Sporadic intragenic inversion of the mitochondrial DNA MTND1 gene causing fatal infantile lactic acidosis

Mutations of mitochondrial DNA (mtDNA) are an important cause of genetic disease, yet rarely present in the neonatal period. Here we report the clinical, biochemical, and molecular genetic findings of an infant who died at the age of 1 mo with marked biventricular hypertrophy, aortic coarctation, and severe lactic acidosis due to a previously described but unusual mtDNA mutation, a 7-bp intragenic inversion within the mitochondrial gene encoding ND1 protein of complex I (MTND1). In direct contrast to the previous case, an adult with exercise intolerance who only harbored the mutation in muscle, the MTND1 inversion in our patient was present at high levels in several tissues including the heart, muscle, liver, and cultured skin fibroblasts. There was no evidence of the mutation or respiratory complex I defect in a muscle biopsy from the patient's mother. Transmitochondrial cytoplasmic hybrids (cybrids) containing high mutant loads of the inversion expressed the biochemical defect but apparently normal levels of the assembled complex. Our report highlights the enormous phenotypic diversity that exists among pathogenic mtDNA mutations and reemphasizes the need for appropriate genetic counseling for families affected by mtDNA disease.     

Mutation screening in patients with isolated cytochrome c oxidase deficiency

Cytochrome c oxidase (COX) deficiency has been associated with a variety of clinical conditions and can be due to mutations in nuclear or mitochondrial genes. Despite recent progress in our understanding of the molecular bases of COX deficiency, the genetic defect remains elusive in many cases. We performed mutation screening in 30 patients with biochemical evidence of isolated COX deficiency and heterogeneous clinical phenotypes. Sixteen patients had various forms of encephalomyopathy, and six of these had the neuroradiological features of Leigh syndrome. Four patients had encephalohepatopathy, six had hypertrophic cardiomyopathy, and four had other phenotypes. We studied the three mtDNA genes encoding COX subunits, the 22 mtDNA tRNA genes, and seven COX assembly genes: SCO1, SCO2, SURF1, COX10, COX11, COX15, and COX17. We report two novel pathogenic SURF1 mutations in a patient with Leigh syndrome and one novel SCO2 mutation in a patient with hypertrophic cardiomyopathy. These data show that heterogeneous clinical phenotypes are associated with COX deficiency, that mutations in mtDNA COX genes are rare, and that mutations in additional genes remain to be identified.     

线粒体呼吸链复合物Ⅰ缺陷导致幼儿肝内胆汁淤积症

线粒体呼吸链复合物缺陷是导致儿童线粒体病的主要原因。本文就1例线粒体呼吸链复合物Ⅰ缺陷导致的幼儿胆汁淤积症患者的临床经过、生化特点、线粒体呼吸链复合物活性分析及基因突变进行回顾性研究。患儿,男,自1岁1个月起腹泻,体重下降,伴无力、进行性黄疸、肝损害。经多种检查、尿液有机酸分析及血液氨基酸、酯酰肉碱谱分析未见特异性改变。外周血白细胞线粒体呼吸链复合物Ⅰ活性降低,线粒体基因分析发现患儿及其母亲tRNA 5821G>A突变,证实患儿存在线粒体呼吸链复合物Ⅰ缺陷。患儿疾病进展迅速,治疗无效,于1岁5个月时夭折。复合物Ⅰ缺陷是线粒体呼吸链缺陷中最常见的类型,本研究首次诊断了1例线粒体呼吸链复合物Ⅰ缺陷所导致的中国儿童患者,其临床表现为胆汁淤积症。线粒体肝病是导致儿童代谢性肝病的主要原因之一,生化分析、线粒体呼吸链复合物活性测定及基因分析是病因诊断的关键。     

Exome sequencing identifies a CHKB mutation in Spanish patient with Megaconial Congenital Muscular Dystrophy and mtDNA depletion

BackgroundCholine kinase beta gene (CHKB) mutations have been identified in Megaconial Congenital Muscular Dystrophy (MDCMC) patients, but never in patients with an additional combined deficiency of complexes I, III and IV and mitochondrial DNA (mtDNA) depletion.AimsTo report mutations in carry genes for MDCMC with respiratory chain defects and mtDNA depletion.MethodsWhole-exome sequencing (WES) was used to identify the carry genes in a Spanish child with muscle weakness, mild hypotonia at lower limb muscles, mildly elevated creatine kinase (CK), enlarged mitochondria in the periphery of the fibers, combined deficiency of complex I, III and IV and depletion of mtDNA.ResultsWith WES data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The first filter of WES data with the nuclear-encoded mitochondrial genes (MitoCarta) did not get any candidate. However, the analysis of whole exome uncovered a homozygous nonsense pathogenic mutation in CHKB gene (NM_005198.4:c.810T>A, p.Tyr270*).ConclusionsOur data confirm the role of CHKB in MDCMC and point to this gene as unique candidate for the combined deficiency of respiratory chain and mtDNA depletion observed in this patient.     

