Analysis Of Mitochondrial DNA Mutations Research Articles

Analysis of mitochondrial DNA mutations in Pakistani population diagnosed with cardiovascular diseases.

Heart and blood vessel disorders, such as coronary heart disease, brain vessel disease, rheumatic heart disease, and others, are together referred to as cardiovascular disease (CVD). In this study, we sought to determine how mitochondrial Leucine Transfer RNA genes and CVDs are related (MT-L1 and MT-L2). From CVD patients in Peshawar, a total of 27 saliva samples were taken. Leu-tRNA genes expressed by mitochondria were amplified using polymerase chain reaction after DNA was removed. Ten samples were sent for sequencing after PCR and gene cleaning. We obtained all of the sequenced results, which were subsequently aligned and evaluated against the mitochondrial revised Cambridge Reference Sequence (rCRS). However, in our sequenced samples, Leu-tRNA MT-L1 and MT-L2 genes were determined to be unaltered. Thus, it is suggested that a large population be taken into account while screening for mutations in the mitochondrial encoded Leu-tRNA MT-L1 and MT-L2 genes of cardiac patients in areas of Pakistan. Additionally, it is recommended that patients with cardiac problems should also have other mitochondrial encoded genes checked for potential mutations. This could result in the identification of genetic markers that could be used for early CVD screening in Pakistan.

Mitochondrial DNA mutation analysis from exome sequencing-A more holistic approach in diagnostics of suspected mitochondrial disease.

Diagnostics for suspected mitochondrial disease (MD) can be challenging and necessitate invasive procedures like muscle biopsy. This is due to the extremely broad genetic and phenotypic spectrum, disease genes on both nuclear and mitochondrial DNA (mtDNA), and the tissue specificity of mtDNA variants. Exome sequencing (ES) has revolutionized the diagnostics for MD. However, the nuclear and mtDNA are investigated with separate tests, increasing costs and duration of diagnostics. The full potential of ES is often not exploited as the additional analysis of "off-target reads" deriving from the mtDNA can be used to analyze both genomes. We performed mtDNA analysis by ES of 2111 cases in a clinical setting. We further assessed the recall rate and precision as well as the estimation of heteroplasmy by ES data by comparison with targeted mtDNA next generation sequencing in 49 cases. ES identified known pathogenic mtDNA point mutations in 38 individuals, increasing the diagnostic yield by nearly 2%. Analysis of mtDNA variants by ES had a high recall rate (96.2 ± 5.6%) and an excellent precision (99.5 ± 2.2%) when compared to the gold standard of targeted mtDNA next generation sequencing. ES estimated heteroplasmy levels with an average difference of 6.6 ± 3.8%, sufficient for clinical decision making. Taken together, the mtDNA analysis from ES is of sufficient quality for clinical diagnostics. We therefore propose ES, investigating both nuclear and mtDNA, as first line test in individuals with suspected MD. One should be aware, that a negative result does not exclude MD and necessitates further test (in additional tissues).

Mutational Landscape of Spontaneous Base Substitutions and Small Indels in Experimental Caenorhabditis elegans Populations of Differing Size.

Experimental investigations into the rates and fitness effects of spontaneous mutations are fundamental to our understanding of the evolutionary process. To gain insights into the molecular and fitness consequences of spontaneous mutations, we conducted a mutation accumulation (MA) experiment at varying population sizes in the nematode Caenorhabditis elegans, evolving 35 lines in parallel for 409 generations at three population sizes (N = 1, 10, and 100 individuals). Here, we focus on nuclear SNPs and small insertion/deletions (indels) under minimal influence of selection, as well as their accrual rates in larger populations under greater selection efficacy. The spontaneous rates of base substitutions and small indels are 1.84 (95% C.I. ± 0.14) × 10-9 substitutions and 6.84 (95% C.I. ± 0.97) × 10-10 changes/site/generation, respectively. Small indels exhibit a deletion bias with deletions exceeding insertions by threefold. Notably, there was no correlation between the frequency of base substitutions, nonsynonymous substitutions, or small indels with population size. These results contrast with our previous analysis of mitochondrial DNA mutations and nuclear copy-number changes in these MA lines, and suggest that nuclear base substitutions and small indels are under less stringent purifying selection compared to the former mutational classes. A transition bias was observed in exons as was a near universal base substitution bias toward A/T. Strongly context-dependent base substitutions, where 5'-Ts and 3'-As increase the frequency of A/T → T/A transversions, especially at the boundaries of A or T homopolymeric runs, manifest as higher mutation rates in (i) introns and intergenic regions relative to exons, (ii) chromosomal cores vs. arms and tips, and (iii) germline-expressed genes.

