Heteroplasmic Point Mutations Research Articles

Use of Next-Generation Sequencing for Identifying Mitochondrial Disorders.

Mitochondria are major contributors to ATP synthesis, generating more than 90% of the total cellular energy production through oxidative phosphorylation (OXPHOS): metabolite oxidation, such as the β-oxidation of fatty acids, and the Krebs’s cycle. OXPHOS inadequacy due to large genetic lesions in mitochondrial as well as nuclear genes and homo- or heteroplasmic point mutations in mitochondrially encoded genes is a characteristic of heterogeneous, maternally inherited genetic disorders known as mitochondrial disorders that affect multisystemic tissues and organs with high energy requirements, resulting in various signs and symptoms. Several traditional diagnostic approaches, including magnetic resonance imaging of the brain, cardiac testing, biochemical screening, variable heteroplasmy genetic testing, identifying clinical features, and skeletal muscle biopsies, are associated with increased risks, high costs, a high degree of false-positive or false-negative results, or a lack of precision, which limits their diagnostic abilities for mitochondrial disorders. Variable heteroplasmy levels, mtDNA depletion, and the identification of pathogenic variants can be detected through genetic sequencing, including the gold standard Sanger sequencing. However, sequencing can be time consuming, and Sanger sequencing can result in the missed recognition of larger structural variations such as CNVs or copy-number variations. Although each sequencing method has its own limitations, genetic sequencing can be an alternative to traditional diagnostic methods. The ever-growing roster of possible mutations has led to the development of next-generation sequencing (NGS). The enhancement of NGS methods can offer a precise diagnosis of the mitochondrial disorder within a short period at a reasonable expense for both research and clinical applications.

Mutational Analysis and mtDNA Haplogroup Characterization in Three Serbian Cases of Mitochondrial Encephalomyopathies and Literature Review.

Mitochondrial encephalomyopathies (MEMP) are heterogeneous multisystem disorders frequently associated with mitochondrial DNA (mtDNA) mutations. Clinical presentation varies considerably in age of onset, course, and severity up to death in early childhood. In this study, we performed molecular genetic analysis for mtDNA pathogenic mutation detection in Serbian children, preliminary diagnosed clinically, biochemically and by brain imaging for mitochondrial encephalomyopathies disorders. Sanger sequencing analysis in three Serbian probands revealed two known pathogenic mutations. Two probands had a heteroplasmic point mutation m.3243A>G in the MT-TL1 gene, which confirmed mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode syndrome (MELAS), while a single case clinically manifested for Leigh syndrome had an almost homoplasmic (close to 100%) m.8993T>G mutation in the MT-ATP6 gene. After full mtDNA MITOMASTER analysis and PhyloTree build 17, we report MELAS’ association with haplogroups U and H (U2e and H15 subclades); likewise, the mtDNA-associated Leigh syndrome proband shows a preference for haplogroup H (H34 subclade). Based on clinical–genetic correlation, we suggest that haplogroup H may contribute to the mitochondrial encephalomyopathies’ phenotypic variability of the patients in our study. We conclude that genetic studies for the distinctive mitochondrial encephalomyopathies should be well-considered for realizing clinical severity and possible outcomes.

MITOCHONDRIAL DISEASES (Posters): P.191Single muscle fiber analysis of extraocular and skeletal muscles in a CPEO patient harboring a pathogenic point mutation in the MT-TN gene

Chronic progressive external ophthalmoplegia (CPEO) is a frequent feature of mitochondrial disorders and usually associated with mitochondrial DNA (mtDNA) mutations. Skeletal muscle (SKM) tissue is most frequently used for biochemical analyses and mitochondrial genome testing. However, extraocular muscle (EOM) is the clinically most affected tissue but usually not available for routine work-up. Consequently, systematic data on EOM is limited and the reason for preferential clinical affection remains unclear. We addressed this unsolved question by histochemical and genetic analyses of EOM and SKM single muscle fibers in a patient with isolated CPEO caused by a heteroplasmic point mutation in the MT-TN gene. The histochemical analysis showed higher absolute numbers of cytochrome c oxidase (COX)-deficient EOM fibers compared to COX-deficient SKM fibers. However, genetic analyses by restriction fragment length polymorphism revealed no significant difference in the mutation loads between COX-negative single muscle fibers in EOM compared to SKM. Quantitative single fiber real-time PCR revealed higher mtDNA copy numbers in single muscle fibers of EOM compared to SKM. COX-negative single muscle fibers of EOM and SKM showed significantly higher mtDNA copy numbers compared to COX-positive fibers suggestive of a compensatory mtDNA proliferation. We show that high loads of the MT-TN mutation correlate with a biochemical loss of COX activity in single muscle fibers of EOM and SKM at a similar threshold. The higher absolute numbers of COX-negative fibers in EOM compared to SKM might be caused by facilitated segregation of the mutation into the EOM providing thereby a possible explanation of the preferential ocular manifestation in CPEO.