Mitochondrial dysfunction in skeletal muscle of children with cardiomyopathy

OBJECTIVES: This study sought to examine skeletal muscle of children with cardiomyopathy (CM) for changes in mitochondrial enzyme activities and in mitochondrial DNA (mtDNA). BACKGROUND: Heart mitochondrial enzymatic activity defects have been often found in dilated and hypertrophic CM. The defects primarily involve the activities of the electron transport system and oxidative phosphorylation pathway including respiratory complexes I, III, IV, and V. METHODS: Skeletal muscle biopsies of 8 children with CM were examined for specific mitochondrial enzyme activities, mtDNA copy number and the presence of pathogenic mutations and deletions in mtDNA. RESULTS: A marked deficiency in specific mitochondrial enzyme activities was found in 6 of 8 patients in skeletal muscle as well as in 2 of 3 hearts of those in whom cardiac tissue was available. Specific activity defects were found in complex I (2 cases), complex III (5 cases), complex IV (3 cases), and complex V (4 cases). Complex II and citrate synthase activities were unaffected. None of the previously reported pathogenic mutations associated with CM were detected, nor was there any evidence of mtDNA depletion. The incidence of defective respiratory complex activities in skeletal muscle was similar to the incidence of defective complex activities previously reported in cardiac tissue. CONCLUSIONS: Mitochondrial analysis of skeletal muscle is warranted in the overall clinical evaluation of children with CM, and particularly before consideration for cardiac transplantation.     

Defective assembly of the respiratory chain

A functional respiratory chain is dependent on protein components encoded by both mtDNA and nuclear DNA. Isolated cytochrome c oxidase (COX) deficiency is often caused by mutations in nuclear genes regulating the assembly of the 13 protein subunits of this complex. The accompanying paper by Zeman and co-workers reports that mutations in SCO2 are common in infantile COX deficiency and are associated with a very poor prognosis.

Conclusion: Molecular diagnosis is often feasible in patients with COX deficiency and particular attention should be paid to mutations in COX assembly genes.     

ND3基因10191T>C突变导致的线粒体呼吸链复合物Ⅰ缺陷

本文报道1例由于ND3基因突变导致线粒体呼吸链复合物 Ⅰ 缺陷的患儿。该患儿自6岁起出现眼睑下垂、无力、癫癎及运动倒退,呈进行性加重。血液乳酸、丙酮酸增高,脑MRI示双侧基底节对称性损害,符合Leigh综合征诊断。为明确病因,提取患儿和父母的外周血白细胞线粒体蛋白,进行氧化磷酸化酶复合物 Ⅰ～V活性测定,并提取DNA,分析编码线粒体呼吸链复合物 Ⅰ 的7个线粒体结构基因。结果显示患儿线粒体呼吸链复合物 Ⅰ 活性为33.1 nmol/min?毫克线粒体总蛋白（正常对照44.0±5.4 nmol/min?毫克线粒体总蛋白）,复合物 Ⅰ 与柠檬酸合酶活性比值为19.8%（正常对照48.1%±11.0%）,均降低。复合物 Ⅱ～V活性正常。患儿线粒体ND3基因10191T>C突变。其父母线粒体基因及呼吸链复合物酶活性正常。治疗后,现患者16岁,癫癎控制良好,双下肢痉挛性瘫痪,智力正常。通过外周血白细胞线粒体氧化磷酸化酶复合物活性测定及基因分析,本研究首次诊断了编码线粒体呼吸链复合物Ⅰ亚基的ND3基因10191T>C突变导致复合物Ⅰ缺陷,为Leigh综合征的发病原因提供依据。     

An unusual patient with the neonatal Marfan phenotype and mitochondrial complex I deficiency

We report the case of a 16-month-old male with the neonatal appearance of Marfan syndrome (NMS), with dolichocephaly, a long midface, deep-set eyes, arachnodactyly, dislocated lenses and carciovascular abnormalities. The presence of persistent lactic acidosis led to studies which disclosed mitochondrial complex I deficiency. We speculate that this unusual association may be due to the combination of an inherited mutation affecting complex I activity along with a de novo mutation disrupting the corresponding locus and an adjacent NMS locus on the homologous autosome. The possibility that the phenotype observed in this patient is directly due to the mitochondrial defect cannot be excluded.     

