Search

We report on a method to analyze length heteroplasmy within the human mitochondrial genome in which there are polycytosine [poly(C)] stretches. These poly(C) tracts induce heteroplasmy with the resultant inherent problems of accurate sequence designations. In this study, 20 samples that exhibited length heteroplasmy due to variation in the C-tracts within hypervariable region I (HVI) were treated with bisulfite, and one or more cytosine bases in these C-tracts were converted randomly to uracil. This resulted in an accurate sequence designation for nearly all samples. The only exceptions in which the DNA sequence could still not be determined occurred when there was total conversion, or a lack of conversion, of the cytosine bases. Replicate tests on the same samples showed that individual cytosine bases were randomly converted to uracil. This simple method was useful for investigating length heteroplasmy due to 16189C and 310C transitions in the mitochondrial-DNA control region. It is valuable for medical and forensic investigations.

Heteroplasmy is the presence of more than one mitochondrial DNA (mtDNA) variant within a cell, tissue, or individual. In this study, sequence variation was investigated in the control region (CR) of mitochondrial DNA (mtDNA) from 135 individuals belonging to five primary domestic Bactrian camel breeds in China and Mongolia. Due to variation of the repeat unit G(T/C)(AC)n, length heteroplasmy was detected within each camel by direct sequencing and fragment analysis. A high occurrence of mtDNA heteroplasmy, up to 100 percentages was observed in five camel populations. Our study provides the first evidence of extensive length heteroplasmy in Bactrian camels. [ABSTRACT FROM PUBLISHER]

Accurate sequencing of the control region of the mitochondrial genome is notoriously difficult due to the presence of polycytosine bases, termed C-tracts. The precise number of bases that constitute a C-tract and the bases beyond the poly cytosines may not be accurately defined when analyzing Sanger sequencing data separated by capillary electrophoresis. Massively parallel sequencing has the potential to resolve such poor definition and provides the opportunity to discover variants due to length heteroplasmy. In this study, the control region of mitochondrial genomes from 20 samples was sequenced using both standard Sanger methods with separation by capillary electrophoresis and also using massively parallel DNA sequencing technology. After comparison of the two sets of generated sequence, with the exception of the C-tracts where length heteroplasmy was observed, all sequences were concordant. Sequences of three segments 16184–16193, 303–315 and 568–573 with C-tracts in HVI, II and III can be clearly defined from the massively parallel sequencing data using the program SEQ Mapper. Multiple sequence variants were observed in the length of C-tracts longer than 7 bases. Our report illustrates the accurate designation of all the length variants leading to heteroplasmy in the control region of the mitochondrial genome that can be determined by SEQ Mapper based on data generated by massively parallel DNA sequencing. [ABSTRACT FROM AUTHOR]

We report on a method to analyze length heteroplasmy within the human mitochondrial genome in which there are polycytosine [poly(C)] stretches. These poly(C) tracts induce heteroplasmy with the resultant inherent problems of accurate sequence designations. In this study, 20 samples that exhibited length heteroplasmy due to variation in the C-tracts within hypervariable region I (HVI) were treated with bisulfite, and one or more cytosine bases in these C-tracts were converted randomly to uracil. This resulted in an accurate sequence designation for nearly all samples. The only exceptions in which the DNA sequence could still not be determined occurred when there was total conversion, or a lack of conversion, of the cytosine bases. Replicate tests on the same samples showed that individual cytosine bases were randomly converted to uracil. This simple method was useful for investigating length heteroplasmy due to 16189C and 310C transitions in the mitochondrial-DNA control region. It is valuable for medical and forensic investigations. [ABSTRACT FROM AUTHOR]

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined. • Simultaneous analysis of autosomal and Y-chromosomal STRs as well as mitochondrial targets. • Useful screening tool particularly for low template samples. • Assistance with decision for downstream analysis. • Conservation of valuable sample extract. [ABSTRACT FROM AUTHOR]

