mtDNA Coding Region Research Articles

Novel mutations of SLC26A4 in Chinese patients with nonsyndromic hearing loss

Conclusions: This study demonstrated high prevalence of GJB2, SLC26A4, and mtDNA A1555G mutations in Chinese patients with nonsyndromic hearing loss and discovered eight novel mutations in SLC26A4. Most of these novel mutations were predicted pathogenic variants. Objectives: Nonsyndromic hearing loss is the most common neurosensory deafness where the majority of patients have highly diversified genetic defects. This study aimed to define the genetic profile of deafness in a Chinese population with potential to discover novel mutations. Methods: A total of 227 segregating deaf students and 200 individuals with normal hearing were enrolled. With the Sanger sequencing chemistry, direct sequencing was performed on entire coding regions of GJB2, GJB3, SLC26A4, and mtDNA m.C1494T and m.A1555G. Results: Direct sequencing analysis revealed that 53 (23.35%) of 227 patients carried at least 1 mutant allele in GJB2, 40 (17.62%) patients in SLC26A4, 5 (2.20%) patients in mtDNA A1555G, and 1 (0.44%) patient in mtDNA C1494T mutations. Four patients carried three unclassified mutations in GJB3 genes. Overall 38 mutant variants were detected in this cohort of patients, including 8 novel mutations in SLC26A4. The eight novel variants were six missense substitutions (p.V163L, p.G222S, p.A456D, p.N457I, p.C466Y, p.F667L), one nonsense mutation (p.W472X), and one frameshift (p.Asn612Ilefs×23).

Somatic mitochondrial DNA mutations in Chinese patients with osteosarcoma

Somatic mutations in mitochondrial DNA (mtDNA) have been long proposed to drive the pathogenesis and progression of human malignancies. Previous investigations have revealed a high frequency of somatic mutations in the D-loop control region of mtDNA in osteosarcoma. However, little is known with regard to whether or not somatic mutations also occur in the coding regions of mtDNA in osteosarcoma. To test this possibility, in the present study we screened somatic mutations over the full-length mitochondrial genome of 31 osteosarcoma tumour tissue samples, and corresponding peripheral blood samples from the same cohort of patients. We detected a sum of 11 somatic mutations in the mtDNA coding regions in our series. Nine of them were missense or frameshift mutations that have the potential to hamper mitochondrial respiratory function. In combination with our earlier observations on the D-loop fragment, 71.0% (22/31) of patients with osteosarcoma carried at least one somatic mtDNA mutation, and a total of 40 somatic mutations were identified. Amongst them, 29 (72.5%) were located in the D-loop region, two (5%) were in the sequences of the tRNA genes, two (5%) were in the mitochondrial ATP synthase subunit 6 gene and seven (17.5%) occurred in genes encoding components of the mitochondrial respiratory complexes. In addition, somatic mtDNA mutation was not closely associated with the clinicopathological characteristics of osteosarcoma. Together, these findings suggest that somatic mutations are highly prevalent events in both coding and non-coding regions of mtDNA in osteosarcoma. Some missense and frameshift mutations are putatively harmful to proper mitochondrial activity and might play vital roles in osteosarcoma carcinogenesis.

Somatic mutations of the mitochondrial genome in Chinese patients with Ewing sarcoma

Somatic mutations in mitochondrial DNA (mtDNA) have been long proposed to drive initiation and progression of human malignancies. Our previous study revealed a high prevalence of somatic mutations in the D-loop region of mtDNA in Ewing sarcoma (EWS). However, it is unclear whether somatic mutations also occur in the coding regions of mtDNA in EWS. To test this possibility, in the present study, we sequenced the whole mitochondrial genome from 20 cases of EWS specimens and their corresponding peripheral blood samples. We identified a total of 6 somatic mutations in the mtDNA coding regions in our EWS series, and 5 of them were missense or frame-shift mutations that have the potential to directly influence proper mitochondrial function. In combination with our earlier observations on the D-loop fragment, 70% (14/20) of EWS tissues appeared to harbor somatic mtDNA mutations. Among the identified 25 somatic mutations, 19 (76%) were located in the D-loop control region, 1 (4%) was in the sequence of the tRNA(Val) gene, 1 (4%) was in the mitochondrial ATP synthase subunit 6 gene, and 4 (16%) occurred in genes encoding components of the mitochondrial respiratory complexes. In addition, patients carrying somatic mtDNA mutations did not show significant association with their clinicopathologic characteristics. Together, these findings suggest that somatic mtDNA mutations occur in both protein coding and noncoding regions of mtDNA, which may play critical roles in the pathogenesis of EWS and should be further explored for its possible use as a novel marker for monitoring EWS occurrence and advancement.

