Results of the 1999–2000 collaborative exercise and proficiency testing program on mitochondrial DNA of the GEP-ISFG: an inter-laboratory study of the observed variability in the heteroplasmy level of hair from the same donor

Alonso, Antonio; Salas, Antonio; Albarrán, Cristina; Arroyo, Eduardo; Castro, Azucena; Crespillo, Manuel; Lonardo, A.M. Di; Lareu, Marı́a Victoria; Cubrı́a, Carlos López; Soto, Manuel López; Lorente, José A.; Semper, Marta Montesino; Palacio, Ana; Paredes, Manuel; Pereira, Luı́sa; Lezaun, Anna Pérez; Pestano, J.; Sala, Andrea; Vide, M.C.; Whittle, M.R.; Londoño, Juan José Yunis; Gómez, Josefina

doi:10.1016/s0379-0738(01)00602-8

Cited by 48 publications

(32 citation statements)

References 19 publications

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“…In view of the high mutational rate of mtDNA and the large number of mtDNA copies per organelle and per cell, the probability of the existence of mtDNA with alternate primary structure(s) in every individual is likely to approach to the level of 1. However, a necessary proviso would be that the determination of the homoplasmic or heteroplasmic state depends strongly on the technical approach employed for DNA analysis [42]. More sensitive and discrete techniques on the one hand and the prevalence of mtDNA sequence variants on the other hand would yield higher incidence of revealed heteroplasmy [43,44].…”

Section: Structure and Biological Properties Of Mtdnamentioning

confidence: 99%

Mitochondrial DNA and Human Thyroid Diseases

2004

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

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Section: Structure and Biological Properties Of Mtdnamentioning

confidence: 99%

Mitochondrial DNA and Human Thyroid Diseases

2004

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“…For the amplification of the hair DNA extracts, prepared in the previous study [24,41], a direct PCR approach was used, amplifying the HV1 in two overlapping fragments (hereinafter called "direct PCR"). This technique is widely employed by forensic mtDNA laboratories [14,28,[31][32][33]. The following two sets of primers were used for reanalysis: L15990 (5' TTA ACT CCA CCA TTA GCA CC 3'); H16239 (5' TGG CTT TGG AGT TGC AGT TG 3'); L16209 (5' CCC CAT GCT TAC AAG CAA GT 3'); H16391 (5' GAG GAT GGT GGT CAA GGG AC 3').…”

Section: Amplification and Sequencing Conditionsmentioning

confidence: 99%

“…There is a considerable evidence that mtDNA sequence heteroplasmy exists at an appreciable frequency and segregates differentially in different tissues [6][7][8][9][10]. Furthermore, it was demonstrated that in some tissues, including hair, heteroplasmy tends to occur at highly variable levels [11][12][13][14]. In a majority of cases, new heteroplasmic mutations are observed preferentially at hypervariable sites of the human mtDNA control region [15].…”

Section: Introductionmentioning

confidence: 99%

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High levels of mitochondrial DNA heteroplasmy in single hair roots: Reanalysis and revision

et al. 2003

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The present study demonstrates a reinvestigation of the mitochondrial DNA sequence heteroplasmy, which was previously found by the use of nested polymerase chain reaction (PCR) technique in single hairs of 13 individuals. The direct PCR approach was used for the amplification of mitochondrial DNA and a phylogenetic analysis was applied to both data sets for the verification of the authenticity of sequences. The comparative analysis of the sequencing results obtained from the same hair DNA extracts - but using two different techniques - shows that direct mitochondrial DNA amplification results in a considerably lower number of mixed positions. The majority of the confirmed heteroplasmic variants preferentially occurs in mitochondrial DNA hypervariable sites (mutational hotspots). However, the pattern of heteroplasmic mutations observed in four extracts after nested PCR significantly differs from the pattern of natural mutations. Some of these rare polymorphisms should be revised as inconsistent with phylogenetic expectations. The results of the present study contribute to the earlier reports by indicating that phylogenetic analysis is an effective tool in a posteriori quality check of mitochondrial DNA data.