Retrospective, multicentric study of 180 children with cytochrome C oxidase deficiency

A retrospective, multicenter study of 180 children with cytochrome c oxidase (COX) deficiency analyzed the clinical features, prognosis, and molecular bases of the COX deficiency. Clinical symptoms including failure to thrive, encephalopathy, hypotony, Leigh syndrome, cardiac involvement, and hepatopathy appeared in most patients early after birth or in early childhood. Two thirds of all children died. Biochemical examination revealed an isolated COX deficiency in 101 children and COX deficiency combined with disturbances of other respiratory chain complexes in 79 children. Blood and cerebrospinal fluid lactate increased in 85% and 81% of examined cases, respectively. Pathogenic mutations in mitochondrial or nuclear DNA were established in 75 patients. Mutations in surfeit locus protein 1 gene (SURF1) were found in 47 children with Leigh syndrome; 2bp deletion 845-846delCT was found in 89% of independent alleles. Mutations in a mitochondrial copper-binding protein (SCO2) gene were found in nine children with encephalomyopathy and/or cardiomyopathy; all of them were homozygotes or heterozygotes for 1541G>A mutation. Different mitochondrial DNA (mtDNA) deletion or depletion were found in nine children, mtDNA mutation 3243A>G in six, mtDNA mutation 8363G>A in two children with Leigh syndrome and mtDNA mutations 8344A>G, and 9205-9206delTA in one child each. COX deficiency represents a heterogeneous group of diseases with unfavorable prognosis. Marked prevalence of two nuclear DNA mutations (845-846delCT in the SURF1 gene and 1541G>A in the SCO2 gene) associated with COX deficiency in a Slavonic population suggests the existence of regional differences in the genetic basis of COX deficiency.     

Cytochrome c oxidase deficiency in a child with isolated myopathy

Cytochrome c oxidase (COX) deficiency is the most commonly recognized respiratory chain defect in childhood. The disease is clinically heterogeneous with phenotypes including Leigh syndrome, hepatic failure and myopathies. COX deficiency has been associated with mitochondrial DNA mutations in COX I, II, and III with large-scale deletions of the mitochondrial genome and with point mutations in mitochondrial tRNA genes. Here we report on a 3.5-year-old girl with a rare type of isolated myopathy due to COX deficiency.     

A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion

Isolated mitochondrial myopathies (IMM) are either due to primary defects in mtDNA, in nuclear genes that control mtDNA abundance and structure such as thymidine kinase 2 (TK2), or due to CoQ deficiency. Defects in the TK2 gene have been found to be associated with mtDNA depletion attributed to a depleted mitochondrial dNTP pool in non-dividing cells. We report an unusual case of IMM, homozygous for the H90N mutation in the TK2 gene but unlike other cases with the same mutation, does not demonstrate mtDNA depletion. The patient's clinical course is relatively mild and a muscle biopsy showed ragged red muscle fibers with a mild decrease in complexes I and an increase in complexes IV and II activities. This report extends the phenotypic expression of TK2 defects and suggests that all patients who present with an IMM even with normal quantities of mtDNA should be screened for TK2 mutations.     

Clinical characteristics of patients with non-specific and non-categorized mitochondrial diseases

Aim:  Mitochondrial disease is a heterogeneous disorder entity induced by defects in mitochondrial respiratory chain complex (MRC). A significant portion of patients with MRC defect will not conform to a specific, known syndrome. We have analysed the clinical features of 108 Korean paediatric patients with non-specific and non-categorized mitochondrial disease.
Methods:  We retrospectively reviewed the clinical and laboratory features of 108 paediatrics patients with non-specific and non-categorized mitochondrial diseases who showed defects in MRC activity, confirmed by spectrophotometric biochemical enzyme assay of their muscles.
Results:  Neuromuscular involvement was noted in all patients, with developmental delay and seizure accounting for 92.6% and 77.8% of total patients respectively. Various extraneurological symptoms were observed. Most patients exhibited MRC I defect, accounting for 100 (92.6%) patients. The most common brain magnetic resonance imaging (MRI) finding was diffuse cerebral atrophy. However, in 23.1% of patients, no notable changes were visible on MRI.
Conclusions:  Mitochondrial respiratory chain complex I defect was the most common finding in this study. Though neuromuscular symptoms predominated, with presence of numerous extraneurological findings, we could not find any novel symptoms that might be unique to this category of mitochondrial disease. But, comparatively, more patients presented with unremarkable birth histories and normal brain MRI findings.     