Analysis of eight mtDNA coding region polymorphisms for characterization of the female lineages ancestry in Alagoas, Brazil

In this work we evaluated eight mitochondrial DNA coding region polymorphisms (8281–8289d, 1736, 13263, 4883, 3594, 10873, 10400, and 12705) in order to identify lineages of African, Amerindian and European origin in Alagoas, Northeastern Brazil. Seven of these polymorphisms were detected by multiplex minisequencing with SnaPshot. For validation purposes a sample previously characterized by sequencing of the HVI and HVII regions was analyzed. Good agreement was observed between the phylogenetic results obtained in this work and the HVI and HVII regions sequencing. The results obtained suggest that the set of markers evaluated in this study can be used in ancestry studies of Brazilian populations.

New strategies for the study of polymorphisms in mitochondrial DNA coding region based on denaturing high-performance liquid chromatography

The purpose of this study is to establish a novel method for the detection of mitochondrial DNA (mtDNA) polymorphism based on denaturing high-performance liquid chromatography (DHPLC) and to explore the new mitochondrial DNA polymorphisms in coding region in order to improve the discrimination power of mtDNA in forensic DNA typing. MtDNA coding region loci were amplified by the polymerase chain reaction (PCR). Using a technique of DNA pools and performing the analysis of pairwise combining samples, two strategies that based on the most homologous and retention times of cartridge were evaluated and optimized. The new strategies were the foundation for exploring polymorphism in entire mitochondrial genome and were expected to be important methods for forensic mtDNA identification.

Analysis of European case-control studies suggests that common inherited variation in mitochondrial DNA is not involved in susceptibility to amyotrophic lateral sclerosis

While some cases of familial ALS can be entirely attributed to known inherited variation, the majority (∼ 90%) are sporadic, where the cause(s) are not entirely understood. Both genetic and environmental factors may contribute to susceptibility. Mitochondrial damage, a common feature of neurodegenerative disease, is observed in most patients and inherited polymorphism in the mitochondrial genome has been suggested as a contributing factor. We used an economic and efficient method to test whether such involvement is probable. We genotyped 22 mtDNA coding region SNPs and sequenced the mtDNA hypervariable region 1 to determine the position of each mitochondrial genome within the genealogy of mitochondrial haplotypes in samples of ALS patients (n = 700) and controls (n = 462) from two European populations. We compared haplotype and haplogroup distribution in cases and controls drawn from the same populations. No statistical difference was observed between cases and controls at either the haplogroup or haplotype level (p = ≥ 0.2). In conclusion, it is unlikely that common, shared genetic variants in the mitochondrial genome contribute substantially to ALS. Combining the data with other studies will allow meta-analysis to look for variants with modest effect sizes. The sequencing of complete mitochondrial genomes will be required to assess the role of rare mutations.

Genetic evidence for recent range fragmentation and severely restricted dispersal in the critically endangered Sierra Madre Sparrow, Xenospiza baileyi

Assessing patterns of genetic structure and diversity of threatened species has become an essential tool for determining conservation status and designing management strategies. We examine the genetic structure of the Sierra Madre sparrow (Xenospiza baileyi), a species restricted to fragmented patches of subalpine bunchgrass in three small isolated areas of northwestern and central Mexico. Coding and non-coding regions of mtDNA (1,878 bp) from individuals of the only three known populations revealed the existence of a single major lineage, with closely related haplotypes being shared between populations across the range. The sharing of haplotypes between the distant northwest and central populations (~800 km) suggests a recent fragmentation of a formerly contiguous population. Despite a lack of large-scale phylogeographic structure, haplotype frequencies at local scales revealed significant genetic differentiation and high FST values between all three remaining populations, even between localities separated by less than 12 km. These results suggest restricted gene flow and limited dispersal, likely due to the species’ inability to cross areas of unsuitable habitat. On the basis of genetic interchange and ecological equivalence criteria, we recommend that the species be managed as a single unit, permitting the strengthening of the small population in the northwest with individuals from central Mexico, and/or the translocation of individuals to new areas of suitable habitat.

