Infantile-onset spinocerebellar ataxia and mitochondrial recessive ataxia syndrome are associated with neuronal complex I defect and mtDNA depletion

Infantile-onset spinocerebellar ataxia (IOSCA) is a severe neurodegenerative disorder caused by the recessive mutation in PEO1, leading to an Y508C change in the mitochondrial helicase Twinkle, in its helicase domain. However, no mitochondrial dysfunction has been found in this disease. We studied here the consequences of IOSCA for the central nervous system, as well as the in vitro performance of the IOSCA mutant protein. The results of the mtDNA analyses were compared to findings in a similar juvenile or adult-onset ataxia syndrome, mitochondrial recessive ataxia syndrome (MIRAS), caused by the W748S mutation in the mitochondrial DNA polymerase (POLG). We show here that IOSCA brain does not harbor mtDNA deletions or increased amount of mtDNA point mutations, whereas MIRAS brain shows multiple deletions of mtDNA. However, IOSCA, and to a lesser extent also MIRAS, show mtDNA depletion in the brain and the liver. In both diseases, especially large neurons show respiratory chain complex I (CI) deficiency, but also CIV is decreased in IOSCA. Helicase activity, hexamerization and nucleoid structure of the IOSCA mutant were, however, unaffected. The lack of in vitro helicase defect or cell culture phenotype suggest that Twinkle-Y508C dysfunction affects mtDNA maintenance in a highly context and cell-type specific manner. Our results indicate that IOSCA is a new member of the mitochondrial DNA depletion syndromes.     

Clinical,biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.     

Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number

Mechanisms of mitochondrial DNA (mtDNA) maintenance have recently gained wide interest owing to their role in inherited diseases as well as in aging. Twinkle is a new mitochondrial 5'-3' DNA helicase, defects of which we have previously shown to underlie a mitochondrial disease, progressive external ophthalmoplegia with multiple mtDNA deletions. Mouse Twinkle is highly similar to the human counterpart, suggesting conserved function. Here, we have characterized the mouse Twinkle gene and expression profile and report that the expression patterns are not conserved between human and mouse, but are synchronized with the adjacent gene MrpL43, suggesting a shared promoter. To elucidate the in vivo role of Twinkle in mtDNA maintenance, we generated two transgenic mouse lines overexpressing wild-type Twinkle. We could demonstrate for the first time that increased expression of Twinkle in muscle and heart increases mtDNA copy number up to 3-fold higher than controls, more than any other factor reported to date. Additionally, we utilized cultured human cells and observed that reduced expression of Twinkle by RNA interference mediated a rapid drop in mtDNA copy number, further supporting the in vivo results. These data demonstrate that Twinkle helicase is essential for mtDNA maintenance, and that it may be a key regulator of mtDNA copy number in mammals.     

Sequence-specific stalling of DNA polymerase γ and the effects of mutations causing progressive ophthalmoplegia

A large number of mutations in the gene encoding the catalytic subunit of mitochondrial DNA polymerase γ (POLγA) cause human disease. The Y955C mutation is common and leads to a dominant disease with progressive external ophthalmoplegia and other symptoms. The biochemical effect of the Y955C mutation has been extensively studied and it has been reported to lower enzyme processivity due to decreased capacity to utilize dNTPs. However, it is unclear why this biochemical defect leads to a dominant disease. Consistent with previous reports, we show here that the POLγA:Y955C enzyme only synthesizes short DNA products at dNTP concentrations that are sufficient for proper function of wild-type POLγA. In addition, we find that this phenotype is overcome by increasing the dNTP concentration, e.g. dATP. At low dATP concentrations, the POLγA:Y955C enzyme stalls at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The POLγA:Y955C enzyme will compete with wild-type POLγA for primer utilization, and this will result in a heterogeneous population of short and long DNA replication products. In addition, there is a possibility that POLγA:Y955C is recruited to nicks of mtDNA and there enters an idling mode preventing ligation. Our results provide a novel explanation for the dominant mtDNA replication phenotypes seen in patients harboring the Y955C mutation, including the existence of site-specific stalling. Our data may also explain why mutations that disturb dATP pools can be especially deleterious for mtDNA synthesis.     