Previously, the mitochondrial control region of 214 Belgian dogs was sequenced. Analysis of this data indicated length heteroplasmy of the polyT stretch in the polyC-polyT-polyC stretch from positions 16661 to 16674. Nine polyC-polyT-polyC haplotype combinations were observed, consisting of seven major haplotypes (highest signal intensity) combined with minor haplotypes (lower signal intensity) one T shorter than the major haplotype in all but three dogs. The longer the polyT stretch, the smaller was the difference in signal intensity between the major and minor haplotype peaks. Additional sequencing, cloning, and PCR trap experiments were performed to further study the intra-individual variation of this mitochondrial DNA (mtDNA) region. Cloning experiments demonstrated that the proportion of clones displaying the minor haplotypes also increased with the length of the polyT stretch. Clone amplification showed that in vitro polymerase errors might contribute to the length heteroplasmy of polyT stretches with at least 10 Ts. Although major and minor polyC-polyT-polyC haplotypes did not differ intra-individually within and between tissues in this study, interpretation of polyT stretch variation should be handled with care in forensic casework. [ABSTRACT FROM AUTHOR]

Abstract: In forensic genetics mitochondrial DNA (mtDNA) is usually analyzed by direct Sanger-type sequencing (STS). This method is known to be laborious and sometimes prone to human error. Alternative methods have been proposed that lead to faster results. Among these are methods that involve mass-spectrometry resulting in base composition profiles that are, by definition, less informative than the full nucleotide sequence. Here, we applied a highly automated electrospray ionization mass spectrometry (ESI-MS) system (PLEX-ID) to an mtDNA population study to compare its performance with respect to throughput and concordance to STS. We found that the loss of information power was relatively low compared to the gain in speed and analytical standardization. The detection of point and length heteroplasmy turned out to be roughly comparable between the technologies with some individual differences related to the processes. We confirm that ESI-MS provides a valuable platform for analyzing mtDNA variation that can also be applied in the forensic context. [Copyright &y& Elsevier]

Abstract: Length heteroplasmy (LH) in mitochondrial (mt)DNA is usually observed in homopolymeric tracts and manifest as mixture of various length variants. The generally used difference-coded annotation to report mtDNA haplotypes does not express the degree of LH variation present in a sample, even more so, it is sometimes difficult to establish which length variants are present and clearly distinguishable from background noise. It has therefore become routine practice for some researchers to call the dominant type, the “major molecule”, which represents the LH variant that is most abundant in a DNA extract. In the majority of cases a clear single dominant variant can be identified. However, in some samples this interpretation is difficult, i.e. when (almost) equally quantitative LH variants are present or when multiple sequencing primers result in the presentation of different dominant types. To better understand those cases we designed amplicon sizing assays for the five most relevant LH regions in the mtDNA control region (around ntps 16,189, 310, 460, 573, and the AC-repeat between 514 and 524) to determine the ratio of the LH variants by fluorescence based amplicon sizing assays. For difficult LH constellations derived by Sanger sequencing (with Big Dye terminators) these assays mostly gave clear and unambiguous results. In the vast majority of cases we found agreement between the results of the sequence and amplicon analyses and propose this alternative method in difficult cases. [Copyright &y& Elsevier]

There are several areas within mitochondrial DNA that show length heteroplasmy. If the heteroplasmy pattern is unique and consistent for each person, it may be used to support an interpretation of exclusion in identity testing. We investigated whether the length heteroplasmy pattern would be consistent under different amplification conditions. We also determined whether various amplification parameters would affect the homopolymeric cytosine stretches (C-stretch) in HV1. Monoclonal samples tended to be heteroplasmic after amplification. After several repetitions, C-stretch patterns of all samples were inconsistent even under the same amplification conditions. Increased PCR cycles and high template concentrations resulted in a more frequent heteroplasmic tendency. These amplification parameters seem to have little effect if samples are not long enough in C-stretch or total length of the segment from nt 16180 to nt 16193. It is suggested that the pattern of length heteroplasmy cannot be used as an additional polymorphic marker. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate mitochondrial DNA (mtDNA) hypervariable segment-I (HVS-I) C-stretch variations and explore the significance of these variations in forensic and population genetics studies. The C-stretch sequence variation was studied in 919 unrelated individuals from 8 Chinese ethnic groups using both direct and clone sequencing approaches. Thirty eight C-stretch haplotypes were identified, and some novel and population specific haplotypes were also detected. The C-stretch genetic diversity ( GD) values were relatively high, and probability ( P) values were low. Additionally, C-stretch length heteroplasmy was observed in approximately 9% of individuals studied. There was a significant correlation ( r=−0.961, P<0.01) between the expansion of the cytosine sequence length in the C-stretch of HVS-I and a reduction in the number of upstream adenines. These results indicate that the C-stretch could be a useful genetic maker in forensic identification of Chinese populations. The results from the Fst and dA genetic distance matrix, neighbor-joining tree, and principal component map also suggest that C-stretch could be used as a reliable genetic marker in population genetics. [ABSTRACT FROM AUTHOR]

