Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability,Nucleotide Hydrolysis,and Helicase Activity

Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg²⁺ ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.     

Molecular analysis for mitochondrial DNA disorders

In this article, we review the current methodologies used for the molecular diagnosis of mitochondrial DNA defects. Definition of mitochondrial disorders at the molecular level has been difficult because of both clinical and genetic heterogeneity. Direct DNA analysis for common point mutations and large mtDNA deletions is readily performed and can be done routinely. However, a large number of patients who have the clinical manifestations and muscle pathology findings consistent with mitochondrial DNA disorders do not have detectable common mutations. Additional mutation screening methods are required for the detection of rare and previously undescribed mutations in the mitochondrial genome.     

Genetics and mitochondrial abnormalities in autism spectrum disorders: a review

We review the current status of the role and function of the mitochondrial DNA (mtDNA) in the etiology of autism spectrum disorders (ASD) and the interaction of nuclear and mitochondrial genes. High lactate levels reported in about one in five children with ASD may indicate involvement of the mitochondria in energy metabolism and brain development. Mitochondrial disturbances include depletion, decreased quantity or mutations of mtDNA producing defects in biochemical reactions within the mitochondria. A subset of individuals with ASD manifests copy number variation or small DNA deletions/duplications, but fewer than 20 percent are diagnosed with a single gene condition such as fragile X syndrome. The remaining individuals with ASD have chromosomal abnormalities (e.g., 15q11-q13 duplications), other genetic or multigenic causes or epigenetic defects. Next generation DNA sequencing techniques will enable better characterization of genetic and molecular anomalies in ASD, including defects in the mitochondrial genome particularly in younger children.     

Gene therapy for diseases of the nervous system.

Advances in molecular biology and recombinant DNA technologies have contributed to our understanding of the molecular basis of many diseases. Now the possibility of gene transfer into normal cells to produce a gene product of therapeutic potential, or into diseased cells to correct the pathologic alteration, promises to revolutionize medical practice. In contemporary medicine, many therapeutic strategies focus on the link between a biochemical deficiency and the ensuing disorder. The treatment of noninfectious disease is often based on replacement therapy; medication is given to compensate for biochemical defects and to prevent or reverse the progression of disease. Although conventional therapies seldom alter the fundamental cause of a disease, gene therapy potentially could correct, at a molecular level, the genetic abnormalities contributing to its pathogenesis. Treatment directed at specific molecular alterations associated with the development of neurologic disease provides expectations of more effective and less toxic therapy. The development of gene therapy for nervous system tumors has progressed rapidly and may be prototypical in the development of therapies for inherited and acquired disorders of the nervous system. We describe possible strategies for using gene therapy to treat nervous system disorders, and we review recent advances in gene therapy for nervous system tumors.     

Structure,function and evolution of the animal mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is essential for animal mitochondrial DNA (mtDNA) maintenance. Deleterious mutations in the gene that encodes it cause mitochondrial dysfunction manifested in developmental delays, defects and arrest, limited life span, and a number of human pathogenic phenotypes that are recapitulated in animals across taxa. In fact, the replicative mtDNA helicase was discovered with the identification of human disease mutations in its nuclear gene, and based upon its deduced amino acid sequence homology with bacteriophage T7 gene 4 protein (T7 gp4), a bi-functional primase-helicase. Since that time, numerous investigations of its structure, mechanism, and physiological relevance have been reported, and human disease alleles have been modeled in the human, mouse, and Drosophila systems. Here, we review this literature and draw evolutionary comparisons that serve to shed light on its divergent features.     

The oxidative phosphorylation (OXPHOS) system: nuclear genes and human genetic diseases

The ubiquitous nature of mitochondria, the dual genetic foundation of the respiratory chain in mitochondrial and nuclear genome, and the peculiar rules of mitochondrial genetics all contribute to the extraordinary heterogeneity of clinical disorders associated with defects of oxidative phosphorylation (mitochondrial encephalomyopathies). Here, we review recent findings about nuclear gene defects in isolated OXPHOS enzyme complex deficiency. This information should help in identifying patients with mitochondrial disease and defining a biochemical and molecular basis of the disorder found in each patient. This knowledge is indispensable for accurate genetic counseling and prenatal diagnosis, and is a prerequisite for the development of rational therapies, which are still, at present, woefully inadequate.     