Thymidine kinase 2 mutations in autosomal recessive progressive external ophthalmoplegia with multiple mitochondrial DNA deletions

Autosomal-inherited progressive external ophthalmoplegia (PEO) is an adult-onset disease characterized by the accumulation of multiple mitochondrial DNA (mtDNA) deletions in post-mitotic tissues. Mutations in six different genes have been described to cause the autosomal dominant form of the disease, but only mutations in the DNA polymerase gamma gene are known to cause autosomal recessive PEO (arPEO), leaving the genetic background of arPEO mostly unknown. Here we used whole-exome sequencing and identified compound heterozygous mutations, leading to two amino acid alterations R225W and a novel T230A in thymidine kinase 2 (TK2) in arPEO patients. TK2 is an enzyme of the mitochondrial nucleotide salvage pathway and its loss-of-function mutations have previously been shown to underlie the early-infantile myopathic form of mtDNA depletion syndrome (MDS). Our TK2 activity measurements of patient fibroblasts and mutant recombinant proteins show that the combination of the identified arPEO variants, R225W and T230A, leads to a significant reduction in TK2 activity, consistent with the late-onset phenotype, whereas homozygosity for R225W, previously associated with MDS, leads to near-total loss of activity. Our finding identifies a new genetic cause of arPEO with multiple mtDNA deletions. Furthermore, MDS and multiple mtDNA deletion disorders are manifestations of the same pathogenic pathways affecting mtDNA replication and repair, indicating that MDS-associated genes should be studied when searching for genetic background of PEO disorders.     

Non-C282Y familial iron overload: evidence for locus heterogeneity in haemochromatosis.

Haemochromatosis (HC) is an autosomal recessive disease with progressive iron overload leading to midlife onset of clinical complications. The causal gene (HFE) maps to 6p, in close linkage with the HLA class I genes. An HFE candidate gene recently identified has two missense mutations (C282Y and H63D) associated with the disease. Here we document the phenotypic and genetic analysis of a nuclear family comprising two sibs with symptomatic and massive iron overload before the age of 25. The disease seemed to be recessively transmitted and fitted the agreed criteria for haemochromatosis, but was neither associated with the C282Y and H63D mutations nor linked with HLA markers. Our data strongly support locus heterogeneity in haemochromatosis by showing evidence that the gene responsible for juvenile haemochromatosis (JH) does not map to 6p. In the absence of clear cut phenotypic distinction from typical haemochromatosis, patients below 30 years of age and C282Y negative should be considered as putative juvenile cases. This has practical implications in genetic counselling and family management.     

A large homozygous deletion in the SAMHD1 gene causes atypical Aicardi-Goutiéres syndrome associated with mtDNA deletions

Aicardi-Goutiéres syndrome (AGS) is a genetic neurodegenerative disorder with clinical symptoms mimicking a congenital viral infection. Five causative genes have been described: three prime repair exonuclease1 (TREX1), ribonucleases H2A, B and C, and most recently SAM domain and HD domain 1 (SAMHD1). We performed a detailed clinical and molecular characterization of a family with autosomal recessive neurodegenerative disorder showing white matter destruction and calcifications, presenting in utero and associated with multiple mtDNA deletions. A muscle biopsy was normal and did not show any evidence of respiratory chain dysfunction. Southern blot analysis of tissue from a living child and affected fetuses demonstrated multiple mtDNA deletions. Molecular analysis of genes involved in mtDNA synthesis and maintenance (POLGα, POLGβ, Twinkle, ANT1, TK2, SUCLA1 and DGOUK) revealed normal sequences. Sequencing of TREX1 and ribonucleases H2A, B and C failed to reveal any mutations. Whole-genome homozygosity mapping revealed a candidate region containing the SAMHD1 gene. Sequencing of the gene in the affected child and two affected fetuses revealed a large deletion (9 kb), spanning the promoter, exon1 and intron 1. The parents were found to be heterozygous for this deletion. The identification of a homozygous large deletion in the SAMHD1 gene causing atypical AGS with multiple mtDNA deletions may add information regarding the involvement of mitochondria in self-activation of innate immunity by cell intrinsic components.     