Identification of Azores islands haplogroups by mitochondrial DNA analysis

Mitochondrial DNA (mtDNA) has enormous potential in population genetics allowing the correlation and identification of the origin of individuals in a population. In forensic genetics mtDNA enables the identification of the matrilineal lineage from degraded samples. Genetic testing can be performed using mtDNA noncoding region and/or mtDNA coding region. However, to apply this inclusive approach as evidence in legal practice is necessary to determine the genetic structure of populations. Traditional mtDNA sequencing and Single Nucleotide Polymorphisms (SNP) analysis techniques were combined to establish the mtDNA variability of the Azorean population and classify the haplotypes into haplogroups. Forty-eight haplotypes were identified. All mtDNA haplotypes were included into specific haplogroups: forty-three haplotypes belonging to macrohaplogroup R; three haplotypes belonging to macrohaplogroup N; and two haplotypes belonging to macrohaplogroup M.

Mitochondrial DNA haplogroup R in the Han population and recovery from septic encephalopathy

To determine whether the main mitochondrial DNA (mtDNA) haplogroups of the Han people are associated with neurological recovery from septic encephalopathy. We studied 137 individuals with septic encephalopathy who were sequentially admitted to the intensive care unit or the emergency intensive care unit at the First Affiliated Hospital, College of Medicine, Zhejiang University, and the People's Hospital of Zhejiang Province. Demographic and clinical data were recorded along with clinical outcome over 28days. The Glasgow coma scale (GCS) score was calculated daily until it reached 15 or until the patient died during the 28-day period. Follow-up was completed for all study participants. We then determined the mtDNA haplogroups of the patients by analyzing sequences of hypervariable mtDNA segments and testing diagnostic polymorphisms in the mtDNA coding region with DNA probes. MtDNA haplogroup R, one of the main mtDNA haplogroups of the Han people, was a strong independent predictor of outcome following septic encephalopathy, conferring a 4.053-fold (95% CI 1.803-9.110, p=0.001) increased chance of neurological recovery within 28days compared with those with a non-R mtDNA haplogroup. In the Han population, mtDNA haplogroup R is a strong independent predictor of the outcome of septic encephalopathy, conferring an increased chance of neurological recovery compared with individuals with a non-R haplogroup. Our results provide potential insights into the mechanisms involved in septic encephalopathy, and reveal that the mtDNA haplogroup R is an independent predictor of the outcome of septic encephalopathy.

Somatic mutations of the mitochondrial genome in human breast cancers

Somatic mutations in mitochondrial DNA (mtDNA) have been identified in various tumors, including breast cancer. However, their clinicopathological impact on breast cancer still remains unclear. In this study, we re-sequenced the entire mtDNA from breast cancer samples together with paired non-tumorous breast tissues from 58 Taiwanese patients. We identified 19 somatic mutations in the mtDNA coding region of 16 breast cancers. Out of these mutations, 12 of the 19 mutations (63%) are missense or frame-shift mutations that have the potential to cause mitochondrial dysfunction. In combination with our previously study on the D-loop region of mtDNA, we found that 47% (27/58) of the breast cancers harbored somatic mtDNA mutations. Among a total of 40 somatic mutations, 53% (21/40) were located in the D-loop region of the mtDNA, 5% (2/40) were in the ribosomal RNA genes, 5% (2/40) were in the tRNA genes, and 38% (15/40) occurred in mRNA genes. The occurrence of these somatic mtDNA mutations is associated with an older onset age (≥ 50-year old, P = 0.039), a higher TNM stage (P = 0.027), and a higher histological grade (P = 0.012). Multiple logistic regression analysis revealed that an older onset age (P = 0.029) and a higher histological grade (P = 0.006) are significantly correlated with patients having somatic mutations in the mtDNA in their breast cancer sample. In conclusion, our results suggest that somatic mtDNA mutations may play a critical role in the progression of breast cancer.