The other human genome.

In the past 13 years, a new chapter of human genetics, "mitochondrial genetics", has opened up and is becoming increasingly important in differential diagnosis. Although the clinical manifestations of disorders related to mitochondrial DNA (mtDNA) are extremely variable, recent advances in genetic testing aid in the identification of patients. Muscle morphology can give important clues for diagnosis, but histological features alone cannot define a specific disorder. Biochemical analysis may reveal a single enzyme defect, or when multiple activities are affected, suggest an mtDNA mutation. However, definitive diagnosis often requires DNA analysis and documentation of a specific mtDNA abnormality. Disorders associated with mtDNA mutations are associated with a wide variety of syndromes, and owing to the properties and characteristics of mtDNA, these are often transmitted by maternal inheritance. Although therapy for mitochondrial diseases is limited, identification of the molecular defect is important for genetic counseling.     

线粒体tRNAser(UCN)基因突变致线粒体脑肌病1例并文献复习

目的分析1例线粒体细胞病患儿的临床表现及其基因突变特点。方法对1例临床诊断为线粒体脑肌病患儿归纳总结其临床表现及实验室检查结果，并运用PCR法扩增患儿外周血线粒体基因3243、8344和8993热点突变及已报道的62个常见突变位点所在片段，对扩增片段采用直接测序方法检测突变。在某医院年度体检中选择70名无血缘关系的健康成人作为正常对照，采用PCR RFLP方法进行多态性分析。结果 男性患儿，1岁9个月时出现持续高乳酸血症、反复严重代谢性酸中毒和高氨血症，头颅CT扫描显示双侧额顶叶对称性空泡样低密度灶，考虑线粒体性脑肌病；脑萎缩。2岁时死亡。患儿外周血线粒体基因3243、8344和8993热点突变及已报道的62个常见突变位点均未见突变，患儿线粒体tRNAser(UCN)基因存在7496 T→C突变。为证实在正常人群中线粒体tRNAser(UCN)基因是否存在7496 T→C突变，70名正常对照组皆未发现这一位点突变。结论线粒体脑肌病可以表现为代谢紊乱和神经损伤，应提高警惕。线粒体tRNAser(UCN)基因7496 T→C突变可能导致线粒体细胞病。该突变尚未见报道。     

Cytochrome c oxidase deficiency

Cytochrome c oxidase (COX) is a complex enzyme composed of 13 subunits, three of which are encoded by the mitochondrial DNA (mtDNA). The other 10 subunits are encoded by the nuclear DNA, synthesized in the cytoplasm, and transported into the mitochondria. The complexity of the enzyme and its dual genetic control explain the heterogeneity of clinical phenotypes associated with COX deficiency. There are two major syndromes, one characterized by muscle involvement (fatal infantile or benign infantile myopathy), the other dominated by brain disease (Leigh syndrome, myoclonic epilepsy with ragged red fibers, Menkes' disease). Partial defects of COX have been shown in muscle of patients with progressive external ophthalmoplegia, either alone (ocular myopathy) or as part of Kearns-Sayre syndrome. Biochemical studies have documented either muscle-specific or generalized defects of COX; COX deficiency is reversible in the benign infantile myopathy. Immunologically detectable protein may be normal (benign myopathy) or variably decreased (fatal myopathy, Leigh syndrome). The subunit pattern of COX is normal by immunoblot in patients with fatal myopathy and Leigh syndrome; a disproportionate decrease of subunit II was seen in a patient with myoclonic epilepsy with ragged red fibers. Availability of the three mtDNA genes and of complementary DNA probes for eight of the 10 nuclear DNA-encoded subunits makes it possible to investigate the different diseases at the molecular level. Large deletions of mtDNA have been found in patients with ocular myopathy and Kearns-Sayre syndrome: the deleted mtDNA appear to be transcribed but not translated, thus explaining the partial COX deficiency.     