Genetic and chemical rescue of the Saccharomyces cerevisiae phenotype induced by mitochondrial DNA polymerase mutations associated with progressive external ophthalmoplegia in humans

The human POLG gene encodes the catalytic subunit of mitochondrial DNA polymerase gamma (pol gamma). Mutations in pol gamma are associated with a spectrum of disease phenotypes including autosomal dominant and recessive forms of progressive external ophthalmoplegia, spino-cerebellar ataxia and epilepsy, and Alpers-Huttenlocher hepatocerebral poliodystrophy. Multiple deletions, or depletion of mtDNA in affected tissues, are the molecular hallmarks of pol gamma mutations. To shed light on the pathogenic mechanisms leading to these phenotypes, we have introduced in MIP1, the yeast homologue of POLG, two mutations equivalent to the human Y955C and G268A mutations, which are associated with dominant and recessive PEO, respectively. Both mutations induced the generation of petite colonies, carrying either rearranged (rho-) or no (rho0) mtDNA. Mutations in genes that control the mitochondrial supply of deoxynucleotides (dNTP) affect the mtDNA integrity in both humans and yeast. To test whether the manipulation of the dNTP pool can modify the effects of pol gamma mutations in yeast, we have overexpressed a dNTP checkpoint enzyme, ribonucleotide reductase, RNR1, or deleted its inhibitor, SML1. In both mutant strains, the petite mutability was dramatically reduced. The same result was obtained by exposing the mutant strains to dihydrolipoic acid, an anti-oxidant agent. Therefore, an increase of the mitochondrial dNTP pool and/or a decrease of reactive oxygen species can prevent the mtDNA damage induced by pol gamma mutations in yeast and, possibly, in humans.     

Depletion of the other genome-mitochondrial DNA depletion syndromes in humans

We present the current knowledge on the genetic and phenotypic aspects of mitochondrial DNA depletion syndromes. The human mitochondrial DNA encodes 13 of the 82 structural proteins of the mitochondrial electron transport chain. The replication and maintenance of the mtDNA require a large number of nuclear encoded enzymes and balanced nucleotide pools. Mitochondrial nucleotide synthesis is of major importance because of the constant need for nucleotides for mtDNA maintenance even in quiescent cells. As de novo enzymes are not present in the mitochondria, synthesis is accomplished via the salvage pathway. Defective mtDNA synthesis and maintenance manifest by multiple deletions or by depletion of the mitochondrial genome. Patients with multiple deletions typically present with progressive external ophthalmoplegia, ptosis and, exercise intolerance after the first decade of life. mtDNA depletion is usually an infantile disease characterized by severe muscle weakness, hepatic failure, or renal tubulopathy with fatal outcome. Linkage analysis in families with multiple mtDNA deletions reveal mutations in proteins that participate in mtDNA replication, the mitochondrial DNA polymerase gene, and the Twinkle gene, a putative mitochondrial helicase and in factors which play a role in mitochondrial nucleotide metabolism, the adenine nucleotide translocator, and the thymidine phosphorylase gene. We have recently identified mutations in an additional two essential proteins in the nucleotide salvage pathway, the mitochondrial deoxyribonucleoside kinases. The phenotype was distinctive for each gene, with hepatic failure and encephalopathy associated with mutations in the deoxyguanosine kinase gene and isolated devastating myopathy as the sole manifestation of thymidine kinase 2 deficiency. The tissue selectivity of these disorders and especially the exclusive muscle involvement in thymidine kinase 2 mutations is puzzling. The normal sequence of the remaining mtDNA copies in spite of a serious mitochondrial nucleotide imbalance is also unexpected. We propose several tissue-specific protective mechanisms and a time window, likely encompassing fetal life and even early infancy, during which nuclear nucleotide synthesis provides mitochondrial needs in all organs. We also speculate on future genes to be discovered in other phenotypes of mtDNA depletion.     

Decreased mtDNA, oxidative stress, cardiomyopathy, and death from transgenic cardiac targeted human mutant polymerase gamma

POLG is the human gene that encodes the catalytic subunit of DNA polymerase gamma (Pol gamma), the replicase for human mitochondrial DNA (mtDNA). A POLG Y955C point mutation causes human chronic progressive external ophthalmoplegia (CPEO), a mitochondrial disease with eye muscle weakness and mtDNA defects. Y955C POLG was targeted transgenically (TG) to the murine heart. Survival was determined in four TG (+/-) lines and wild-type (WT) littermates (-/-). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy defined the phenotype. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol gamma, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.     