Investigation of frequent somatic mutations of MTND5 gene in gastric cancer cell lines and tissues

The present study investigated the single nucleotide variants (SNVs) in mitochondrial DNA (mtDNA) of 13 paired gastric cancer tissue samples and seven gastric cancer cell lines using direct sequencing analysis of the MTND5 region. Results showed that nuclear mitochondrial pseudogenes (NUMTs) and mitochondrial copy number affected the detection of the SNV frequency in gastric cancer tissue and cell line samples using high-throughput sequencing technique. The heteroplasmic point mutation C12474T and G12835A happened in AGS and BGC823 cell lines, respectively. A total of seven SNVs were found in three paired gastric cancer tissue samples, including five heteroplasmic point mutations (A12406G, C12705T, T12882C, G12501A, and A12584G) and two homoplasmic point mutations (G12561A and C13590T). Gastric cancer tissue sample 16 exhibited the highest SNVs frequency with four SNVs (np 12406, np 12705, np 12882, and np 12501), whereas no SNVs or SNPs were detected in the tissue sample 4. SNP 12705 turned out to be an SNV in gastric cancer tissue sample 16. SNV 12338 detected by exome sequencing approach appeared to be an SNP in this study.

A New Mitochondrial tRNA(Met)—Gene Mutation in a 12-Year-Old Boy with MELAS: Clinical Presentation and Effects of Arginine Treatment

Introduction: MELAS syndrome is a mitochondrial (mt) DNA disorder with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes as main features. Fourteen different point mutations in six different mitochondrial tRNA genes have been identified so far. The clinical course is progressive leading to mental deterioration and neurological deficits. We report on the first identification of a heteroplasmic point mutation in the mitochondrial tRNA gene for Methionine.

Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs

The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but arenot detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, andmany mutations were non-synonymous or resided in RNA coding genes and thus can lead torespiratory defects. Our results highlight a need tomonitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine themetabolic status of iPSCs destined for clinical applications.

A new mitochondrial point mutation in the transfer RNALys gene associated with progressive external ophthalmoplegia with impaired respiratory regulation

We report a novel heteroplasmic point mutation G8299A in the gene for mitochondrial tRNALys in a patient with progressive external ophthalmoplegia complicated by recurrent respiratory insufficiency. Biochemical analysis of respiratory chain complexes in muscle homogenate showed a combined complex I and IV deficiency. The transition does not represent a known neutral polymorphism and affects a position in the tRNA acceptor stem which is conserved in primates, leading to a destabilization of this functionally important domain. In vitro analysis of an essential maturation step of the tRNA transcript indicates the probable pathogenicity of this mutation. We hypothesize that there is a causal relationship between the novel G8299A transition and progressive external ophthalmoplegia with recurrent respiratory failure due to a depressed respiratory drive.

Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells

To investigate the role of ROS in the helicobacter pylori (Hp) induced mtDNA mutations, AGS cells were treated by extracts of Hp11638 or Hp11638M. The ROS levels, cytochrome C reductions, and intracellular ATP levels were measured. The coding region and the D-Loop region were amplified and sequenced. Results showed the ROS levels, cytochrome C reduction and mtDNA mutations were markedly increased and cell viability decreased after treatment with both Hp extracts, and 616 mutations were detected in D-Loop region and 3 heteroplasmic point mutations in the Cytb gene. No mutations were found in the coding region. The mutation rates of mtDNA D-Loop region were positively correlated with the ROS levels and negatively to the ATP levels.

