbers of yeast cells (0.5× 105 to 4.0× 105 cells) transformed with wild-type hCOQ2. Next, the mean basal OCRs of 1.0× 105 yeasts with wild-type and mutant hCOQ2 were compared using two-tailed t test withBonferroni’s correction. P values less than 0.05 were considered to indicate a statistical significance. The linearity of the OCR measurement was confirmed in the range of 0.5× 105 to 4.0× 105 cells/well (R2= 0.9998; Fig. 1 a). The mean basal OCRs of wild-type, V393A, S146N, and M128V were determined to be 148.7±12.6, 103.4±17.6, 75.7±14.6, and 98.7±24.7 pmol/min, respectively (Fig. 1 b; mean±SD).