A New Strategy for the Discrimination of Mitochondrial DNA Haplogroups in Han Population

Mitochondrial DNA (mtDNA) haplogroup discrimination is interesting not only for phylogenetic and clinical but also for forensic studies. We discriminated the mtDNA haplogroups of 570 healthy unrelated Han people from Zhejiang Province, Southeast China, by comprehensive analysis mutations of the hypervariable segments-I sequence and diagnostic polymorphisms in mtDNA coding region using real-time polymerase chain reaction (RT-PCR), which was compared with the widely used PCR and restriction fragment length polymorphism (PCR-RFLP) method. The results showed that in superhaplogroup M, haplogroup D was the most common haplotype within this assay to 24.6%, and in the other superhaplotype N, haplogroup B and F were the most common groups. Samples re-identified by PCR-RFLP showed the consistent results that were got with RT-PCR. In conclusion, the RT-PCR strategy appears to be an accurate, reproducible, and sensitive technique for the discrimination of mtDNA haplogroups, especially for mass screenings quickly and economically.

Southeast Asian diversity: first insights into the complex mtDNA structure of Laos

BackgroundVast migrations and subsequent assimilation processes have shaped the genetic composition of Southeast Asia, an area of close contact between several major ethnic groups. To better characterize the genetic variation of this region, we analyzed the entire mtDNA control region of 214 unrelated donors from Laos according to highest forensic quality standards. To detail the phylogeny, we inspected selected SNPs from the mtDNA coding region. For a posteriori data quality control, quasi-median network constructions and autosomal STR typing were performed. In order to describe the mtDNA setup of Laos more thoroughly, the data were subjected to population genetic comparisons with 16 East Asian groups.ResultsThe Laos sample exhibited ample mtDNA diversity, reflecting the huge number of ethnic groups listed. We found several new, so far undescribed mtDNA lineages in this dataset and surrounding populations. The Laos population was characteristic in terms of haplotype composition and genetic structure, however, genetic comparisons with other Southeast Asian populations revealed limited, but significant genetic differentiation. Notable differences in the maternal relationship to the major indigenous Southeast Asian ethnolinguistic groups were detected.ConclusionsIn this study, we portray the great mtDNA variety of Laos for the first time. Our findings will contribute to clarify the migration history of the region. They encourage setting up regional and subpopulation databases, especially for forensic applications. The Laotian sequences will be incorporated into the collaborative EMPOP mtDNA database http://www.empop.org upon publication and will be available as the first mtDNA reference data for this country.

MtGenome reference population databases and the future of forensic mtDNA analysis

Mitochondrial DNA (mtDNA) testing in the forensic context requires appropriate, high quality population databases for estimating the rarity of questioned haplotypes. Currently, however, available forensic mtDNA reference databases only include information from the mtDNA control region. While this information is obviously strengthening the foundation upon which current mtDNA identification efforts are based, these data do not adequately prepare the field for recent and rapid advancements in mtDNA typing technologies. Novel tools that quickly and easily permit access to mtDNA coding region data for increased discrimination are now available in the form of single nucleotide polymorphism assays, sequence specific oligonucleotide probes, mass spectrometry instrumentation and next generation sequencing technologies. However, the randomly sampled entire mtGenome reference population data required for statistical interpretation of coding region data are lacking. As a result, in the near future, it seems that routine use of mtDNA coding region data in forensic case work will depend more upon the availability of high-quality entire mtGenome population reference data than the ease with which coding region data can be generated from evidence specimens. Until mtGenome reference databases are available, the utility of novel mtDNA typing technologies and the benefits of recovering mtDNA coding region information from forensic specimens will be limited. Thus, future mtDNA databasing efforts are needed for the development of entire mtDNA genome reference population data suitable for forensic comparisons.