MtDNA mutations are a common cause of severe disease phenotypes in children with Leigh syndrome

Leigh syndrome is a common clinical manifestation in children with mitochondrial disease and other types of inborn errors of metabolism. We characterised clinical symptoms, prognosis, respiratory chain function and performed extensive genetic analysis of 25 Swedish children suffering from Leigh syndrome with the aim to obtain insights into the molecular pathophysiology and to provide a rationale for genetic counselling. We reviewed the clinical history of all patients and used muscle biopsies in order to perform molecular, biochemical and genetic investigations, including sequencing the entire mitochondrial DNA (mtDNA), the mitochondrial DNA polymerase (POLGA) gene and the surfeit locus protein 1 (SURF1) gene. Respiratory chain enzyme activity measurements identified five patients with isolated complex I deficiency and five with combined enzyme deficiencies. No patient presented with isolated complex IV deficiency. Seven patients had a decreased ATP production rate. Extensive sequence analysis identified eight patients with pathogenic mtDNA mutations and one patient with mutations in POLGA. Mutations of mtDNA are a common cause of LS and mtDNA analysis should always be included in the diagnosis of LS patients, whereas SURF1 mutations are not a common cause of LS in Sweden. Unexpectedly, age of onset, clinical symptoms and prognosis did not reveal any clear differences in LS patients with mtDNA or nuclear DNA mutations.     

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes,a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.     

Mitochondrial encephalomyopathies: Clinical and molecular analysis

The classification of mitochondrial encephalomyopathies relied upon clinical, biochemical, and histological features until the discovery of mitochondrial DNA defects in 1988. Since then, an outburst of molecular genetic information has aided our understanding of the pathogenesis and the classification of these heterogeneous disorders. Novel concepts of maternal inheritance, mitochondrial DNA (mtDNA) heteroplasmy, tissue distribution, and threshold have explained many of the clinical characteristics. The discovery of point mutations, large-scale mtDNA deletions, duplications, and autosomally inherited disorders with multiple mtDNA deletions have revealed new genetic phenomena. Despite our rapidly expanding understanding of the molecular genetic defects, many questions remain to be explored to fill the gap in our knowledge of the relationship between genotype and clinical phenotype.     

Mitochondrial DNA disease—molecular insights and potential routes to a cure

Mitochondrial DNA diseases are common neurological conditions caused by mutations in the mitochondrial genome or nuclear genes responsible for its maintenance. Current treatments for these disorders are focussed on the management of the symptoms, rather than the correction of biochemical defects caused by the mutation. This review focuses on the molecular effects of mutations, the symptoms they cause and current work focusing on the development of targeted treatments for mitochondrial DNA disease.     

Mitochondrial Disease: Mutations and Mechanisms

The mitochondrial diseases encompass a diverse group of disorders that can exhibit various combinations of clinical features. Defects in mitochondrial DNA (mtDNA) have been associated with these diseases, and studies have been able to assign biochemical defects. Deficiencies in mitochondrial oxidative phosphorylation appear to be the main pathogenic factors, although recent studies suggest that other mechanisms are involved. Reactive oxygen species (ROS) generation has been implicated in a wide variety of neurodegenerative diseases, and mitochondrial ROS generation may be an important factor in mitochondrial disease pathogenesis. Altered apoptotic signaling as a consequence of defective mitochondrial function has also been observed in both in vitro and in vivo disease models. Our current understanding of the contribution of these various mechanisms to mitochondrial disease pathophysiology will be discussed.     

A critical approach to the therapy of mitochondrial respiratory chain and oxidative phosphorylation diseases

Taking advantage of a series of questions raised by an association of patients with mitochondrial disease, this review, after a brief overview of basic concepts of mitochondrial bioenergetics and genetics, discusses the pros and cons of a number of practical options in the field of mitochondrial therapy. This makes it clear that, in contrast to the spectacular progress in our understanding of the biochemical and molecular bases of the mitochondrial diseases defined restrictively as disorders due to defects in the mitochondrial respiratory chain, we are still extremely limited in our ability to treat these conditions. We finally discussed the emerging genetic-based strategies that show some promise, even if much work remains to be done.     

Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches

Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE.     

Mitochondrial function in neurodegeneration and ageing.

The mitochondrial respiratory chain and oxidative phosphorylation system are responsible for the production of ATP by aerobic metabolism. Defects of the respiratory chain are increasingly recognised as important causes of human disease, and neurodegenerative disorders in particular. This article will seek to review the clinical and biochemical effects of respiratory chain defects, and summarise what is known about the molecular mechanisms that underlie them. Increasing age is also associated with a decline in mitochondrial function. The biochemical correlates of this dysfunction and the possible molecular defects that may cause it will also be reviewed.     

