Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency.
Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency.
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DOI：
10.1038/s41598-017-14623-2
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发表时间：
2017-11-15
影响因子：
4.6
通讯作者：
Taylor RW
中科院分区：
文献类型：
--
作者：
Ahmed ST;Alston CL;Hopton S;He L;Hargreaves IP;Falkous G;Oláhová M;McFarland R;Turnbull DM;Rocha MC;Taylor RW
孤立复合体I(CI)缺乏症是最常见的线粒体呼吸链生化缺陷，影响OXPHOS的最大成分。CI具有遗传异质性；致病变异体影响38个核编码亚基之一、7个线粒体DNA(MtDNA)编码亚基或14个已知CI组装因子。实验室诊断依赖于线粒体丰富的组织匀浆中酶活性的分光光度分析，这需要至少50 mg骨骼肌，因为没有可靠的组织化学方法来直接评估组织冰冻切片中的CI活性。我们已经评估了一种有效的四重免疫荧光素氧合酶(IHC)检测方法，用于在诊断环境中检测CI缺陷，使用来自25名经基因证实的致病CI变异的患者的10 微米横肌切片。我们观察到，在所有突变影响核编码结构亚单位和组装因子的患者中，NDUFB8免疫反应性丧失，而在影响mtDNA编码结构亚单位突变的10名患者中，只有3名患者表现出NDUFB8缺失，BN-PAGE对CI组装和使用另一种商用CI(NDUFS3)抗体进行的IHC分析证实了这一点。IHC分析具有明确的诊断潜力，可以识别起源于孟德尔的CI缺陷患者，同时突出了在疑似线粒体疾病患者的诊断工作中完整线粒体基因组测序的必要性。
Isolated Complex I (CI) deficiency is the most commonly observed mitochondrial respiratory chain biochemical defect, affecting the largest OXPHOS component. CI is genetically heterogeneous; pathogenic variants affect one of 38 nuclear-encoded subunits, 7 mitochondrial DNA (mtDNA)-encoded subunits or 14 known CI assembly factors. The laboratory diagnosis relies on the spectrophotometric assay of enzyme activity in mitochondrially-enriched tissue homogenates, requiring at least 50 mg skeletal muscle, as there is no reliable histochemical method for assessing CI activity directly in tissue cryosections. We have assessed a validated quadruple immunofluorescent OXPHOS (IHC) assay to detect CI deficiency in the diagnostic setting, using 10 µm transverse muscle sections from 25 patients with genetically-proven pathogenic CI variants. We observed loss of NDUFB8 immunoreactivity in all patients with mutations affecting nuclear-encoding structural subunits and assembly factors, whilst only 3 of the 10 patients with mutations affecting mtDNA-encoded structural subunits showed loss of NDUFB8, confirmed by BN-PAGE analysis of CI assembly and IHC using an alternative, commercially-available CI (NDUFS3) antibody. The IHC assay has clear diagnostic potential to identify patients with a CI defect of Mendelian origins, whilst highlighting the necessity of complete mitochondrial genome sequencing in the diagnostic work-up of patients with suspected mitochondrial disease.
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影响因子：
7.3
作者：
Alston, Charlotte L.;Rocha, Mariana C.;Lax, Nichola Z.;Turnbull, Doug M.;Taylor, Robert W.
通讯作者：
Taylor, Robert W.
影响因子：
15.9
作者：
Kirby, DM;Salemi, R;Thorburn, DR
通讯作者：
Thorburn, DR
影响因子：
2.8
作者：
Alston, Charlotte L.;Morak, Monika;Taylor, Robert W.
通讯作者：
Taylor, Robert W.
影响因子：
29
作者：
Guerrero-Castillo, Sergio;Baertling, Fabian;Nijtmans, Leo
通讯作者：
Nijtmans, Leo
影响因子：
15.9
作者：
MARIOTTI, C;TIRANTI, V;ZEVIANI, M
通讯作者：
ZEVIANI, M