Novel mutation in DGUOK in hepatocerebral mitochondrial DNA depletion syndrome associated with cystathioninuria

Mitochondrial depletion syndrome (MDS) refers to a heterogeneous group of mitochondrial disorders characterized by a reduction of the mtDNA copy number in affected tissues. Mutations in DGUOK encoding deoxyguanosine kinase (MIM 601465) cause the hepatocerebral form of MDS (MIM 251880). Cystathioninuria (MIM 219500) can result from mutations in CTH encoding cystathionine gamma lyase (MIM 607657) or can be a secondary finding in several diverse clinical conditions. We present three patients from two apparently unrelated old colony Mennonite families, each of whom had the hepatocerebral form of MDS together with cystathioninuria. Each affected child was homozygous for the novel DGUOK p.D255Y mutation, but had no CTH mutation, indicating that the hepatocerebral form of MDS might be associated with secondary cystathioninuria.     

The role of saposin C in Gaucher disease

Saposin C is one of four homologous proteins derived from sequential cleavage of the saposin precursor protein, prosaposin. It is an essential activator for glucocerebrosidase, the enzyme deficient in Gaucher disease. Gaucher disease is a rare autosomal recessive lysosomal storage disorder caused by mutations in the GBA gene that exhibits vast phenotypic heterogeneity, despite its designation as a "simple" Mendelian disorder. The observed phenotypic variability has led to a search for disease modifiers that can alter the Gaucher phenotype. The PSAP gene encoding saposin C is a prime candidate modifier for Gaucher disease. In humans, saposin C deficiency due to mutations in PSAP results in a Gaucher-like phenotype, despite normal in vitro glucocerebrosidase activity. Saposin C deficiency has also been shown to modify phenotype in one mouse model of Gaucher disease. The role of saposin C as an activator required for normal glucocerebrosidase function, and the consequences of saposin C deficiency are described, and are being explored as potential modifying factors in patients with Gaucher disease.     

Twenty-two novel mutations in the lysosomal alpha-glucosidase gene (GAA) underscore the genotype-phenotype correlation in glycogen storage disease type II

Patients with glycogen storage disease type II (GSDII, Pompe disease) suffer from progressive muscle weakness due to acid alpha-glucosidase deficiency. The disease is inherited as an autosomal recessive trait with a spectrum of clinical phenotypes. We have investigated 29 cases of GSDII and thereby identified 55 pathogenic mutations of the acid alpha-glucosidase gene (GAA) encoding acid maltase. There were 34 different mutations identified, 22 of which were novel. All of the missense mutations and two other mutations with an unpredictable effect on acid alpha-glucosidase synthesis and function were transiently expressed in COS cells. The effect of a novel splice-site mutation was investigated by real-time PCR analysis. The outcome of our analysis underscores the notion that the clinical phenotype of GSDII is largely dictated by the nature of the mutations in the GAA alleles. This genotype-phenotype correlation makes DNA analysis a valuable tool to help predict the clinical course of the disease.     

Novel and recurrent mutations in lamin A/C in patients with Emery-Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EDMD) is characterized by slowly progressive muscle wasting and weakness; early contractures of the elbows, Achilles tendons, and spine; and cardiomyopathy associated with cardiac conduction defects. Clinically indistinguishable X-linked and autosomal forms of EDMD have been described. Mutations in the STA gene, encoding the nuclear envelope protein emerin, are responsible for X-linked EDMD, while mutations in the LMNA gene encoding lamins A and C by alternative splicing have been found in patients with autosomal dominant, autosomal recessive, and sporadic forms of EDMD. We report mutations in LMNA found in four familial and seven sporadic cases of EDMD, including seven novel mutations. Nine missense mutations and two small in-frame deletions were detected distributed throughout the gene. Most mutations (7/11) were detected within the LMNA exons encoding the central rod domain common to both lamins A/C. All of these missense mutations alter residues in the lamin A/C proteins conserved throughout evolution, implying an essential structural and/or functional role of these residues. One severely affected patient possesed two mutations, one specific to lamin A that may modify the phenotype of this patient. Mutations in LMNA were frequently identified among patients with sporadic and familial forms of EDMD. Further studies are needed to identify the factors modifying disease phenotype among patients harboring mutations within lamin A/C and to determine the effect of various mutations on lamin A/C structure and function.     