Segregation of point mutation heteroplasmy in the control region of dog mtDNA studied systematically in deep generation pedigrees

Heteroplasmy, the presence of two or more variants in an organism, may render mitochondrial DNA (mtDNA)-based individual identification challenging in forensic analysis. However, the variation of heteroplasmic proportions and the segregation of heteroplasmic variants through generations and within families have not been systematically described at a large scale in animals such as the domestic dog. Therefore, we performed the largest study to date in domestic dogs and screened a 582-bp-long fragment of the mtDNA control region in 180 individuals in 58 pedigrees for signs of heteroplasmy. We identified three pedigrees (5.17%) with heteroplasmic point mutations. To follow the segregation of the point mutations, we then analyzed 131 samples from these three independent pedigrees and found significant differences in heteroplasmy between generations and among siblings. Frequently (10% of cases), the proportion of one base changed from 0–10% to 80–90% (as judged from Sanger electropherograms) between generations and varied to a similar extent among siblings. We included also a literature review of heteroplasmic and potential mutational hot spot positions in the studied region which showed that all heteroplasmic positions appear to be mutational hot spots. Thus, although heteroplasmy may be used to increase the significance of a match in forensic case work, it may also cause erroneous exclusion of related individuals because of sharp switches from one state to the other within a single generation or among siblings especially in the presented mutational hot spots.Electronic supplementary materialThe online version of this article (doi:10.1007/s00414-010-0524-7) contains supplementary material, which is available to authorized users.

Mitochondrial tRNALeu(UUR) gene mutation and maternally inherited diabetes mellitus in Pakistani population

Background and objectivesMaternally Inherited Diabetes Mellitus and Deafness (MIDD) occurs due to the mutations in mitochondrial DNA (mtDNA). The most common heteroplasmic point mutation reported, is in the tRNALeu(UUR) gene, i.e., A3243G, accompanied with deafness. The objectives of the present study were to determine whether the cause of MIDD in selected Pakistani population, is also the mutation of A3243G in mitochondrial tRNALeu(UUR) gene or not, and is there any genotype–phenotype correlation for the MIDD in this population. Subjects and methodsThe present study was conducted in the Department of Biochemistry and Molecular Biology, Army Medical College, Rawalpindi, Pakistan, during the period November 2005 to November 2007. The patients and control subjects were randomly selected from the two cities; Rawalpindi and Multan (both rural and urban areas) and were divided into three groups. (1) Fifty patients with T2DM and maternal history with feature(s) of MIDD. (2) Fifty non-diabetic first-degree relatives of patients with T2DM. (3) Fifty non-diabetic controls with no maternal history of T2DM. The patients and control subjects were scanned for the detection of potential mutations in mitochondrial DNA tRNALeu(UUR) gene (np 3035–3456, 422 bp fragment). ResultsOn the basis of a polymerase chain reaction, electrophoresis and mtDNA sequencing along with insulin dependence, degree of deafness in patients and subjects, it is proved that there is no A-to-G mutation at np 3243 of mitochondrial leucine tRNA gene in any of the groups studied. ConclusionIt is concluded that in the Pakistani population, selected for the present study, the MIDD is not due to A3243G mutation in mitochondrial tRNALeu(UUR) gene.

Environmental Mutagens Induced Transversions but not Transitions in Regulatory Region of Mitochondrial DNA

One of the long-term objectives of the research in our laboratory was to determine whether mitochondrial DNA (mtDNA) mutations were generated in cell lines exposed to a variety of known mutagens. Many of these mutagens are known to increase oxidative stress in the cell, and one potential outcome of this would be an increased incidence of point mutations in mtDNA. Recently, there has been some controversy regarding the validity of point mutations in the regulatory region of mtDNA as a predictive or causative marker for carcinogenesis. Studies were undertaken to assess whether nuclear mutagens such as arsenic (As), asbestos, and ultraviolet (UV) and γ-radiation, induced both heteroplasmic and homoplasmic point mutations in mtDNA. A direct sequencing approach was used to reduce the occurrence of experimental errors and cross-checked all base changes with databases of known polymorphisms. Our results showed that, while base changes did occur, there was no marked difference between the number of changes in treated and untreated cells. Furthermore, in human lymphocyte samples from subjects exposed to As, most of these base changes were previously reported. Interestingly, there was an increase in the number of transversions (purine ( pyrimidine) in smokers from a human population study, but as with the findings in cell culture samples, there was no difference in the total number of base changes. Data suggest that only a change in the number of rare transversions would be indicative of an increase in point mutations in mtDNA after exposure to mutagens.

Progressive myoclonus epilepsies: an electroclinical, biochemical, morphological and molecular genetic study of 17 cases.

