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At least 19 recordsLinked to original sources
PubMed2006

[Heteroplasmy in human mtDNA control region].

OBJECTIVE: To observe the length heteroplasmy and point heteroplasmy in human mtDNA control region. METHODS: The peripheral blood, buccal cell, and single hair shaft from 50 individuals and 16 family members, related in their maternallineage were analyzed by direct sequencing, and clones from 20 individuals whose mtDNA sequences have a T-C transition at 16189 nt were sequenced. RESULTS: No point heteroplasmy were observed in peripheral blood, buccal cell, single hair shaft from the same individual, neither in maternally related individuals. Length heteroplasmy was observed in those individuals with a homopolymeric tract and the different clones from the same individual has different proportions of length variants, but the hair shafts from the same individual were very similar to the measurements made from blood DNA. No length heteroplasmy was observed between different tissues from the same individual. CONCLUSION: mtDNA sequences have a characteristic of high consistency and genetic stability, mtDNA sequencing is a suitable tool for forensic applications such as individual identification.

Base Sequence↗
PubMed2005

Understanding differences between phylogenetic and pedigree-derived mtDNA mutation rate: a model using families from the Azores Islands (Portugal).

We analyzed the control region of the mitochondrial DNA (mtDNA) from maternally related individuals originating from the Azores Islands (Portugal) in order to estimate the mutation rate of mtDNA and to gain insights into the process by which a new mutation arises and segregates into heteroplasmy. Length and/or point heteroplasmies were found at least in one individual of 72% of the studied families. Eleven new point substitutions were found, all of them in heteroplasmy, from which five appear to be somatic mutations and six can be considered germinal, evidencing the high frequency of somatic mutations in mtDNA in healthy young individuals. Different values of the mutation rate according to different assumptions were estimated. When considering all the germinal mutations, the value of the mutation rate obtained is one of the highest reported so far in family studies. However, when corrected for gender (assuming that the mutations present in men have the same evolutionary weight of somatic mutations because they will inevitably be lost) and for the probability of intraindividual fixation, the value for the mutation rate obtained for HVRI and HVRII (0.2415 mutations/site/Myr) was in the upper end of the values provided by phylogenetic estimations. These results indicate that the discrepancy, that has been reported previously, between the human mtDNA mutation rates observed along evolutionary timescales and the estimations obtained using family pedigrees can be minimized when corrections for gender proportions in newborn individuals and for the probability of intraindividual fixation are introduced. The analyses performed support the hypothesis that (1) in a constant, tight bottleneck genetic drift alone can explain different patterns of heteroplasmy segregation and (2) in neutral conditions, the destiny of a new mutation is strictly related to the initial proportion of the new variant. Another important point arising from the data obtained is that, even in the absence of a paternal contribution of mtDNA, recombination may occur between mtDNA molecules present in an individual, which is only observable if it occurs between mtDNA types that differ at two or more positions.

Azores↗
PubMed2004

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts.

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.

DNA, Mitochondrial↗
PubMed2004

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.

DNA, Mitochondrial↗
PubMedDate not supplied

Severe reversible cardiomyopathy in four unrelated infants associated with mitochondrial DNA D-loop heteroplasmy.

Inherited disorders of energy metabolism are increasingly being recognized as important causes of cardiomyopathy in children. We previously reported that heteroplasmic point substitutions in the mitochondrial DNA D-loop were found in 15 of 75 children at risk for mitochondrial disease (vs 0/95 controls). Four of these cases presented with severe cardiomyopathy in congestive failure in addition to other anomalies and are presented here. In each case, myocardial dysfunction greatly improved following supportive therapy aimed at reversing both congestive failure and catabolism. D-loop point heteroplasmy may be a marker for severe, reversible, infantile multisystem disease that can present with cardiomyopathy.

Cardiomyopathies↗
PubMed2003

Mitochondrial DNA heteroplasmy or artefacts--a matter of the amplification strategy?

We compared two different PCR strategies for the amplification of mtDNA hypervariable region 1 (HV1) with regard to the detection and interpretation of point mutation heteroplasmy in human hair roots. We monitored the level of detected heteroplasmy using direct sequence analysis. PCR amplifications were performed in duplicate on each hair root, using 62 cycles of nested PCR versus 35 cycles of direct PCR. As a previous publication reported different sensitivities of heteroplasmy detection based on the number of PCR cycles used, we were interested in whether and how different PCR amplification strategies would impact sequence quality and the detection of point heteroplasmy. We identified 12 out of 93 hair roots as heteroplasmic (7 out of 31 persons) with direct PCR, whereas 2 of these heteroplasmic events could not be identified with the nested PCR approach. Generally, the quality of the sequence electropherograms in terms of background noise was significantly lower for the nested PCR amplification strategy, leading to ambiguous results in some of the nucleotide positions. Thus, the ability to clearly distinguish a genuine mixture of two nucleotides from background noise at a heteroplasmic position was substantially greater with direct PCR amplification, which generally resulted in higher quality sequence electopherograms.