Tumorigenesis from a pathological perspective : Tumor spread and epigenetically regulated genes in bladder cancer

The article describes the tumorigenesis of bladder cancer from apathological perspective in three dimensions: morphology, genetics and epigenetics. Field cancerization and tumor cell migration/seeding are the two main hypotheses used for explaining synchronous and metachronous tumors in the urinary tract. By detailed histological mapping of completely embedded cystectomy specimens we found asingle tumor focus in nearly 2/3of the bladders accompanied by surrounding preinvasive carcinoma in situ. We substantiated our findings by studies analyzing TP53 mutations and loss of heterozygosity in various tumor sites. Identical TP53 mutations suggested aclonal relationship of the tumor foci. In situ lineage tracing via cytochrome C oxidase and succinate dehydrogenase enzyme histochemistry and subsequent mitochondrial DNA mutation analysis for definitive evidence of aclonal relationship in bladder tumors remained inconclusive. We found indications for both theories but intraurothelial migration/seeding was more prominent.Afurther mechanism in tumorigenesis is gene inactivation by epigenetic DNA methylation. We analyzed DNA methylation of various genes, which had previously been found by RNA expression analysis to be downregulated in bladder cancer. Most importantly, epigenetically silenced ITIH5 was associated with early relapse in pT1 high grade tumors and functionally showed an enhanced invasive metastatic phenotype in tumor cells, suggesting aputative tumor suppressive role. Thus, epigenetic gene silencing is an additional mechanism of tumorigenesis especially in tumor progression.

Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.

Mitochondrial D-loop and Cytochrome Oxidase C subunit I polymorphisms among the breast cancer patients of Mizoram, Northeast India

Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations as it is prone to oxidative stress. The aim of the present study is to assess the novel mutations in mitochondrial genes from blood samples among the breast cancer patients from a less studied Northeast Indian population. D, B, L haplogroups were observed in the cancer samples and a total of 44 mtDNA D-loop sequence variations at 42 distinct nucleotide positions were found. All the sequence variations were transitional substitutions and 6 were heteroplasmic states, except for a cytosine copy number change (9C/8C) at np 303e309 in three samples examined. A total of 88 Cytochrome Oxidase C subunit I (COXI) sequence differences with respect to the Revised Cambridge Reference Sequence (rCRS) were identified including 20 missense variants with 100 % sample mutation frequency. All 20 missense mutations are highly conserved with a Cumulate Index of 100 %. Among 88 COXI mutations, 24 (13 were Non-Synonymous and 11 were Synonymous) were not previously reported (novel mutation) in the literature or the public mtDNA mutation databases. Analysis of three-dimensional structure of COXI open reading frame (ORF) predicted the effect of one single codon (96R > C, 217T > I, 224-225GG > EE and 227D > T) mutations located in the signal peptide binding position. Analysis of mitochondrial DNA mutations, as a viable alternative, has the advantage of being capable of detecting inherent risk factors for breast cancer development.

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.

Mitochondrial DNA mutation analysis in 97 Chinese patients with mitochondrial cephalomyopathy

To investigate the characteristics of mitochondrial DNA mutations and genotype-phenotype correlations in mitochondrial encephalomyopathies. Biopsy of skeletal muscle and collection of peripheral blood samples were conducted among 97 patients with mitochondrial encephalomyopathies. Southern blotting, PCR-RFLP and direct sequencing of PCR products were performed to search large scale deletions, and common and uncommon pathological point mutations in the muscle and/or blood mtDNA. Seventy-seven patients were identified to be with mitochondrial DNA mutations, including single large deletion (n = 21), multiple large-scale deletions (n = 4), A3243G point mutation (n = 43), A8344G point mutation (n = 6), T8993G mutation (n = 1), T8993C mutation (n = 1), and T3271C mutation (n = 1). Total mtDNA sequencing revealed 4 different novel point mutations in four unrelated patients with isolated mitochondrial myopathy. The type and frequency of mtDNA mutations in this series of Chinese mitochondrial encephalopathies are consistent with those reported abroad, Although there is some association between the genotype and phenotype, heterogeneity in phenotype and genotype is also a prominent feature seen in this series of patients, especially those with A3243G mutation.