Electroclinical, morphological, biochemical and molecular genetic data from 17 patients affected by progressive myoclonus epilepsies (PME) are reported. Twelve patients were characterized by prominent action myoclonus, sporadic seizures, mild ataxia, lack of dementia and persistence of normal EEG background activity; three patients showed a more rapid worsening of symptomatology, characterized by early mental impairment, massive and action myoclonus, cerebellar signs and tonic clonic seizures; in these patients EEG background activity was slow, even in early stages of the disease. In two patients, previously classified as cryptogenetic PME, a mitochondrial aetiology was recognized by the presence of ragged red fibers in muscle biopsy and by a reduction of the respiratory chains enzymes. Molecular genetical investigation of mtDNA demonstrated the reported heteroplasmic point mutation at nt 8344 of mtDNA in the two MERRF patients, while it was negative in all of the others.

Detection of known base substitution mutations in human mitochondrial DNA of MERRF and MELAS by biochip technology

We developed a DNA biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) and myoclonic epilepsy associated with ragged-red fibers (MERRF). A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was covalently immobilized. Cy5-labeled DNA targets were amplified from sample DNAs containing 31 potential MELAS and/or MERRF mutations by a multiplex PCR method. Detection parameters for the DNA biochip-based assay were accordingly optimized. Seven clinically confirmed patients with MELAS, 5 patients with MERRF, 1 suspected MERRF case and 25 healthy controls were tested using the DNA biochip. For discriminating of homoplasmic and heteroplasmic point mutations in mtDNA, a diagnostic factor based on the ratio between the hybridization signals from the reference and test targets with each probe was used. The results showed that all the cases with MELAS had a causal heteroplasmic A3243G tRNALeu(UUR) mutation. In the MERRF patients, four cases were found to be a homoplasmic A8344G tRNALys mutation and one case was a heteroplasmic T8356C tRNALys mutation. None of the healthy controls carried the potential mutations. The results of the DNA biochip were completely consistent with those by DNA sequencing. Thus, the DNA biochip would potentially become a valuable tool in clinical specific screening of the mtDNA point mutations associated with MELAS and/or MERRF syndrome.

Detection and Quantification of the Age‐Related Point Mutation A189G in the Human Mitochondrial DNA

Mutation analysis in the mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine. Among the difficulties linked to the mtDNA analysis, one can find the detection of heteroplasmy, which can be inherited or somatic. Recently, age-related point mutation A189G was described in mtDNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe, and peptide nucleic acid (PNA)/real-time PCR combined method on different biological samples. Our results give additional information on the increase in mutation frequency with age in muscle tissue and revealed that the PNA/real-time PCR is a largely more sensitive method than DNA sequencing for heteroplasmy detection. These investigations could be of interest in the detection and interpretation of mtDNA heteroplasmy in anthropological and forensic studies.

A new method for analysis of mitochondrial DNA point mutations and assess levels of heteroplasmy

Determination of mitochondrial DNA (mtDNA) heteroplasmy for the diagnosis of patients with mitochondrial disorders is a difficult task due to the coexistence of wild-type and mutant genomes. We have developed a new method for genotyping and quantification of heteroplasmic point mutations in mtDNA based on the SNaPshot technology. We compared the data of this method with the widely used “last hot-cycle” PCR-RFLP method by studying 15 patients carrying mtDNA mutations. We showed that SNaPshot is an accurate, reproducible, and sensitive technique for the determination of heteroplasmic mtDNA mutations in different tissues from patients, and it is a promising system to be used in prenatal and postnatal diagnosis of mtDNA-associated disorders.

A Mother and a Child with Maternally Inherited Diabetes and Deafness (MIDD) Showing Atrophy of the Cerebrum, Cerebellum and Brainstem on Magnetic Resonance Imaging (MRI)

A mother and a child with maternally inherited diabetes and deafness (MIDD) showing atrophy of the cerebrum, cerebellum and brainstem on magnetic resonance imaging (MRI) were reported. The proband had slowly progressive cerebellar ataxia and her son had depression. Mitochondrial DNA purified from their leucocytes had the heteroplasmic point mutation at position 3243 (A-->G). Involvement of the central nervous system should be considered in MIDD as well as in other mitochondrial diseases.