Artifacts↗
PubMed2002

Critique of interpretation of high levels of heteroplasmy in the human mitochondrial DNA hypervariable region I from hair.

The phenomenon known as heteroplasmy can be operationally observed in some human mitochondrial DNA (mtDNA) samples. Typically, heteroplasmy manifests itself in an individual presenting two mtDNA species that differ at a single base. Heteroplasmy at two, and even possibly three sites, also may occur, but at very low rates. A recent report (Grzybowski, 2000, see ref. [13]) suggests that much higher levels of mtDNA (point substitution) heteroplasmy can occur in hair. This observation is contrary to the experience of the forensic mtDNA community. There are several explanations for the unusual findings of high levels of heteroplasmy. First, the template quantities of DNA are approximately three orders of magnitude higher than required for mtDNA sequencing, and an excessive number of amplification cycles were used. Thus, the protocol used did not follow routine practices by the forensic community. Second, there are misidentifications and tabular errors that call into question the reliability of the findings. Third, by comparing the natural human mtDNA variation with a reference sample population with that observed in the heteroplasmy in hair study, the data are inconsistent with population genetic expectations. The observation of high levels of heteroplasmy may be due to contamination of the samples and/or possibly the amplification of nuclear pseudogenes. The results observed in the heteroplasmy in hair study do not apply to other methods of mtDNA analysis and cannot be used to question the reliability of the current forensic mtDNA practices.

DNA, Mitochondrial↗
PubMed2026

Probability of Mitochondrial DNA heteroplasmy in different tissues from European populations.

Mitochondrial DNA (mtDNA) heteroplasmy complicates genetic analyses due to its variability across individuals and tissues. We analyzed over 400 Spanish blood samples and integrated published Massively Parallel Sequencing (MPS) data from ten additional European tissues. Heteroplasmy was tissue-specific, with skeletal muscle, kidney, and liver showing the highest levels, while the intestines, skin, and cerebellum had the lowest. Blood uniquely displayed more heteroplasmies in coding than non-coding regions. Several conserved positions not previously described as hotspots showed high frequencies. These results establish the first comprehensive tissue-specific heteroplasmic profile of the complete mitochondrial genome in a European population, improving the interpretation of mtDNA variation in forensic and biomedical contexts.

Humans↗
PubMed1995

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA.

Studies in vitro have shown that a respiratory-deficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A-->G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (G-->A) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (rho0) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.

Adolescent↗
PubMed1998

Sequence polymorphism of mitochondrial DNA control region in Japanese.

Sequence polymorphisms of the mitochondrial DNA (mtDNA) control region, hypervariable regions I and II, from 100 unrelated Japanese were determined by PCR amplification and direct sequencing. Sequences of 404 nucleotides for hypervariable region I and 379 nucleotides for region II were obtained. Variable sites (85 and 45) were revealed in region I and region II, respectively, as compared to the reference sequence, and a total of 96 different genetic patterns from both regions I and II were determined. A point mutation heteroplasmy was observed at the ratio of approximately 50:50 from one individual at the sequence position 151 showing a nucleotide transition from C to T. The probability of identity was estimated as 2.3% for region I, 3.9% for region II, and 1.1% combined for both regions. These results suggest that sequence polymorphism of mtDNA control region would be very useful in forensic practice as a marker for individual identification.

Base Sequence↗
PubMed2026

Ambrosia beetle invasions are structured by inbreeding, intraspecific hybridisation, and bridgeheads.

When invasive populations establish in regions far from their origin, they may accumulate deleterious mutations that limit population viability and later expansion. Invasions stemming from such bridgehead populations may experience further sequential bottlenecks. However, deleterious mutations can be masked or eliminated when populations outbreed with other lineages. Here, we analyse global invasions of a species complex of persistently inbreeding ambrosia beetles, using genomic data (N=247) from invasive populations in Africa, North America and Australia, and from native populations in Asia. We mostly focus on one species of this complex (Euwallacea fornicatus) which poses a severe threat to tree species worldwide and is rapidly expanding its global range. We uncover a single lineage of this species across California, South Africa, and Western Australia, involving an invasive bridgehead and containing almost no nuclear genetic variation. In South Africa we identify a second lineage that has repeatedly hybridised with the first lineage. Genetic patterns in the native range indicate that such opportunistic outbreeding may be common. Despite lacking nuclear variation, the first lineage contained two CO1 haplotypes that were also observed in every hybrid lineage, pointing to heteroplasmy and possible hybrid origins of this lineage. Native populations had fewer missense mutations than invasive populations, indicating that opportunistic outbreeding may help purge fixed deleterious mutations when local lineage diversity is high. These findings highlight the importance of outbreeding even when inbreeding is common, and they demonstrate the biosecurity threat posed by subsequent gene flow into invasive populations.

Journal Article↗
PubMed2006

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.

Cell Fusion↗
PubMed1993

[Molecular genetic analysis for myoclonus epilepsy associated with ragged-red fibers (MERRF)].

