Investigating the pathogenesis of CoQ10 deficiencies

描述（由申请人提供）：辅酶Q10（CoQ10）是一种小的亲脂性分子，由苯醌环和疏水性类异戊二烯尾部组成，其存在于几乎所有细胞膜中。 在线粒体呼吸链中，辅酶Q10对于将电子从复合物I和复合物II转运到复合物III至关重要。 它也是一种抗氧化剂、膜稳定剂和细胞凋亡调节剂。 人类辅酶Q10缺乏症与四种临床表型相关。 所有形式的CoQ10缺乏症患者都通过口服补充剂得到改善，因此认识到这种可治疗的遗传性疾病很重要。 在过去的十年中，候选人和她的导师收集了84名患者（71个家庭）的生物样本，这些患者记录了肌肉和/或成纤维细胞中的CoQ10缺乏症，或者根据临床表现以及对CoQ10补充剂的反应怀疑CoQ10缺乏症。 共有54名患者（48个家庭）记录了肌肉，成纤维细胞或两者的辅酶Q10缺乏症。 2006年，研究小组报告了辅酶Q10生物合成基因的第一个突变，COQ2，编码4-对羟基苯甲酸：聚异戊二烯转移酶; PDSS2，编码decaprenyl二磷酸合酶的亚基2。 此外，在一个有4名小脑性共济失调和辅酶Q10缺乏症患者的家庭中，他们发现了APTX基因中的致病突变，该基因编码一种参与单链断裂修复的蛋白质。 因此，这些研究表明CoQ10缺乏症可以是原发性或继发性的。 CoQ10缺乏会导致呼吸链活性的缺陷（复合物I + III和II + III的活性降低），这并不奇怪。 呼吸链缺陷、ROS产生和细胞凋亡在辅酶Q10缺乏症发病机制中的相对重要性尚不清楚。 研究小组研究了严重CoQ10缺乏对含有COQ2和PDSS2突变的培养皮肤成纤维细胞的生物能量学，氧化应激和抗氧化防御的后果。 辅酶Q10生物合成途径前两个关键步骤的缺陷会产生不同的生化改变。PDSS2突变成纤维细胞具有相对于对照细胞的12%CoQ10，并且显著减少ATP合成，但不显示增加的活性氧（ROS）产生、氧化应激的迹象或增加的抗氧化防御标记物。 相比之下，COQ2突变成纤维细胞具有30%的CoQ10，在ATP合成中具有部分缺陷，并且显著增加了ROS的产生以及脂质和蛋白质的氧化。 为了更好地了解CoQ10缺乏症的发病机制，研究小组已经表征了CoQ10缺乏症的不同严重程度对ROS产生和线粒体生物能量学的影响，这些细胞具有CoQ10生物合成的不同遗传缺陷。 他们证实了他们之前的发现，并进一步观察到CoQ10水平与ROS产生之间的相关性遵循抛物线; 10 - 15%的残留CoQ10和60 - 70%与显著的ROS产生无关，而30 - 50%的残留CoQ10与ROS产生的最大增加有关。 此外，活性氧的增加似乎与初始超极化，然后去极化和细胞死亡。 这些数据得到了CoQ10和其他抗氧化剂在CoQ10缺乏患者成纤维细胞中治疗的初步结果的证实。 为了更好地了解人类辅酶Q10缺乏症的发病机制，候选人提出了以下三个具体目标：目标1：确定辅酶Q10缺乏症的新遗传原因。 目标二：为了了解相同遗传背景下不同程度CoQ10缺乏的线粒体生物能量学和氧化应激后果，她将使用RNA干扰（RNAi）调节COQ2和PDSS2表达。 目标3：为了测试ROS清除作为一种潜在的治疗策略，她将在COQ2突变成纤维细胞中过表达超氧化物锰歧化酶（MnSOD），并评估ROS，氧化应激和细胞凋亡的水平。 旁白：线粒体的缺陷导致多种人类疾病。 线粒体疾病的一种亚型是由辅酶Q10（CoQ10）缺乏引起的，辅酶Q10是线粒体参与能量产生的重要成分。 CoQ10缺乏症患者通常会通过补充CoQ10而显着改善。 候选人将研究患有这种疾病的患者，她将试图了解为什么突变会导致CoQ10缺乏症。 了解辅酶Q10缺乏症的原因可能会提高我们对辅酶Q10生物合成的科学知识，并将提供准确的遗传咨询，产前诊断和更快速启动治疗的分子测试。

DESCRIPTION (Provided by Applicant): Coenzyme Q10 (CoQ10) is a small lipophilic molecule composed of a benzoquinone ring and a hydrophobic isoprenoid tail which is present in virtually all cell membranes. In the mitochondrial respiratory chain, CoQ10 is vital for the transport of electrons from complex I and complex II to complex III. It is also an antioxidant, membrane stabilizer, and modulator of apoptosis. Human CoQ10-deficiency has been associated with four clinical phenotypes. Patients with all forms of CoQ10-deficiency have improved with oral supplementation, therefore recognition of this treatable genetic condition is important. In the last decade, the candidate and her mentors have collected biological samples from 84 patients (71 families) with documented CoQ10 deficiency in muscle and/or fibroblasts, or suspected CoQ10 deficiency based on the clinical manifestations as well as the response to CoQ10 supplementation. A total of 54 patients (48 families) have documented CoQ10 deficiency in muscle, fibroblasts, or both. In 2006, the investigative team reported the first mutations in CoQ10 biosynthetic genes, COQ2, which encodes 4-para-hydroxybenzoate: polyprenyl transferase; and PDSS2, which encodes subunit 2 of decaprenyl diphosphate synthase. In addition, in a family with four individuals with cerebellar ataxia and CoQ10 deficiency, they identified a pathogenic mutation in the APTX gene, which encodes a protein involved in single-strand break repair. Thus, these studies have revealed that CoQ10 deficiency can be primary or secondary. Not