DNA analysis of sex chromosomal aberration: curious mutation found in Turner syndrome

The clear evidence that curious fashion of mitochondrial DNA (mtDNA) inheritance was reported in sex chromosomal aberration. Human mtDNA is located outside the nucleus of the cell producing energy for the cell. In the recent years, mtDNA is being used in forensic DNA identification because each cell has many copies of mitochondria, and is easy to be amplified by PCR. Human mtDNA has polymorphic sites in hypervariable regions (HVR) I, II and III. However, human mtDNA evokes some problems, such as heteroplasmic point mutations and questionable maternal inheritance. To investigate this phenomenon, we had a chance to perform a mtDNA testing on 1-month-old infant, diagnosed as Turner Syndrome which was confirmed to be 45XO karyotype by X-STR testing. As a result, two remarkable evidences were obtained. The first, an intermarriage, is seemed to be related in the genesis of the sex chromosomal aberration. The second, the exact maternal inheritance of mtDNA was not maintained in Turner syndrome, as well as in Klinefelter syndrome reported by us previously. In these cases, although paternal influence of mtDNA inheritance was deniable, mutations of mother's mtDNA were excluded or repaired to the usual type in child's mtDNA.

Monitoring the inheritance of heteroplasmy by computer-assisted detection of mixed basecalls in the entire human mitochondrial DNA control region.

The entire mitochondrial DNA control region (approximately 1122 bp) of 270 blood samples (135 mother-child pairs) was determined by direct sequencing. Overall, 135 'generational events' were screened and within these, 20 mother-offspring pairs demonstrated more than 1 mtDNA haplotype. In 13 families, differences in the haplotypes between mother and offspring were detected in the form of heteroplasmic substitutions. Intergenerational comparisons led to the identification of three heteroplasmic point mutations and eight heteroplasmic length mutations affecting the children only. In two cases, a point heteroplasmy of the maternal sequence was resolved to homoplasmy in the corresponding sequence of the child. These discordant maternal-offspring haplotypes suggest that the shift in the mtDNA haplotype was the result of segregation of a limited maternal subpopulation of mtDNA. As technical implement, quality values assigned to basecalls were tested for their application in automated point heteroplasmy detection.

Mitochondrial DNA and human thyroid diseases.

Cells of the thyroid tissue, either diseased or normal, can accumulate altered mitochondrial genomes in primary lesions and in surrounding parenchyma. Depending on the experimental approaches and the extent of the mutational process, it has been possible to demonstrate the occurrence of homoplasmic or heteroplasmic point mutations, presence of a common deletion and random large-scale mtDNA aberrations in various pathological states. Point somatic mutations documented in 5-60% of thyroid tumors do not concentrate in obvious hotspots but tend to cluster in certain regions of the mitochondrial genome and their distribution may differ between carcinomas and controls. Large-scale deletions in mtDNA are quite prevalent in healthy and diseased thyroid; however, the proportion of aberrant mtDNA molecules accounts for a very small part of total mtDNA and does not seem to correlate with pathological characteristics of thyroid tumors. Common deletion is most abundant in Hurthle cell tumors, yet it also occurs in other thyroid diseases as well as in normal tissue. The principal difference between the common deletion and other deletion-type mtDNA molecules is that the former does not depend on the relative mtDNA content in the tissue whereas in a subset of thyroid tumors, such as radiation-associated papillary carcinomas and follicular adenomas, there is a strong correlation between mtDNA levels and prevalence of large-scale deletions. Relative mtDNA levels by themselves are elevated in most thyroid tumors compared to normal tissue. Distinct differential distribution and prevalence of mutational mtDNA burden in normal tissue and thyroid lesions are suggestive of the implication of altered mtDNA in thyroid diseases, especially in cancer.

Inheritance of mitochondrial disorders

Over the last decade there have been major advances in our understanding of the genetic basis of mitochondrial disease, enabling genetic counseling for patients with autosomal dominant and autosomal recessive disorders. Genetic counseling for patients with mitochondrial DNA (mtDNA) mutations is less well established. Approximately one-third of adults with a mtDNA disorder are sporadic cases, usually due to a single deletion of mtDNA. About two-thirds of adults with mtDNA disease harbor a maternally transmitted point mutation. The recurrence risks are well documented for homoplasmic mtDNA mutations causing Leber hereditary optic neuropathy, but the situation is less clear for families with heteroplasmic mtDNA disorders. Two large studies have shown that for some heteroplasmic point mutations there appears to be a relationship between the percentage level of mutant mtDNA in a mother's blood and her risk of having clinically affected offspring. The situation is less clear for other point mutations, some of which may cause sporadic disease. Recent evidence has cast light on the general principles behind the transmission of heteroplasmic mtDNA point mutations, which may be important for genetic counseling in the future.