In vitro supplementation with dAMP/dGMP leads to partial restoration of mtDNA levels in mitochondrial depletion syndromes
In vitro supplementation with dAMP/dGMP leads to partial restoration of mtDNA levels in mitochondrial depletion syndromes
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DOI：
10.1093/hmg/ddp074
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发表时间：
2009-05-01
影响因子：
3.5
通讯作者：
Horvath, Rita
中科院分区：
文献类型：
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作者：
Bulst, Stefanie;Abicht, Angela;Horvath, Rita
线粒体DNA缺失综合征是儿童（肝）脑肌病的常见原因，定义为与核DNA相关的线粒体DNA拷贝数减少。以前的研究表明，在脱氧鸟苷激酶缺陷的成纤维细胞中，补充dAMP/dGMP可以防止mtDNA耗竭。我们研究了诊断为mtDNA缺失的患者的肌管，这些患者携带DGUOK、POLG 1（Alpers综合征）和TYMP致病突变。在TYMP缺乏症患者和对照组的分化肌管中添加不同剂量的dAMP/dGMP或dAMP/dGMP/dCMP，并分析mtDNA/nDNA比值和细胞色素c氧化酶（考克斯）活性。血清剥夺和肌管形成引发DGUOK或POLG 1缺陷型肌管中mtDNA拷贝数减少，但TYMP缺陷型和健康对照组中没有。补充dAMP/dGMP可显著且可重复地挽救DGUOK缺乏症患者的mtDNA耗竭。POLG 1缺陷型肌管也表现出线粒体DNA拷贝数的轻度、不显著的增加。在DGUOK和POLG 1缺陷型肌管中，MtDNA缺失不会导致考克斯染色缺陷。用溴化乙锭处理导致所有细胞类型中非常严重的消耗和考克斯染色的缺乏，并且在补充dAMP/dGMP后没有观察到恢复。我们发现，补充dAMP/dGMP显著增加DGUOK缺陷型肌管的mtDNA拷贝数，并导致POLG 1缺陷型mtDNA耗竭的轻度、非显著改善。体外高剂量补充对mtDNA拷贝数无不良影响。需要进一步的研究来确定补充dAMP/dGMP对体内DGUOK缺乏症的可能治疗意义。
Mitochondrial DNA depletion syndrome, a frequent cause of childhood (hepato)encephalomyopathies, is defined as a reduction of mitochondrial DNA copy number related to nuclear DNA. It was previously shown that mtDNA depletion can be prevented by dAMP/dGMP supplementation in deoxyguanosine kinase-deficient fibroblasts. We investigated myotubes of patients diagnosed with mtDNA depletion carrying pathogenic mutations in DGUOK, POLG1 (Alpers syndrome) and TYMP. Differentiating myotubes of all patients and controls were supplemented with different doses of dAMP/dGMP or dAMP/dGMP/dCMP in TYMP deficiency, and analysed for mtDNA/nDNA ratio and for cytochrome c oxidase (COX) activity. Serum deprivation and myotube formation triggered a decrease in mtDNA copy number in DGUOK or POLG1 deficient myotubes, but not in TYMP deficiency and healthy controls. Supplementation with dAMP/dGMP leads to a significant and reproducible rescue of mtDNA depletion in DGUOK deficiency. POLG1 deficient myotubes also showed a mild, not significant increase in mtDNA copy number. MtDNA depletion did not result in deficient COX staining in DGUOK and POLG1-deficient myotubes. Treatment with ethidium bromide resulted in very severe depletion and absence of COX staining in all cell types, and no recovery was observed after supplementation with dAMP/dGMP. We show that supplementation with dAMP/dGMP increases mtDNA copy number significantly in DGUOK deficient myotubes and, leads to a mild, non-significant improvement of mtDNA depletion in POLG1 deficiency. No adverse effect on mtDNA copy number was observed on high-dose supplementation in vitro. Further studies are needed to determine possible therapeutic implications of dAMP/dGMP supplementation for DGUOK deficiency in vivo.