Diseases caused by nuclear genes affecting mtDNA stability

Diseases caused by nuclear genes that affect mitochondrial DNA (mtDNA) stability are an interesting group of mitochondrial disorders, involving both cellular genomes. In these disorders, a primary nuclear gene defect causes secondary mtDNA loss or deletion formation, which leads to tissue dysfunction. Therefore, the diseases clinically resemble those caused by mtDNA mutations, but follow a Mendelian inheritance pattern. Several clinical entities associated with multiple mtDNA deletions have been characterized, the most frequently described being autosomal dominant progressive external ophthalmoplegia (adPEO). MtDNA depletion syndrome (MDS) is a severe disease of childhood, in which tissue-specific loss of mtDNA is seen. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients may have multiple mtDNA deletions and/or mtDNA depletion. Recent reports of thymidine phosphorylase mutations in MNGIE and adenine nucleotide translocator mutations in adPEO have given new insights into the mechanisms of mtDNA maintenance in mammals. The common mechanism underlying both of these gene defects could be disturbed mitochondrial nucleoside pools, the building blocks of mtDNA. Future studies on MNGIE and adPEO pathogenesis, and identification of additional gene defects in adPEO and MDS will provide further understanding about the mammalian mtDNA maintenance and the crosstalk between the nuclear and mitochondrial genomes.     

A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2)

Progressive myoclonus epilepsy of the Lafora type or Lafora disease (EPM2; McKusick no. 254780) is an autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration and glycogen-like intracellular inclusion bodies (Lafora bodies). A gene for EPM2 previously has been mapped to chromosome 6q23- q25 using linkage analysis and homozygosity mapping. Here we report the positional cloning of the 6q EPM2 gene. A microdeletion within the EPM2 critical region, present inhomozygosis in an affected individual, was found to disrupt a novel gene encoding a putative protein tyrosine phosphatase (PTPase). The gene, denoted EPM2, presents alternative splicing in the 5' and 3' end regions. Mutational analysis revealed that EPM2 patients are homozygous for loss-of-function mutations in EPM2. These findings suggest that Lafora disease results from the mutational inactivation of a PTPase activity that may be important in the control of glycogen metabolism.      

POLG基因突变在人类疾病中作用的研究

DNA聚合酶γ(DNA polymerase γ,pol γ)由核基因组的POLG基因编码,该基因定位于染色体15q24-q26,有23个外显子.pol γ对于线粒体DNA的复制和修复起到了十分重要的作用.近年的研究表明POLG基因的突变是人类疾病的一个重要致病因素.迄今为止,已发现约150种POLG基因的致病突变和9种非致病性多态性与人类疾病相关,如进行性外眼肌麻痹(progressive external ophhalmo-plegia,PEO)、阿尔佩尔综合征(Alpers syndrome)、感觉性共济失调、神经病变性运动性构音障碍及外眼肌麻痹(sensory ataxia neuropathy dysarthria and ophthalmoplegia,SANDO)、帕金森综合征(Parkinson's djsease,PD)和男性不育(male infertility)等等.近年来转基因小鼠模型的建立以及pol γ生化性质方面的研究使人们对POLG基因突变与人类疾病的发病机制的相关性有了进一步的认识.本文对近年来POLG基因突变与人类疾病相天性的研究进展作一综述.     

Spondyloepimetaphyseal dysplasia with neurodegeneration associated with AIFM1 mutation – a novel phenotype of the mitochondrial disease

In 1999, based on a single family, spondyloepimetaphyseal dysplasia (SEMD) with mental retardation (MR) was described as a novel syndrome with probably X‐linked recessive inheritance and unknown molecular defect (MIM 300232). Our purpose was to search for the causative defect in the originally described family and in an independently ascertained second family. All patients had slowly progressive neurodegeneration with central and peripheral involvement and identical skeletal dysplasia. Whole exome sequencing performed in two subjects showed a single plausible candidate – the p.Asp237Gly variant in AIFM1 (chr. Xq26.1). The p.Asp237Gly segregated with disease as indicated by linkage analysis [maximum logarithm of odds score (LOD) score at theta 0 for the two families was 3.359]. This variant had not been previously reported and it was predicted to be pathogenic by Polyphen2, SIFT, MutationTaster and Mutation Assessor. AIFM1 encodes mitochondria associated apoptosis‐inducing factor. The AIFM1 gene has been linked with COXPD6 encephalomyopathy (MIM 300816), Cowchock syndrome (MIM 310490) and X‐linked deafness with neuropathy (DFNX5, MIM 300614), none of which are similar to SEMD‐MR. Our results place SEMD as the third instance of a skeletal phenotype associated with a mitochondrial disease (the others being EVEN‐PLUS syndrome caused by mutations of HSPA9 and CODAS syndrome due to LONP1 mutations).