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.     

Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome

Mitochondrial deoxyguanosine kinase (dGK) catalyzes the initial phosphorylation of purine deoxynucleosides. Mutations in the dGK gene leading to deficiency in dGK activity is one of the causes of severe mitochondrial DNA depletion diseases. We used site-directed mutagenesis to introduce the clinically observed genetic alterations in the dGK gene and characterized the recombinant enzymes. The R142K enzyme had very low activity with deoxyguanosine and no activity with deoxyadenosine. The E227K mutant enzyme had unchanged K(m) values for all its substrates but very low V(max) values. C-terminal truncated dGK proteins were inactive. These results may help to define the role of dGK in mitochondrial DNA (mtDNA) precursor synthesis.     

High frequency of mitochondrial DNA D-loop mutations in Ewing’s sarcoma

Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing’s sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations’ possible involvement in the initiation and/or progression of this rare malignancy.     

Specific point mutations may not accumulate with aging in the mouse mitochondrial DNA control region

Increasing evidence suggests that mitochondrial function declines during aging in various tissues and in a wide range of organisms. This correlates with an age-dependent large accumulation of specific point mutations in the mtDNA control region that was reported recently in human fibroblast and skeletal muscle. However, evaluations of aging-related mtDNA mutations in other model animal systems. In this study, we analyzed mtDNA control regions of brain, skeletal muscle, heart, and other tissues from aged mice, in search of specific point mutations. A 948-bp fragment covering the entire mtDNA control region from various tissues of mice at the age of 25–26 months was sequenced. The sequence analysis was accomplished with a newly developed program Mutation Quantifier, which was able to accurately detect mutations with frequencies as low as 3%. Probably due to the relative shorter life-span, unlike what has been reported in human mtDNA, our results indicated there might be no significant accumulation of specific mutations in mouse mtDNA control region during aging.     

Novel point mutations in the mitochondrial DNA detected in patients with dilated cardiomyopathy by screening the whole mitochondrial genome

Dilated cardiomyopathy (DCM) is widely accepted as a pluricausal or multifactorial disease. Because of the linkage between energy metabolism in the mitochondria and cardiac muscle contraction, it is reasonable to assume that mitochondrial abnormalities may be responsible for some forms of DCM. We analysed the whole mitochondrial genome in a series of 45 patients with DCM for alterations and compared the findings with those of 62 control subjects. A total of 458 sequence changes could be identified. These sequence changes were distributed among the whole mitochondrial DNA (mtDNA). An increased number of novel missense mutations could be detected nearly in all genes encoding for protein subunits in DCM patients. In genes coding for NADH dehydrogenase subunits the number of mtDNA mutations detected in patients with DCM was significantly increased (p < 0.05) compared with control subjects. Eight mutations were found to occur in conserved amino acids in the above species. The c.5973G > A (Ala-Trp) and the c.7042T > G (Val-Asp) mutations were located in highly conserved domains of the gene coding for cytochrome c oxidase subunit. Two tRNA mutations could be detected in the mtDNA of DCM patients alone. The T-C transition at nt 15,924 is connected with respiratory enzyme deficiency, mitochondrial myopathy, and cardiomyopathy. The c.16189T > C mutation in the D-loop region that is associated with susceptibility to DCM could be detected in 15.6% of patients as well as in 9.7% of controls. Thus, mutations altering the function of the enzyme subunits of the respiratory chain can be relevant for the pathogenesis of dilated cardiomyopathy.     