In this work, we present the results of the screening of human mitochondrial DNA (mtDNA) heteroplasmy in the control region of mtDNA from 210 unrelated Spanish individuals. Both hypervariable regions of mtDNA were amplified and sequenced in order to identify and quantify point and length heteroplasmy. Of the 210 individuals analyzed, 30% were fully homoplasmic and the remaining presented point and/or length heteroplasmy. The prevalent form of heteroplasmy was length heteroplasmy in the poly(C) tract of the hypervariable region II (HVRII), followed by length heteroplasmy in the poly(C) tract of hypervariable region I (HVRI) and, finally, point heteroplasmy, which was found in 3.81% of the individuals analyzed. Moreover, no significant differences were found in the proportions of the different kinds of heteroplasmy in the population when blood and buccal cell samples were compared. The pattern of heteroplasmy in HVRI and HVRII presents important differences. Moreover, the mutational profile in heteroplasmy seems to be different from the mutational pattern detected in population. The results suggest that a considerable number of mutations and, particularly, transitions that appear in heteroplasmy are probably eliminated by drift and/or by selection acting at different mtDNA levels of organization. Taking as a whole the results reported in this work, it is mandatory to perform a broad-scale screening of heteroplasmy to better establish the heteroplasmy profile which would be important for medical, evolutionary, and forensic proposes. [ABSTRACT FROM AUTHOR]

Abstract: Sequence analysis of HV2 in mitochondrial DNA has been performed as a tool for forensic identification, in addition to that of HV1. HV2 contains length heteroplasmy, which shows high variability within an individual or in maternal relatives. In this study, we used cloning analysis and PCR direct sequencing to compare, between mothers and their children, HV2 length heteroplasmic profiles in different tissues. For two mother–child pairs, different types of variant distribution were observed by cloning analysis. In pair 1, length heteroplasmic patterns in most tissues were similar (predominantly 9 and 10Cs variants), but different length heteroplasmic levels, with shifts in predominant genotype, were observed for some hairs in both mother and child. In pair 2, genotype distribution was similar for all tissues, with a predominant 8Cs genotype, but varying in the proportion of minor component. The proportion of one minor length variant (9Cs) in blood from the child was significantly higher than that from the mother, but the proportions of minor components (7 and/or 9Cs) in other tissue samples decreased from mother to child. Moreover, we could confirm that sequence types of PCR products were reflected by the distribution of length variants, which were observed especially in high proportion, in cloning analysis. Our results reveal variable changes in length heteroplasmic level in various tissues between generations. Variability between tissues, especially among hairs, within an individual would result in complicated differences in genotype distribution between maternal generations, and correlate with longer length of Cs for predominant variants. [Copyright &y& Elsevier]

To investigate mitochondrial DNA (mtDNA) distribution within tissues during life, we observed length heteroplasmy in a polycytosine tract of the mitochondrial HV2 region by size-based separation of PCR products, using a mutagenic primer which was designed to avoid stutter production. Blood, brain, heart, liver, skeletal muscle and hair shaft samples were collected during autopsies of 25 individuals. Here, we demonstrate differences in the level of mtDNA length heteroplasmy both within and between individuals and tissues. We also show that mtDNA is distributed randomly in varying proportions in various somatic tissues during growth, resulting in an imbalance in the composition of mtDNA pools among tissues. This mtDNA distribution appears not to be strictly random, and can be explained by the random somatic segregation of nucleoids. On the other hand, significant qualitative/quantitative mtDNA peak pattern variations in hair shafts are thought to be a result of the different developmental origins of hairs. Each hair shaft may have a restricted or clonal set of mtDNA molecules derived from a discrete group of stem cells. [ABSTRACT FROM AUTHOR]