Mitochondrial hepatopathies in the newborn period

Mitochondrial disorders recognized in the neonatal period usually present as a metabolic crisis combined with one or several organ manifestations. Liver disorder in association with a respiratory chain deficiency may be overlooked since liver dysfunction is common in severely sick newborn infants. Lactacidosis, hypoglycemia, elevated serum transaminases and conjugated bilirubin are common signs of mitochondrial hepatopathy. Hepatosplenomegaly may occur in severe cases. A clinical picture with fetal growth restriction, postnatal lactacidosis, hypoglycemia, coagulopathy, and cholestasis, especially in combination with neurological symptoms or renal tubulopathy, should alert the neonatologist to direct investigations on mitochondrial disorder. A normal lactate level does not exclude respiratory chain defects. The most common liver manifestation caused by mutated mitochondrial DNA (deletion) is Pearson syndrome. Recently, mutations in several nuclear DNA genes have been identified that lead to mitochondrial hepatopathy, e.g. mitochondrial depletion syndrome caused by DGUOK, MPV17, SUCLG1, POLG1, or C10ORF2 mutations. A combination of lactacidosis, liver involvement, and Fanconi type renal tubulopathy is common when the complex III assembly factor BCS1L harbors mutations, the most severe disease with consistent genotype-phenotype correlation being the GRACILE syndrome. Mutations in nuclear translation factor genes (TRMU, EFG1, and EFTu) of the respiratory chain enzyme complexes have recently been identified. Diagnostic work-up of neonatal liver disorder should include assessment of function and structure of the complexes as well as mutation screening for known genes. So far, treatment is mainly symptomatic.     

Multiple oxidative phosphorylation deficiencies in severe childhood multi-system disorders due to polymerase gamma (POLG1) mutations

Failure to thrive, feeding difficulties, variable forms of infantile epilepsy or psychomotor developmental delay and hypotonia were the most frequent clinical disease presentations in eight children with combined oxidative phosphorylation enzyme complex deficiencies carrying mutations in the polymerase gamma (POLG1) gene. Five out of eight patients developed severe liver dysfunction during the course of the disease. Three of these patients fulfilled the disease criteria for Alpers syndrome. Most children showed deficiencies of respiratory chain enzyme complexes I and III, in combination with complex II, complex IV and/or PDHc in muscle, whereas in fibroblasts normal enzyme activities were measured. All children carried homozygous or compound heterozygous mutations in the POLG1 gene, including two novel mutations in association with mtDNA depletion. Conclusion We suggest performing POLG1 mutation analysis in children with combined oxidative phosphorylation deficiencies in muscle, even if the clinical picture is not Alpers syndrome.     

线粒体呼吸链酶复合物V缺陷与线粒体病

线粒体呼吸链酶复合物V,也称为ATP合酶,是位于线粒体内膜上的大蛋白复合体,由2个功能性蛋白复合物F0及F1构成。复合物V是线粒体呼吸链的最后一个复合物,在线粒体中通过电化学梯度传递质子,以ADP、Pi及Mg2+为原料合成ATP,为细胞供能。大多数患者新生儿期发病,导致严重脑损害或多脏器损害,病死率很高。主要临床表现为神经肌肉病、心肌病、高乳酸血症及3 甲基戊烯二酸尿症等,因受累器官的不同导致显著的临床异质性。复合物V由16个亚基组成,由线粒体基因与核基因共同编码。迄今,国内外已报道了MT-ATP6、MT-ATP8、ATPAF2、TMEM70、ATP5E基因突变导致的复合物V缺陷。本文总结了线粒体呼吸链复合物V的结构及功能,并对复合物V缺陷的病理、临床表现、诊断、治疗及分子遗传学研究进展进行了综述。     

A new family with the mitochondrial tRNAGLU gene mutation m.14709T>C presenting with hydrops fetalis.

BACKGROUND: In the heterogeneous group of mitochondrial disorders, patients with the same genotype can show different phenotypes and the same phenotype can be caused by different genotypes. We describe a family with the m.14709T>C mutation and a clinical presentation of hydrops fetalis, in contrast to previous reports in which patients presented with myopathy and/or diabetes mellitus. AIM: To identify a mutation in the mtDNA of a family with a heterogeneous clinical presentation. METHODS: Both biochemical and molecular analyses were performed. RESULTS: Biochemical results showed a decreased complex I and IV activity in muscle tissue of the patients. A mosaic-staining pattern for complex I in the patients' fibroblasts was revealed using immunocytochemistry. Molecular analyses identified the m.14709T>C mutation in the mitochondrial encoded tRNA(Glu) gene. CONCLUSION: We report 2 siblings with the m.14709T>C mutation in the mitochondrial tRNA(Glu) gene. The first patient showed hydrops fetalis, a new presentation within the clinical spectrum of this mutation, and the other a known presentation namely mild myopathy.