Two mutations in tRNA(Lys) gene of mitochondrial DNA were detected as the causes of this disease. We reviewed our previous studies and the recent literatures. We analyzed the mtDNA nucleotide sequence of a MERRF patient, the original case of MERRF described by Fukuhara et al., and identified a point mutation of 8,344 in tRNA(Lys) gene. This mutation detected in all 8 MERRF patients from 6 independent families, and not detected in 15 controls by polymerase chain reaction using a mismatched primer. We also quantitated the degrees of heteroplasmy of the point mutation at nt 8,344 of tRNA(Lys) in various postmortem tissues from two patients with MERRF. The percentages of the mutant mtDNA were similar in both clinically affected and unaffected tissues.

Base Sequence↗
PubMed2005

Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing.

Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations.

Base Sequence↗
PubMed1995

Quantitative determination of heteroplasmy in Leber's hereditary optic neuropathy by single-strand conformation polymorphism.

PURPOSE: The maternal inheritance of Leber's hereditary optic neuropathy (LHON) is caused by defects in the genes of mitochondrial DNA (mtDNA). The most prevalent mtDNA mutation, present in 40% to 90% of families with this disease, is a G to A substitution at nucleotide position 11778. The rapid and accurate quantification of heteroplasmy of this mutation will help determine the relative risk for disease expression. METHODS: The authors conducted screening tests for heteroplasmy in 44 visually affected patients with the 11778 mutation and 34 unaffected members of 36 Japanese families with LHON using the single-strand conformation polymorphism analysis. This method can detect even a single base difference between the sequences of wild type and mutant DNA strands. The percentage of mutant mtDNA was calculated using an image analyzer. RESULTS: Single-strand conformation polymorphism analysis allowed the detection of heteroplasmy ranging from 5% to 95%. Five (14%) of the 36 families showed heteroplasmy, and 14 (18%) of the 78 persons tested had heteroplasmy ranging from 10% to 94%. Seven patients with heteroplasmy with visual loss had mutant mtDNA ranging from 62% to 94%. CONCLUSIONS: Single-strand conformation polymorphism analysis is rapid, efficient, and accurate for detecting point mutations and quantifying heteroplasmy in mtDNA. Individuals with heteroplasmy with less than 60% of mutant mtDNA in circulating leukocytes are probably at lesser risk for developing optic atrophy.

Adult↗
PubMed2002

Mitochondrial DNA mutations in focal segmental glomerulosclerosis lesions.

Glomerular epithelial cells are primary pathogenic sites in focal segmental glomerulosclerosis (FGS) lesions. Glomerular epithelial cells are regarded as terminally differentiated cells that do not proliferate. These characteristics are also noted for neurons and muscular cells, which are major sites of mitochondrial DNA (mtDNA) mutation accumulation. Screening for mtDNA mutations was performed with renal biopsy specimens from patients with primary FGS and patients with IgA nephropathy (as subjects with secondary FGS and as control subjects). mtDNA extracted from kidney biopsy specimens was amplified with appropriate primer pairs for study of the mtDNA point mutations 3243A-->G, 3271T-->C, 8344A-->G, and 8993T-->G/C, as well as the common deletion (a 4977-bp deletion spanning mtDNA nucleotide pairs 8469 to 13447). In situ amplification of both total mtDNA and the common deletion was also performed. Two patients with FGS demonstrated the 3243A-->G point mutation; 12 patients with FGS and seven patients with IgA nephropathy accompanied by glomerulosclerotic lesions exhibited the common deletion in their kidney tissue. No patient demonstrated the mtDNA mutations 3271T-->C, 8344A-->G, or 8993T-->G/C. The degree of heteroplasmy for the 3243A-->G point mutation was >85%; however, the heteroplasmy for the common deletion was <1%. As determined with in situ PCR, normal mtDNA was mainly distributed in the tubular epithelium and mtDNA with the common deletion was mainly distributed among glomerular epithelial cells. In conclusion, it is suggested that mtDNA mutations are distributed in glomerular epithelial cells among some patients with primary FGS or secondary FGS with IgA nephropathy. These mutations may be related to glomerular epithelial cell damage.

Adolescent↗
PubMed1994

The 8993 mtDNA mutation: heteroplasmy and clinical presentation in three families.

The point mutation at bp 8993 of human mtDNA in the ATPase 6 gene is associated with neurogenic weakness, ataxia and retinitis pigmentosa, and with subacute necrotizing encephalomyelopathy (Leigh disease) when present at high copy number. In this study we describe three new multiplex families with the ATPase 8993 mtDNA mutation and demonstrate a correlation between the percentage heteroplasmy of this mutation and the clinical phenotype. By combining this study with previous data we produce a graph of age of onset of symptoms versus percentage heteroplasmy of the mutation. Finally, we determine that ATP synthesis with NAD-linked substrates in cultured lymphoblast mitochondria from three patients with Leigh disease who had a high percentage heteroplasmy was on average 66% of the rate seen in control lymphoblast mitochondria. Similar rates are observed in lymphoblast mitochondria isolated from patients with Leigh disease due to complex I deficiency. This percentage appears to be independent of the rate of electron transport in mitochondria from patient cell lines with the mtDNA 8993 mutation.

Adenosine Triphosphatases↗