Doubly uniparental inheritance of mtDNA (DUI) is commonly observed in several genera of bivalves. Under DUI, female offspring inherit mtDNA from their mothers, while male offspring inherit mtDNA from both parents but preferentially transmit the paternally inherited mtDNA to their sons. Several studies have shown that the female- and male-specific mtDNA lineages in blue mussels, Mytilus spp., vary by upward of 20% at the nucleotide level. In addition to high levels of nucleotide substitution, the present study observed substantial gender-based length polymorphism in the presumptive mitochondrial control region (=large unassigned region; LUR) of North American M. trossulus. In this species, female lineage LUR haplotypes are over 2 kb larger than male lineage LUR haplotypes. Analysis of sequence data for these length variants indicates that the F LUR haplotypes of North American M. trossulus contain sequences similar to the F lineage control region in the congeners M. edulis and M. galloprovincialis. Relative to the F LUR in the latter two species, however, the F lineage LUR haplotypes in M. trossulus contain two large sequence insertions, each nearly 1 kb in size. One of these insertions has high sequence similarity to the male lineage LUR of M. trossulus. The tandem arrangement of F and M control region sequences in the F lineage LUR of M. trossulus is most likely the result of nonhomologous recombination between the male and the female mitochondrial genomes in M. trossulus, a finding that has important implications regarding the transmission and evolution of blue mussel mitochondrial genomes. [ABSTRACT FROM AUTHOR]

Abstract: Sequencing of the homopolymeric tract of cytosines (C-stretch) in human mitochondrial HVI region usually results in a blurred pattern beyond it when a T/C transition at nt 16189 occurs: it depends on a length heteroplasmy probably arising through a replication slippage. This study aims to investigate the distribution of heteroplasmic length variants within three related individuals along maternal lineage by cloning approach. Sequencing of multiple independent clones (12–14) is sufficient to yield heteroplasmic profiles. In addition, we illustrate a direct correlation between expansion of heteroplasmy modal length and reduction of the number of adenines preceding the homopolymeric tract; this association may be useful in pedigree analysis and in forensic field for tissues comparison, single hair sample included. [Copyright &y& Elsevier]

We describe a method to obtain reliable mitochondrial DNA (mtDNA) sequences downstream of the homopolymeric stretches with length heteroplasmy in the sequencing direction. The method is based on the use of junction primers that bind to a part of the homopolymeric stretch and the first 2–4 bases downstream of the homopolymeric region. This junction primer method gave clear and unambiguous results using samples from 21 individuals with length heteroplasmy in the hypervariable regions HV1, HV2 or both. The method is of special value for forensic casework, because sequencing of both strands of an mtDNA region is preferable in order to reduce ambiguities in sequence determination. [Copyright &y& Elsevier]

The sequences of the two hypervariable regions of the mitochondrial DNA control region (HV1 and HV2) from close maternal relatives (mother-child pairs) were compared to determine the frequency of mutations between two generations. A total of 68 blood samples were sequenced only in HV1 and 86 were analysed for HV1 and HV2. The intergenerational comparison led to the identification of six heteroplasmic point mutations affecting the children only. In one case a heteroplasmy of the maternal sequence was resolved to homoplasmy in the corresponding sequence of the child. [ABSTRACT FROM AUTHOR]

Abstract: The variability in np16184–16193 HVI region presents two different types of polymorphism: sequence variability and length variability with length heteroplasmy. It is important to define properly the HVI homopolimeric regions for a correct classification of homopolimeric track defining the number of As and Cs and the various population types identified by adjacent bases to the homopolimeric track. [Copyright &y& Elsevier]

Abstract: The applicability of liquid chromatography online hyphenated to electrospray ionization time-of-flight mass spectrometry to the characterization of length heteroplasmic samples was demonstrated. Two segments within the first and second hypervariable regions (HV1=16144…16237; HV2=220…381) of the mitochondrial DNA (mtDNA) control region were selected as targets. The two mtDNA regions were simultaneously amplified within a single PCR and analyzed. 90 maternally unrelated mother–offspring pairs served as Austrian population sample. The plausibility of ICEMS results was checked by sequencing. [Copyright &y& Elsevier]