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“…Because of the high level of heteroplasmy, the latter report has been questioned and can be regarded as controversial (18,19) and reanalysis of Grzybowski's study showed fewer heteroplasmic positions in hairs without using nested PCR (20). Alonso et al also found various levels of heteroplasmy and several mutations in hairs from a single individual during an interlaboratory study (21). A denaturing gradient-gel electrophoresis (DGGE) assay has been demonstrated to be a sensitive tool for detecting heteroplasmy (6,22).…”

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Mitochondrial DNA Heteroplasmy Among Hairs from Single Individuals

Sekiguchi

Sato

Kasai

2004

Journal of Forensic Sciences

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A denaturing gradient gel electrophoresis (DGGE) assay was used to detect mitochondrial DNA (mtDNA) sequence heteroplasmy in 160 hairs from each of three individuals. The HV1 and HV2 heteroplasmic positions were then identified by sequencing. In several hairs, the heteroplasmic position was not evident by sequencing and dHPLC separation of the homoduplex/heteroduplex species was carried out with subsequent reamplification and sequencing to identify the site. The overall detection frequency of sequence heteroplasmy in these hairs was 5.8% (28/480) with DGGE and 4.4% (21/280) with sequencing. Sequence heteroplasmy of hair was observed even when the reference blood sample of the individual was homoplasmic. The heteroplasmic positions were not necessarily observed at sites where high rates of substitution have been reported. In two hairs, a complete single base change from the reference blood sample was observed with sequencing, while the heteroplasmic condition at that site in the hair was observed using DGGE. The DGGE results in such samples would serve as an aid in considering the possibility of match significance. In a forensic case, this situation would lead to the possibility of a failure to exclude rather than to be inconclusive. KEYWORDS:forensic science, DNA typing, mitochondrial DNA, hair DNA, heteroplasmy, denaturing gradient gel electrophoresis MtDNA analysis is a useful tool for the identification of old or degraded biological evidentiary samples and is also important in the forensic comparison of hair samples. Heteroplasmy, the presence of two or more types of mtDNA in a single individual, was first observed in humans in association with a mitochondrial disorder (1). However, heteroplasmy in mtDNA has been reported, not only in association with mitochondrial disorders, but in the non-coding control region as well. Heteroplasmy in the control region was initially not considered to be significant (2), but it now appears to be a more frequent phenomenon than had originally been thought (3-6). Inter-and intra-generational heteroplasmy has been reported and the proportion of heteroplasmy appears to vary among different family members (5,7,8). Intra-individual heteroplasmy has also been reported in several studies (9,10). Length heteroplasmy in the HV1 and HV2 region, near nucleotide positions 16189 and 309-315, was found among hairs within a single individual (11,12) and the mechanism involved in this length heteroplasmy has been investigated (13,14). Site heteroplasmy, at a single nucleotide position in the control region of mtDNA in hairs, has also been reported and its proportions varied among hairs within a single individual (5,15). The frequency of site heteroplasmy among hairs from an individual with a homoplasmic blood sample has also been reported (16,17). Hühne et al. reported that no heteroplasmy was detected in an analysis of 15 hairs from each of 10 individuals, whereas Grzybowski reported numerous heteroplasmic positions in an analysis of 2-6 hairs each from each of 13 individuals. Be...

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Results of the 1999–2000 collaborative exercise and proficiency testing program on mitochondrial DNA of the GEP-ISFG: an inter-laboratory study of the observed variability in the heteroplasmy level of hair from the same donor

Cited by 48 publications

References 19 publications

Mitochondrial DNA and Human Thyroid Diseases

Mitochondrial DNA and Human Thyroid Diseases

High levels of mitochondrial DNA heteroplasmy in single hair roots: Reanalysis and revision

Mitochondrial DNA Heteroplasmy Among Hairs from Single Individuals

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