Sequence analysis of the complete mitochondrial genome in patients with mitochondrial encephaloneuromyopathies lacking the common pathogenic DNA mutations

The purpose of this study was to identify novel mitochondrial deoxyribonucleic acid (mtDNA) mutations in a series of patients with clinical and/or morphological features of mitochondrial dysfunction, but still no genetic diagnosis. A heterogeneous group of clinical disorders is caused by mutations in mtDNA that damage respiratory chain function of cell energy production. We developed a method to systematically screen the entire mitochondrial genome. The sequence-data were obtained with a rapid automated system. In the six mitochondrial genomes analysed we found 20 variants of the revised Cambridge reference sequence [Nat. Genet. 23 (1999) 147]. In skeletal muscle nineteen novel mtDNA variants were homoplasmic, suggesting secondary pathogenicity or co-responsibility in determination of the disease. In one patient we identified a novel heteroplasmic mtDNA mutation which presumably has a pathogenic role. This screening is therefore useful to extend the mtDNA polymorphism database and should facilitate definition of disease-related mutations in human mtDNA.     

Molecular phylogeny of gibbons inferred from mitochondrial DNA sequences: Preliminary report

We analyzed the 896 base-pair (bp) mitochondrial DNA (mtDNA) sequences for seven gibbons, representative of three out of four subgenera. The result from our molecular analysis is consistent with previous studies as to the monophyly of subgenus Hylobates species, yet the relationship among subgenera remains slightly ambiguous. A striking result of the analysis is the phylogenetic location of Kloss's gibbon (H. klossii). Kloss's gibbon has been considered to be an initial off-shoot of the subgenus Hylobates because of its morphological primitiveness. However, our molecular data strongly suggest that Kloss's gibbon speciated most recently within the subgenus Hylobates. Correspondence to: S. Horai     

Mitochondrial DNA mutations and ageing

The mechanism by which we age has sparked a huge number of theories, and is an area of intense debate. As the elderly population rises, the importance of elucidating these mechanisms is becoming more apparent as age is the single biggest risk factor for a number of diseases such as cancer, diabetes and neurodegenerative disease. Mitochondrial DNA (MtDNA) mutations have been shown to accumulate in cells and tissues during the ageing process; however the question as to whether these mutations have a causal role in the ageing process remains an area of uncertainty. Here we review the current literature, and discuss the evidence for and against a causal role of mtDNA mutations in ageing and in the pathogenesis of age-related disease.     

Molecular analysis of genomic stability of mitochondrial DNA in tissue cultured cells of maize

Summary Mitochondrial DNA (mtDNA) of the Black Mexican sweet line of maize isolated from tissue cultured cell suspension cultures and young seedlings was examined. Restriction fragments generated by two endonucleases were comparatively analyzed by visualization of ethidium bromide stained gels as well as by membrane hybridization with nick-translated DNA probes of plasmid-like S1 and S2 DNA. Although no major molecular alterations were seen in tissue cultured cells, the samples were clearly not identical. The variation was mainly in the stoichiometry of several restriction fragments. Hybridization analyses with S1 and S2 probes show no evidence of molecular rearrangement in this part of the genome in tissue cultured cells. Minor variations in restriction patterns could reflect alterations in frequency of circular mtDNA molecules, perhaps related to nuclear alterations during the extended period of culture.     

Quantification of random mutations in the mitochondrial genome

Mitochondrial DNA (mtDNA) mutations contribute to the pathology of a number of age-related disorders, including Parkinson disease [A. Bender et al., Nat. Genet. 38 (2006) 515,Y. Kraytsberg et al., Nat. Genet. 38 (2006) 518], muscle-wasting [J. Wanagat, Z. Cao, P. Pathare, J.M. Aiken, FASEB J. 15 (2001) 322], and the metastatic potential of cancers [K. Ishikawa et al., Science 320 (2008) 661]. The impact of mitochondrial DNA mutations on a wide variety of human diseases has made it increasingly important to understand the mechanisms that drive mitochondrial mutagenesis. In order to provide new insight into the etiology and natural history of mtDNA mutations, we have developed an assay that can detect mitochondrial mutations in a variety of tissues and experimental settings [M. Vermulst et al., Nat. Genet. 40 (2008) 4, M. Vermulst et al., Nat. Genet. 39 (2007) 540]. This methodology, termed the Random Mutation Capture assay, relies on single-molecule amplification to detect rare mutations among millions of wild-type bases [J.H. Bielas, L.A. Loeb, Nat. Methods 2 (2005) 285], and can be used to analyze mitochondrial mutagenesis to a single base pair level in mammals.     