We describe a method to estimate the proportion of length variants in samples carrying mtDNA homopolymeric C-stretch (303 to 315) originated by a T310C transition. Sequencing analysis of these samples produces blurred electropherograms and the exact number of C-residues remains ambiguous. This has important consequences in forensic casework, since one of the aims in the interpretation of mtDNA profiles is the determination of the existence of a match or a mismatch between two or more sequences under study. HV-2B region (positions 172 to 408) was amplified with two sets of labelled primers. Electrophoresis of fluorescently labelled amplicons was performed on an ABI310 or ABI377. Results were reproducible in repeated PCR experiments, using the two sets of primers and using either capillary or slab-gel electrophoresis. [Copyright &y& Elsevier]

Advancements in sequencing technologies allow for rapid and efficient analysis of mitochondrial DNA (mtDNA) in forensic laboratories, which is particularly beneficial for specimens with limited nuclear DNA. Next generation sequencing (NGS) offers higher throughput and sensitivity over traditional Sanger-type sequencing (STS) as well as the ability to quantitatively analyze the data. Changes in sample preparation, sequencing method and analysis required for NGS may alter the mtDNA haplotypes compared to previously generated STS data. Thus, the present study aimed to characterize the impact of different sequencing workflows on the detection and interpretation of length heteroplasmy (LHP), a particularly complicated aspect of mtDNA analysis. Whole mtDNA genome (mitogenome) data were generated for 16 high-quality samples using well-established Illumina and Ion methods, and the NGS data were compared to previously-generated STS mtDNA control region data. Although the mitogenome haplotypes were concordant with the exception of length and low-level variants (<30 % variant frequency), LHP in the hypervariable segment (HVS) polycytosine regions (C-tracts) differed across sequencing methods. Consistent with previous studies, LHP in HVS1 was observed in samples with nine or more consecutive cytosines (Cs) and eight Cs in the HVS2 region in the STS data. The Illumina data produced a similar pattern of LHP as the STS data, whereas the Ion data were noticeably different. More complex LHP (i.e. more length molecules) was observed in the Ion data, as length variation occurred in multiple homopolymer stretches within the targeted HVS regions. Further, the STS dominant or major molecule (MM) differed from the Ion MM in 11 (37 %) of the 30 regions evaluated and six instances (20 %) in Illumina data. This is of particular interest, as the MM is used by many forensic laboratories to report the HVS C-tract in the mtDNA haplotype. In general, the STS MMs were longer than the Illumina MMs, while the Ion MMs were the shortest. The higher rate of homopolymer indels in Ion data likely contributed to these differences. Supplemental analysis with alternative approaches demonstrated that the LHP pattern may also be altered by the bioinformatic tool and workflow used for data interpretation. The broader application of NGS in forensic laboratories will undoubtedly result in the use of varying sample preparation and sequencing methods. Based on these findings, minor LHP differences are expected across sequencing workflows, and it will be important that C-tract indels continue to be ignored for forensic queries and comparisons., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Abstract: Length heteroplasmy in the hypervariable region HVIII of mitochondrial DNA (mtDNA) was analyzed by direct sequencing, fragment analysis and cloning. Samples from 19 of 437 unrelated Japanese individuals (4.3%) showed length heteroplasmy in the homopolymeric tract of cytosines (nt 568–573). Every length heteroplasmic sample found in this study was composed predominantly of 9, 10 or 11 cytosines in the homopolymeric tract. [Copyright &y& Elsevier]

Abstract: The mitochondrial DNA has been used in forensic identification areas due to its maternal inheritance, high mutation rate, and the high number of copies. Even the highly polymorphic regions, HVI/HVII, sometimes are of low discriminate value due to the presence of common population polymorphisms. Our aim is to evaluate the inheritance of these polymorphisms and heteroplasmies in HVI/HVII using known pairs of mother/child analyzed in blind-test, and characterize the Brazilian population. DNA sequencing was performed in 98 pairs to HVI, with match of 71.4%, and in 88 pairs to HVII with 45.5% of match. For the joint analysis (HVI+HVII=88 pairs) the match was of 86.4%, with 82 haplotypes: 76 (92.7%) unique and 6 seen in 2 individuals. Only HVII showed point heteroplasmies. HVI showed discriminate power much higher than HVII; the joint analysis was able to match only six pairs more than HVI. In a real situation of identification the choice of HVI to initial screening becomes obvious. [Copyright &y& Elsevier]