Characterization of Retinal and Blood Mitochondrial DNA from Age-Related Macular Degeneration Patients

To determine mitochondrial (mt)DNA variants in AMD and age-matched normal retinas. Total DNA was isolated from retinas (AMD, n = 13; age-matched normal, n = 13), choroid (AMD, n = 3), and blood (AMD, n = 138; normal, n = 133). Long-extension-polymerase chain reaction amplified the full-length ( approximately 16.2 kb) mtDNA genome. Retinal mtDNA was sequenced for nucleotide variants and length heteroplasmy. Pyrosequencing was performed on heteroplasmic mtDNA. PCR amplification and enzyme digestions were used to analyze for nucleotide changes. Retinal mtDNA had a greater number of rearrangements and deletions than did blood mtDNA in normal samples (9.3 +/- 1.78 vs. 3 +/- 1.18, P = 0.019), and AMD samples (14.33 +/- 1.96 vs. 5.2 +/- 0.80, P = 0.0031. Five (55%) of 9 AMD patients had unreported SNPs, and 2 (16.6%) of 12 of the normal group did. The mtDNA coding region had 20 SNPs that produced amino acid changes. The noncoding MT-Dloop region had nucleotide heteroplasmy and length heteroplasmy. There were more SNPs per person in the AMD population than in the older (P = 0.003) and younger (P = 0.05) normal subjects. The C12557T (T-I) in the MT-ND5 gene was present in two AMD subjects (2/138) but was absent in the normal (0/133). Common mutations for Leber's hereditary optic neuropathy (LHON: G11778A; T14484C; and G3460A) were not present in AMD samples. AMD subjects have high levels of large mtDNA deletions/rearrangements in the retinas, unreported and amino acid-changing SNPs in the coding genome, and a greater number of SNPs per person in the noncoding MT-Dloop region. These mtDNA variants could diminish energy production efficiency, alter the mtDNA copy numbers and/or impact transcription in AMD retinas.

Analysis of Canis mitochondrial DNA demonstrates high concordance between the control region and ATPase genes

BackgroundPhylogenetic studies of wild Canis species have relied heavily on the mitochondrial DNA control region (mtDNA CR) to infer species relationships and evolutionary lineages. Previous analyses of the CR provided evidence for a North American evolved eastern wolf (C. lycaon), that is more closely related to red wolves (C. rufus) and coyotes (C. latrans) than grey wolves (C. lupus). Eastern wolf origins, however, continue to be questioned. Therefore, we analyzed mtDNA from 89 wolves and coyotes across North America and Eurasia at 347 base pairs (bp) of the CR and 1067 bp that included the ATPase6 and ATPase8 genes. Phylogenies and divergence estimates were used to clarify the evolutionary history of eastern wolves, and regional comparisons of nonsynonomous to synonomous substitutions (dN/dS) at the ATPase6 and ATPase8 genes were used to elucidate the potential role of selection in shaping mtDNA geographic distribution.ResultsWe found high concordance across analyses between the mtDNA regions studied. Both had a high percentage of variable sites (CR = 14.6%; ATP = 9.7%) and both phylogenies clustered eastern wolf haplotypes monophyletically within a North American evolved lineage apart from coyotes. Divergence estimates suggest the putative red wolf sequence is more closely related to coyotes (DxyCR = 0.01982 ± 0.00494 SD; DxyATP = 0.00332 ± 0.00097 SD) than the eastern wolf sequences (DxyCR = 0.03047 ± 0.00664 SD; DxyATP = 0.00931 ± 0.00205 SD). Neutrality tests on both genes were indicative of the population expansion of coyotes across eastern North America, and dN/dS ratios suggest a possible role for purifying selection in the evolution of North American lineages. dN/dS ratios were higher in European evolved lineages from northern climates compared to North American evolved lineages from temperate regions, but these differences were not statistically significant.ConclusionsThese results demonstrate high concordance between coding and non-coding regions of mtDNA, and provide further evidence that the eastern wolf possessed distinct mtDNA lineages prior to recent coyote introgression. Purifying selection may have influenced North American evolved Canis lineages, but detection of adaptive selection in response to climate is limited by the power of current statistical tests. Increased sampling and development of alternative analytical tools will be necessary to disentangle demographic history from processes of natural selection.

Five coding region polymorphism of mitochondrial DNA in Chinese Han population of Yanbian area

To investigate the mitochondrial DNA (mtDNA) five coding region sequence polymorphisms encompassing positions nt3954-4506, nt5218-5974, nt7942-8711, nt10296-10653, and nt14496-14867 in Chinese Han population of Yanbian area, Jilin province. Polymerase chain reaction (PCR) and direct sequencing method were used to detect the haplotype distribution of mtDNA coding region in 200 unrelated Chinese Han individuals. One hundred and ten haplotypes were observed in the 200 individuals. The gene diversity was 0.9879 and the random match probability was 0.0171. Compared with the Anderson's sequence,81 nucleotide variants were obtained,of which 66 were previously registered in MITOMAP,and 15 were novel. The obtained data suggest that these sequence polymorphisms are valuable genetic markers for personal identification when added to mtDAN control region investigation, and thus could be used as basic data for the forensic application in Chinese Han population.