Single molecule PCR in mtDNA mutational analysis: Genuine mutations vs. damage bypass-derived artifacts

The area of somatic mtDNA mutation measurement is in a crisis because the methods used to quantify mtDNA mutations produce results varying by multiple orders of magnitude. The reason for these discrepancies is not clear, but given that most methods involve PCR, the prime suspect is PCR artifacts (e.g. spontaneous errors by the DNA polymerases used). In addition to simple misincorporation, another important source of artificial mutations is the conversion of chemically modified (e.g. damaged) nucleotides into mutations when bypassed by a thermostable DNA polymerase. These latter mutations are particularly difficult to account for because appropriate controls are not available. Here, we argue that single molecule PCR (smPCR) is uniquely positioned to account for these bypass-related artificial mutations and discuss the methodology involved in employing this technique.     

Mitochondria: Biogenesis and mitophagy balance in segregation and clonal expansion of mitochondrial DNA mutations

Mitochondria are cytoplasmic organelles containing their own multi-copy genome. They are organized in a highly dynamic network, resulting from balance between fission and fusion, which maintains homeostasis of mitochondrial mass through mitochondrial biogenesis and mitophagy. Mitochondrial DNA (mtDNA) mutates much faster than nuclear DNA. In particular, mtDNA point mutations and deletions may occur somatically and accumulate with aging, coexisting with the wild type, a condition known as heteroplasmy. Under specific circumstances, clonal expansion of mutant mtDNA may occur within single cells, causing a wide range of severe human diseases when mutant overcomes wild type. Furthermore, mtDNA deletions accumulate and clonally expand as a consequence of deleterious mutations in nuclear genes involved in mtDNA replication and maintenance, as well as in mitochondrial fusion genes (mitofusin-2 and OPA1), possibly implicating mtDNA nucleoids segregation. We here discuss how the intricacies of mitochondrial homeostasis impinge on the intracellular propagation of mutant mtDNA.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Analysis of mitochondrial DNA mutations in D-loop region in thyroid lesions

Background

Mitochondrial defects have been associated with various human conditions including cancers.

Methods

We analyzed the mutations at the mitochondrial DNA (mtDNA) in patients with different thyroid lesions. In particular, in order to investigate if the accumulation of mtDNA mutations play a role in tumor progression, we studied the highly variable main control region of mtDNA, the displacement-loop (D-loop) in patients with non-tumor nodular goiters, with benign thyroid adenomas, and with malignant thyroid carcinomas. Total thyroid tumor or goiter samples were obtained from 101 patients, matched with nearby normal tissue and blood from the same subject.

Results

Noticeably, mitochondrial microsatellite instability (mtMSI) was detected in 2 of 19 nodular goiters (10.53%), and 8 of 77 (10.39%) malignant thyroid carcinomas. In addition, 6 patients, including 5 (6.49%) with malignant thyroid carcinomas and 1 (5.26%) with nodular goiter, were found to harbor point mutations. The majority of the mutations detected were heteroplasmic.

General significance

Our results indicate that mtDNA alterations in the D-loop region could happen before tumorigenesis in thyroid, and they might also accumulate during tumorigenesis.     

Molecular phylogeny of the Chinese ranids inferred from nuclear and mitochondrial DNA sequences

The phylogeny of representative species of Chinese ranids was reconstructed using two nuclear (tyrosinase and rhodopsin) and two mitochondrial (12S rRNA, 16S rRNA) DNA fragments. Maximum parsimony, Bayesian, and maximum likelihood analyses were employed. In comparison with the results from nuclear and mitochondrial data, we used nuclear gene data as our preferred phylogenetic hypothesis. We proposed two families (Ranidae, Dicroglossidae) for Chinese ranids, with the exception of genus Ingerana. Within Dicroglossidae, four tribes were supported including Dicroglossini, Paini, Limnonectini, and Occidozygini. A broader sampling strategy and evidence from additional molecular markers are required to decisively evaluate the evolutionary history of Chinese ranids.     

Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing

Several types of deletions in mitochondrial DNA (mtDNA) have been recetly identified in various tissues of old humans. In order to determine whether there are differences in the incidence and proportion of deleted mtDNAs in different tissues during human ageing, we examined tha 4,977 bp deletion in mtDNA of various tissues from subjects of different ages. Total DNA was extracted from each of the biopsied tissues and was serially diluted by two-fold with distilled water. A 533 bp DNA fragment was amplified by PCR from total mtDNA using a pair of primers L3304-3323 and H3817-3836, and another 524 bp PCR product was amplified from 4,977 bp deleted mtDNA by identical conditions using another pair of primers L8150-8166 and H13631-13650. The maximum dilution fold of each sample that still allowed the ethidium bromide-stained PCR product (533 bp or 524 bp) in the agarose gel to be visible under UV light illumination was taken as the relative abundance of the mtDNA (wild-type or mutant) in the original sample. By this method, we were able to determine the proportion of deleted mtDNA in human tissues. We found that the 4,977 bp deletion started to appear in the second and third decades of life in human muscle and liver tissues. But the deletion was not detectable in the testis until the age of 60 years. Moreover, the proportion of deleted mtDNA varied greatly in different tissues. Among the tissues examined, muscle was found to harbor higher proportin of deleted mtDNA than the other tissues. The average proportion of the 4,977 bp depleted mtDNA of the muscle from subjects over 70 years old was approximately 0.06%, and that of the liver and the testis was 0.0076% and 0.05%, respectively. These findings suggest that the frequency and proportion of the deleted mtDNA in human tissues increase with age and that the mtDNA deletions occur more frequently and abundantly in high energy-demanding tissues during the ageing process of the human.     

Sequence analysis of the complete mitochondrial genome in patients with Leber's hereditary optic neuropathy lacking the three most common pathogenic DNA mutations

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder characterized by central vision loss in young adults. The majority of LHON cases around the world are associated with mutations in the mitochondrial genome at nucleotide positions (np) 3460, 11,778, and 14,484. Usually, these three mutations are screened in suspected LHON patients. The result is important not only in respect to the diagnosis but also as different LHON mutations lead to variations in expression, severity, and recovery of the disease. There are, however, a significant number of patients without any of these primary mutations. In these situations, genetic counselling of a patient and his family can be difficult. We sequenced the complete mitochondrial DNA (mtDNA) in 14 LHON patients with the typical clinical features but without a primary mtDNA mutation to evaluate the potential of extensive mutation screening for clinical purposes. Our results suggest to include the mutation at np 15,257 in a routine screening as well as the ND6 gene, a hot spot for LHON mutations. Screening for the secondary LHON mutations at np 4216 and np 13,708 may also help in making the diagnosis of LHON as these seem to modify the expression of LHON mutations. Although they do not allow to prove the clinical diagnosis, their presence increases the probability of LHON. Sequencing the complete mitochondrial genome can reveal novel and known rare disease causing mutations. However, considering the effort it adds little value for routine screening.     