DNA polymerase gamma and mitochondrial disease: Understanding the consequence of POLG mutations

DNA polymerase γ is the only known DNA polymerase in human mitochondria and is essential for mitochondrial DNA replication and repair. It is well established that defects in mtDNA replication lead to mitochondrial dysfunction and disease. Over 160 coding variations in the gene encoding the catalytic subunit of DNA polymerase γ (POLG) have been identified. Our group and others have characterized a number of the more common and interesting mutations, as well as those disease mutations in the DNA polymerase γ accessory subunit. We review the results of these studies, which provide clues to the mechanisms leading to the disease state.     

Predictive properties of DNA methylation patterns in primary tumor samples for osteosarcoma relapse status

Osteosarcoma is the most common primary malignant bone tumor in children. Validated biological markers for disease prognosis available at diagnosis are lacking. No genome-wide DNA methylation studies linked to clinical outcomes have been reported in osteosarcoma to the best of our knowledge. To address this, we tested the methylome at over 1.1 million loci in 15 osteosarcoma biopsy samples obtained prior to the initiation of therapy and correlated these molecular data with disease outcomes. At more than 17% of the tested loci, samples obtained from patients who experienced disease relapse were more methylated than those from patients who did not have recurrence while patients who did not experience disease relapse had more DNA methylation at fewer than 1%. In samples from patients who went on to have recurrent disease, increased DNA methylation was found at gene bodies, intergenic regions and empirically-annotated candidate enhancers, whereas candidate gene promoters were unusual for a more balanced distribution of increased and decreased DNA methylation with 6.6% of gene promoter loci being more methylated and 2% of promoter loci being less methylated in patients with disease relapse. A locus at the TLR4 gene demonstrates one of strongest associations between DNA methylation and 5 y event-free survival (P-value = 1.7 × 10^?6), with empirical annotation of this locus showing promoter characteristics. Our data indicate that DNA methylation information has the potential to be predictive of outcome in pediatric osteosarcoma, and that both promoters and non-promoter loci are potentially informative in DNA methylation studies.     

Predictive properties of DNA methylation patterns in primary tumor samples for osteosarcoma relapse status

Osteosarcoma is the most common primary malignant bone tumor in children. Validated biological markers for disease prognosis available at diagnosis are lacking. No genome-wide DNA methylation studies linked to clinical outcomes have been reported in osteosarcoma to the best of our knowledge. To address this, we tested the methylome at over 1.1 million loci in 15 osteosarcoma biopsy samples obtained prior to the initiation of therapy and correlated these molecular data with disease outcomes. At more than 17% of the tested loci, samples obtained from patients who experienced disease relapse were more methylated than those from patients who did not have recurrence while patients who did not experience disease relapse had more DNA methylation at fewer than 1%. In samples from patients who went on to have recurrent disease, increased DNA methylation was found at gene bodies, intergenic regions and empirically-annotated candidate enhancers, whereas candidate gene promoters were unusual for a more balanced distribution of increased and decreased DNA methylation with 6.6% of gene promoter loci being more methylated and 2% of promoter loci being less methylated in patients with disease relapse. A locus at the TLR4 gene demonstrates one of strongest associations between DNA methylation and 5 y event-free survival (P-value = 1.7 × 10⁻⁶), with empirical annotation of this locus showing promoter characteristics. Our data indicate that DNA methylation information has the potential to be predictive of outcome in pediatric osteosarcoma, and that both promoters and non-promoter loci are potentially informative in DNA methylation studies.     

基因治疗20年

1990年9月14日美国NIH临床中心首次采用基因治疗成功治愈腺苷脱氨酶（ADA）基因缺陷而患重度联合免疫缺损和免疫系统功能低下疾患,至今已整整20年,其发展迅速,从单纯的重组技术导入基因DNA发展到了涵盖DNA和RNA两个干预水平、和基因上调（如基因增补、矫正、置换等）及下调（基因失活）的两大策略,近年来的进展使得基因治疗登入《Science》杂志2009年度十大科学进展,我国在基因治疗领域诞生了第一个上市药物,有10多个制剂临床前和多个在临床研究。基因治疗在遗传病治疗中具备巨大潜力,已经成为当代生命科学中最有前景的研究方向之一。