Forensic application of mitochondrial DNA SNPs

Mitochondrial DNA (mtDNA) has enormous potential in forensic genetics, allowing identification of genetic material from degraded samples. Genetic testing can also be performed using mtDNA coding region SNPs. SNPs have a number of characteristics that make them unique for forensic analysis, allowing the successful analysis of degraded samples. The aim of the present study was to evaluate the effectiveness of a mtSNP typing assay on skeletal remains that were buried directly in soil for more than thirty years in adverse climate conditions.

Forensic and phylogeographic characterization of mtDNA lineages from northern Thailand (Chiang Mai)

The immigration of diverse ethnic groups over the past centuries from surrounding countries into Thailand left footprints in the genetic composition of Thai mitochondrial DNA (mtDNA) lineages. The entire mtDNA control region (1,122 bp) was typed in 190 unrelated male volunteers from the northern Thailand province of Chiang Mai following highest quality standards. For a more precise haplogroup classification, selected single nucleotide polymorphisms from the mtDNA coding region were genotyped. We found several new, so far undescribed mtDNA lineages. Quasi-median networks were constructed for visualisation of character conflicts. The data were put into population-genetic relationships with other Southeast Asian populations. Although the frequencies of the Thai haplogroups were characteristic for Southeast Asia in terms of haplotype composition and genetic structure, the Thai population was significantly different from other Southeast Asian populations. This necessitates establishing regional databases, especially for forensic applications. The population data have been submitted to the EMPOP database (www.empop.org) and will be available on publication.

Correcting for Purifying Selection: An Improved Human Mitochondrial Molecular Clock

There is currently no calibration available for the whole human mtDNA genome, incorporating both coding and control regions. Furthermore, as several authors have pointed out recently, linear molecular clocks that incorporate selectable characters are in any case problematic. We here confirm a modest effect of purifying selection on the mtDNA coding region and propose an improved molecular clock for dating human mtDNA, based on a worldwide phylogeny of > 2000 complete mtDNA genomes and calibrating against recent evidence for the divergence time of humans and chimpanzees. We focus on a time-dependent mutation rate based on the entire mtDNA genome and supported by a neutral clock based on synonymous mutations alone. We show that the corrected rate is further corroborated by archaeological dating for the settlement of the Canary Islands and Remote Oceania and also, given certain phylogeographic assumptions, by the timing of the first modern human settlement of Europe and resettlement after the Last Glacial Maximum. The corrected rate yields an age of modern human expansion in the Americas at approximately 15 kya that-unlike the uncorrected clock-matches the archaeological evidence, but continues to indicate an out-of-Africa dispersal at around 55-70 kya, 5-20 ky before any clear archaeological record, suggesting the need for archaeological research efforts focusing on this time window. We also present improved rates for the mtDNA control region, and the first comprehensive estimates of positional mutation rates for human mtDNA, which are essential for defining mutation models in phylogenetic analyses.

Is Interlineage Recombination Responsible for Low Divergence of Mitochondrial nad3 Genes in Mytilus galloprovincialis?

The existence of mtDNA recombination in animals has been confirmed by several case studies. Still, for Mytilus mussels possessing two divergent mitochondrial genomes (M and F), which can recombine, no recombination between coding sequences of highly diverged M and F genomes has been shown. Based on the full sequences of both genomes, it has been suggested that particularly low divergence observed within the mitochondrial nad3 gene of the Mytilus galloprovincialis mussel may be caused by its exceptionally low evolutionary rate. Here, we contribute a new pair of mitochondrial genomes typical for M. galloprovincialis and show that this low divergence is not a sign of evolutionary conservation but is rather caused by the acquisition of an F-related sequence by the published M genome of M. galloprovincialis. The most likely scenario for this apparent mtDNA-coding region recombination case is an assembly artifact.