surprisingly, CoQ10 deficiency causes defects of respiratory chain activities (reduced activities of complexes I+III and II+III). The relative importance of respiratory chain defects, ROS production, and apoptosis in the pathogenesis of CoQ10-deficiency is unknown. The investigative team studied the consequences of severe CoQ10 deficiency on bioenergetics, oxidative stress, and antioxidant defenses in cultured skin fibroblasts harboring COQ2 and PDSS2 mutations. Defects in the first two committed steps of the CoQ10 biosynthetic pathway produce different biochemical alterations. PDSS2 mutant fibroblasts have 12% CoQ10 relative to control cells and markedly reduced ATP synthesis, but do not show increased reactive oxygen species (ROS) production, signs of oxidative stress, or increased antioxidant defense markers. In contrast, COQ2 mutant fibroblasts have 30% CoQ10 with partial defect in ATP synthesis, and significantly increased ROS production and oxidation of lipids and proteins. To better understand the pathogenesis of CoQ10 deficiency, the investigative team has characterized the effects of varying severity of CoQ10 deficiency on ROS production and mitochondrial bioenergetics in cells harboring different genetic defects of CoQ10 biosynthesis. They confirmed their previous findings and further observed that the correlation between level of CoQ10 and ROS production follows a parabolic curve; 10-15% residual CoQ10 and 60-70% are not associated with significant ROS production, whereas 30-50% residual CoQ10 is associated with the maximum increases in ROS production. Moreover, increase in reactive oxygen species appears to be associated with initial hyperpolarization followed by depolarization and cell death. These data are corroborated by preliminary results of treatment with CoQ10 and other antioxidants in fibroblasts from the CoQ10 deficient patients. To better understand the pathogenesis of human CoQ10 deficiency the candidate proposes the following three specific aims: Aim 1: To identify novel genetic causes of CoQ10 deficiency. Aim 2: To understand the mitochondrial bioenergetics and oxidative stress consequences of different degrees of CoQ10 deficiency in the same genetic background, she will modulate COQ2 and PDSS2 expression using RNA interference (RNAi). Aim 3: To test ROS scavenging as a potential therapeutic strategy, she will overexpress the enzyme superoxide manganese dismutase (MnSOD) in COQ2 mutant fibroblasts and will assess level of ROS, oxidative stress, and apoptosis. NARRATIVE: Defects of mitochondria cause diverse human diseases. A subtype of mitochondrial disease is caused by deficiency of coenzyme Q10 (CoQ10), an essential component of the mitochondria involved in energy production. Patients with CoQ10 deficiency often improve dramatically with CoQ10 supplementation. The candidate will study patients with this disease and she will attempt to understand why the mutations cause CoQ10 deficiency. Knowing the cause of CoQ10 deficiency will likely enhance our scientific knowledge of CoQ10 biosynthesis, and will provide molecular tests for accurate genetic counseling, prenatal diagnosis, and more rapid initiation of the therapy.