RFLP analysis of mitochondrial DNA from cytoplasmic male-sterile lines of pearl millet

Mitochondrial DNA (mtDNA) from 13 cytoplasmic male-sterile (cms) lines from diverse sources were characterized by Southern blot hybridization to pearl millet and maize mtDNA probes. Hybridization patterns of mtDNA digested with PstI, BamHI, SmaI or XhoI and probed with 13.6-, 10.9-, 9.7- or 4.7-kb pearl millet mtDNA clones revealed similarities among the cms lines 5141 A and ICMA 1 (classified as the S-A1 type of cytoplasm based on fertility restoration patterns), PMC 30A and ICMA 2. The remaining cms lines formed a distinct group, within which three subgroups were evident. Among the maize mitochondiral gene clones used, the coxI probe revealed two distinct groups of cytoplasms similar to the pearl millet mtDNA clones. The atp9 probe differentiated the cms line 81 A4, derived from P. glaucum subsp. monodii, while the coxII gene probe did not detect any polymorphism among the cms lines studied. MtDNA digested with BamHI, PstI or XhoI and hybridized to the atp6 probe revealed distinct differences among the cms lines. The maize atp6 gene clone identified four distinct cytoplasmic groups and four subgroups within a main group. The mtDNA fragments hybridized to the atp6 gene probe with differing intensities, suggesting the presence of more than one copy of the gene in different stoichiometries. Rearrangements involving the coxI and/or rrn18-rrn5 genes (mapped within the pearl millet clones) probably resulted in the S-A1 type of sterility. Rearrangements involving the atp6 gene (probably resulting in chimeric form) may be responsible for male sterility in other cms lines of pearl millet.     

神经肌肉性疾病患者线粒体DNA突变的分析

为了探讨神经肌肉性疾病的发病与线粒体ＤＮＡ突变的关系,采用ＰＣＲ技术检测了２０例患有不同神经肌肉性疾病儿童的外周血和骨骼肌细胞中的线粒体ＤＮＡ（ｍｔＤＮＡ）,发现其中６例患儿有ｍｔＤＮＡ缺失,其中１例至少有２９６８ｂｐ片段的缺失,另５例至少有２０００ｂｐ片段的缺失,此缺失区位于线粒体呼吸链复合物１、４、５、编码区,表明该突变对神经肌肉性疾病的发生有一定作用。     

Clinical phenotype, prognosis and mitochondrial DNA mutation load in mitochondrial encephalomyopathies

We studied 42 individuals, including 8 patients with either complete or partial syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), 8 patients with either complete or partial syndrome of myoclonic epilepsy with ragged-red fibers (MERRF) and 26 maternal family members who carried either the A3243G or A8344G mutation of mitochondrial DNA (mtDNA). Clinical manifestations and prognosis were followed up in the patients harboring the A3243G or A8344G mutation. The relationship between clinical features and proportions of mutant mtDNAs in muscle biopsies, blood cells and/or hair follicles was studied. In the 8 regularly followed patients with the A3243G mutation, 4 died within 1 month to 7 years due to status epilepticus and/or recurrent stroke-like episodes. Two patients developed marked mental deterioration and 2 remained stationary. All of the patients harboring the A8344G mutation were stable or deteriorated slightly, except for 1 patient who died due to brain herniation after putaminal hemorrhage. The A3243G and A8344G mtDNA mutations were heteroplasmic in the muscle biopsies, blood cells and hair follicles of both the probands and their maternal family members. The mean proportion of A3243G mutant mtDNA in the muscle biopsies of the patients with MELAS syndrome (68.5 ± 21.3%, range 33–92%) was significantly higher than that of the asymptomatic family members (37.1 ± 12.6%, range 0–51%). The average proportions of A8344G mutant mtDNA in the muscle biopsies (90.1 ± 3.9%, range 89–95%) and hair follicles (93.9 ± 6.4%, range 84–99%) of the patients with MERRF syndrome were also significantly higher than those of the asymptomatic family members (muscle: 40.3 ± 39.5%, range 1–80%; hair follicles: 51.0 ± 44.5%, range 0.1–82%). We concluded that measurement of the proportion of mutant mtDNA in muscle biopsies may provide useful information in the identification of symptomatic patients with mitochondrial encephalomyopathies. For patients with the A3243G mutation, the prognosis was related to status epilepticus and the number of recurrent stroke-like episodes and was much worse than for patients with the A8344G mutation of mtDNA, who had stable or slowly deteriorating clinical courses.