Quantitative Mitochondrial DNA Mutation Analysis by Denaturing HPLC

In recent years, denaturing HPLC (DHPLC) has been widely used to screen the whole mitochondrial genome or specific regions of the genome for DNA mutations. The quantification and mathematical modeling of DHPLC results is, however, underexplored. We generated site-directed mutants containing some common mutations in the mitochondrial DNA (mtDNA) tRNA(leu) region with different mutation loads and used PCR to amplify the gene segment of interest in these mutants. We then performed restriction digestion followed by slow reannealing to induce heteroduplex formation and analyzed the samples by use of DHPLC. We observed a quadratic relationship between the heteroduplex peak areas and mutant loads, consistent with the kinetics of heteroduplex formation reported by others. This was modeled mathematically and used to quantify mtDNA mutation load. The method was able to detect a mutation present in a concentration as low as 1% and gave reproducible measurements of the mutations in the range of 2.5%-97.5%. The quantitative DHPLC assay is well suited for simultaneous detection and quantification of DNA mutations.

Diagnostic value of mitochondrial DNA mutation analysis in juvenile unilateral ptosis

To highlight the diagnostic relevance of mitochondrial DNA (mtDNA) mutation analysis in acquired juvenile unilateral upper eyelid ptosis. A 13-year-old boy presented with acquired, slowly progressive unilateral ptosis. We performed ophthalmological and neurological examinations, laboratory testing, skeletal muscle biopsy including histological and histochemical investigations, biochemical analysis of respiratory chain enzymes in skeletal muscle homogenate and molecular genetic testing of skeletal muscle DNA. Though clinical, laboratory, histological and biochemical analyses did not reveal any hints suggesting a mitochondrial cytopathy, molecular genetic testing by Southern blot analysis of total DNA from skeletal muscle tissue showed a 5.8 kb mtDNA deletion thus proving the diagnosis of mitochondrial chronic progressive external ophthalmoplegia (CPEO). In patients with unexplained acquired juvenile unilateral ptosis, an underlying mitochondrial cytopathy should be considered even in cases of inconspicuous ancillary examinations comprising skeletal muscle histology and biochemistry. To establish the diagnosis, molecular genetic testing of DNA derived from skeletal muscle tissue is essential in those patients.

Molecular analysis of mitochondrial DNA mutations from bleomycin-treated rats

In our previous studies, we have shown the mutagenicity of bleomycin (BLM) at the nuclear hprt locus. In the present study we have analyzed mutagenic effects of BLM in mitochondrial DNA (mtDNA) using short extension-PCR (SE-PCR) method for detection of low-copy deletions. Fisher 344 rats were treated with a single dose of BLM and total DNA preparations from splenic lymphocytes were processed in SE-PCR assay. Spontaneous deletions were typically flanked by direct repeats (78.5%), while the in BLM-treated group, direct repeats were found in only 46.6% of breakpoints. The ratio between deletions based on direct repeats and random sequence deletions changed from 3.67 in control group to 0.87 in BLM-treated animals, which corresponds to an approximate 1.7-fold increase in the deletion mutation frequency. Furthermore, 62.5% of deletions not flanked by direct repeats in the treated group contained cleavage sites for BLM. The localization of breakpoints was not entirely random. We have found four clusters containing deletions from both groups indicative of deletion hot spots. The results indicate that BLM exposure may be associated with the induction of mtDNA mutations, and suggest the utility of SE-PCR method for evaluating drug-induced genotoxicity.

Mitochondrial DNA mutation analysis in human skin fibroblasts from fetal, young, and old donors

Multibase deletions in mitochondrial DNA (mtDNA) have been shown to accumulate with age in several tissues, including skin, whereas point mutations have only recently been demonstrated to increase during aging, with several specific mutations occurring at high levels (up to 50%) in skin fibroblasts obtained from old donors [Science 286(1999)774]. We have conducted a survey for a specific deletion and for point mutations in several regions of mtDNA from cultured skin fibroblasts derived from eight fetal (12–20 weeks gestational age), ten young (17–33 years of age) and 11 old (78–92 years of age) human donors. Using PCR analysis, detectable levels of the 4977 basepair (bp) ‘common deletion’ were present in all three age groups, with the highest deletion levels of up to 0.3% of total mtDNA found in several cell lines from old donors, although other old donor cell lines had much lower levels. Single strand conformation polymorphism (SSCP) analysis for point mutations in the non-coding D-loop region and two regions of the cytochrome oxidase 2 gene failed to reveal the presence of any single base mutations. We infer that age-related high level mutational damage in mtDNA from human skin fibroblasts may manifest both sequence and inter-individual specificity.

Influence of dietary antioxidants on the mutagenicity of 7,12-dimethylbenz[a]anthracene and bleomycin in female rats

Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals provide end points that can be used for assessing the antimutagenic or anticarcinogenic properties of putative chemopreventive compounds and for predicting their protective efficacy in humans. In this study, we investigated the ability of the dietary antioxidant Vitamins C, E, β-carotene and the mineral selenium to inhibit the mutant frequency (MF) induced by treatment of rats with 7,12-dimethylbenz[a]anthracene (DMBA), a mammary carcinogen and bleomycin (BLM), an anti-tumor agent that can damage DNA by free radical mechanisms. Both chemicals have been previously shown to be mutagenic in the rat lymphocyte Hprt assay. Adult female Fischer 344 rats were given the antioxidants singly or in a combination 2 weeks prior to mutagen treatment. Antioxidant intake continued for an additional 4 weeks post-mutagen treatment. At sacrifice, spleens were aseptically removed for the isolation of lymphocytes to conduct the mutagenesis assay at the Hprt locus. The DMBA and BLM treatment induced a marked increase in MF, 52.8×10 −6 and 19.2×10 −6, respectively, over the controls. The MFs seen in the individual antioxidants alone (single or mixture) were relatively similar to the controls, with the exception of Vitamins C and E, that had 1.7- and 1.5-fold increase, respectively. The degree of inhibitory response was dependent on the type of mutagen and the particular antioxidant. BLM/antioxidant combination had inhibitions ranging from 44 to 80%, while DMBA/antioxidant system ranged from 60 to 93%, with Vitamins C and E achieving the highest inhibition in both systems. The mixture displayed low inhibitory responses, 44.6% for BLM/mix and 47% DMBA/mix. On the whole, the results indicate that the dietary constituents tested are antimutagenic; however, because of the gradations seen with the responses, the protective efficacy of these antioxidants may depend on the type of mutagen/carcinogen they encounter. Pending molecular analysis of mitochondrial DNA mutations will also indicate whether there is a shift in the mutational spectra produced by the carcinogens in the presence of antioxidants.

Macular Pattern Retinal Dystrophy, Adult-onset Diabetes, and Deafness: A Family Study of A3243G Mitochondrial Heteroplasmy

To correlate mitochondrial DNA (mtDNA) mutation with phenotypic expression in three members of a Finnish family with macroreticular pattern dystrophy, non-insulin-dependent diabetes mellitus, and deafness. A multiplex polymerase chain reaction/allele-specific oligonucleotide method was used to screen 10 mtDNA point mutations known to cause mitochondrial DNA disorders, often characterized by myopathy, retinopathy, or both. Quantitative analysis of mutant mitochondrial DNA was performed in three tissue types in each of three family members by determining the percentage of mutant mtDNA in blood, buccal cells, and hair follicles. A heteroplasmic A3243G mtDNA point mutation was found in each of the three family members studied. Heteroplasmy refers to the coexistence of normal and mutant mitochondria in the same cell. The average percentage of mutant heteroplasmy ranged from 11% to 25%. The severity of disease symptoms did not appear to correlate with the average degree of mutant heteroplasmy in the three tissues analyzed. Molecular confirmation in this family emphasizes the importance of mitochondrial DNA mutation analysis in patients with macular pattern retinal dystrophy and other mitochondrial associated nonocular disease, such as non-insulin-dependent diabetes mellitus and deafness. The detection of a disease-associated mitochondrial DNA mutation warrants genetic counseling, appropriate patient follow-up, and possibly the molecular testing of other at-risk family members.

Specific heart muscle disease

Molecular techniques and ultrastructural analysis are beginning to provide insight into the etiologies of some specific heart muscle disorders. Studies using gene probes have suggested a role, in patients infected with HIV, for humoral- or cellular-mediated cardiac injury triggered by latent cytomegalovirus or other viral infections in the development of AIDS-associated cardiomyopathy. In other such patients, structural and functional abnormalities of cardiac mitochondria, induced by antiretroviral agents, may play an important role. However, the likely multifactorial causes of this cardiomyopathy must be kept in mind. Substance abuse can impair ventricular function, as can antimicrobial agents, such as pentamidine and ganciclovir, used to treat opportunistic infections in patients with acquired immunodeficiency syndrome. Advances in gene technology, especially the polymerase chain reaction, have allowed detailed analysis of mitochondrial DNA mutations. These mutations appear to be associated with the development of a wide range of cardiac disease, including specific heart muscle disease, such as that seen in Kearns-Sayre syndrome. An increasing variety of mitochondrial myopathies with prominent cardiomyopathic features are being reported. Tissue characterization by ultrasonography appears to be a promising technique in the early detection of diabetic cardiomyopathy, before clinical symptoms or frank abnormalities of systolic or diastolic function occur. In myotonic dystrophy, the patchy myocardial involvement, although not affecting contractile function, may serve as a substrate for malignant ventricular arrhythmias. The use of cardioverter-defibrillator devices as primary therapy for patients with sarcoidosis and ventricular tachycardia has been proposed.