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Heteroplasmy of the Human mtDNA Control Region Remains Constant during Life

2001; Elsevier BV; Volume: 68; Issue: 5 Linguagem: Inglês

10.1086/320115

1537-6605

Maria Lagerström‐Fermér, Charlotta Olsson, Lars Forsgren, Ann‐Christine Syvänen,

Forensic and Genetic Research

In a longitudinal, retrospective study, we monitored the level of heteroplasmy at nucleotide position (nt) 309 and nt 16189 of the control region of human mtDNA. As a unique source of DNA, we analyzed multiple cervical-cell samples collected, during 1 or 2 decades, from four women with heteroplasmy at either nt 309 or nt 16189. According to accurate, quantitative analysis by solid-phase minisequencing, the level of heteroplasmy remained stable in the cervical-cell samples from all four women during the time studied. We also analyzed autopsy samples from several different tissues, all containing nt 309 in heteroplasmic form, of one of the women, who was deceased. On the basis of our results, heteroplasmy in the control region of mtDNA seems to be inherited and is not the result of somatic age-related accumulation. In a longitudinal, retrospective study, we monitored the level of heteroplasmy at nucleotide position (nt) 309 and nt 16189 of the control region of human mtDNA. As a unique source of DNA, we analyzed multiple cervical-cell samples collected, during 1 or 2 decades, from four women with heteroplasmy at either nt 309 or nt 16189. According to accurate, quantitative analysis by solid-phase minisequencing, the level of heteroplasmy remained stable in the cervical-cell samples from all four women during the time studied. We also analyzed autopsy samples from several different tissues, all containing nt 309 in heteroplasmic form, of one of the women, who was deceased. On the basis of our results, heteroplasmy in the control region of mtDNA seems to be inherited and is not the result of somatic age-related accumulation. Disease-causing mutations in mtDNA are typically heteroplasmic, with normal and mutant sequences coexisting at variable levels in the same individual (Wallace Wallace, 1999Wallace DC Mitochondrial diseases in man and mouse.Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2612) Google Scholar). Heteroplasmy at neutral polymorphic nucleotide positions of human mtDNA has been observed only sporadically (Comas et al. Comas et al., 1995Comas D Pääbo S Bertranpetit J Heteroplasmy in the control region of human mitochondrial DNA.Genome Res. 1995; 5: 89-90Crossref PubMed Scopus (80) Google Scholar; Bendall et al. Bendall et al., 1996Bendall KE Macaulay VA Baker JR Sykes BC Heteroplasmic point mutations in the human mtDNA control region.Am J Hum Genet. 1996; 59: 1276-1287PubMed Google Scholar; Ivanov et al. Ivanov et al., 1996Ivanov PL Wadhams MJ Roby RK Holland MM Weedn VW Parsons TJ Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II.Nat Genet. 1996; 12: 417-420Crossref PubMed Scopus (240) Google Scholar; Jazin et al. Jazin et al., 1996Jazin EE Cavelier L Eriksson I Oreland L Gyllensten U Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA.Proc Natl Acad Sci USA. 1996; 93: 12382-12387Crossref PubMed Scopus (138) Google Scholar; Parsons et al. Parsons et al., 1997Parsons TJ Muniec DS Sullivan K Woodyatt N Alliston-Greiner R Wilson MR Berry DL et al.A high observed substitution rate in the human mitochondrial DNA control region.Nat Genet. 1997; 15: 363-368Crossref PubMed Scopus (382) Google Scholar). Recent studies have, however, suggested that heteroplasmy of neutral mtDNA sequence variants may be more common than has been postulated (Calloway et al. Calloway et al., 2000Calloway CD Reynolds RL Herrin Jr, GL Anderson WW The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age.Am J Hum Genet. 2000; 66: 1384-1397Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar; Howell and Smejkal Howell and Smejkal, 2000Howell N Smejkal CB Persistent heteroplasmy of a mutation in the human mtDNA control region: hypermutation as an apparent consequence of simple-repeat expansion/contraction.Am J Hum Genet. 2000; 66: 1589-1598Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar; Tully et al. Tully et al., 2000Tully LA Parsons TJ Steighner RJ Holland MM Marino MA Prenger VL A sensitive denaturing gradient-gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region.Am J Hum Genet. 2000; 67: 432-443Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and that the frequency of heteroplasmic polymorphisms increases with age, at least when studied either in cell cultures (Michikawa et al. Michikawa et al., 1999Michikawa Y Mazzucchelli F Bresolin N Scarlato G Attardi G Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication.Science. 1999; 286: 774-779Crossref PubMed Scopus (621) Google Scholar) or in population groups at different ages (Calloway et al. Calloway et al., 2000Calloway CD Reynolds RL Herrin Jr, GL Anderson WW The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age.Am J Hum Genet. 2000; 66: 1384-1397Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). To address this question in vivo, we performed a longitudinal retrospective study in which we monitored the fluctuation of heteroplasmy levels of sequence variants in the human mtDNA control region in the same four individuals during 1 or 2 decades of life. Cervical-cell samples that are collected every 3d year from Swedish women, to screen for cervical cancer, served as a unique source of DNA for the study. Initially, mtDNA in leukocytes from 36 women was screened for heteroplasmy by solid-phase minisequencing (Syvänen et al. Syvänen et al., 1993Syvänen A-C Sajantila A Lukka M Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing.Am J Hum Genet. 1993; 52: 46-59PubMed Google Scholar) at eight polymorphic nucleotide positions (nt) reported, in previous studies (Bendall and Sykes Bendall and Sykes, 1995Bendall KE Sykes BC Length heteroplasmy in the first hypervariable segment of the human mtDNA control region.Am J Hum Genet. 1995; 57: 248-256PubMed Google Scholar; Parsons et al. Parsons et al., 1997Parsons TJ Muniec DS Sullivan K Woodyatt N Alliston-Greiner R Wilson MR Berry DL et al.A high observed substitution rate in the human mitochondrial DNA control region.Nat Genet. 1997; 15: 363-368Crossref PubMed Scopus (382) Google Scholar), to occur in heteroplasmic form. The eight analyzed polymorphic variants were G185A, A189G, del309C, T16089C, C16092T, T16093C, C16189T, and C16192T. PCR and minisequencing primer sequences and reaction conditions are available from the authors, on request. The minisequencing method allows quantitative determination of heteroplasmy of mtDNA, at a level of ⩾1%–2% (Suomalainen et al. Suomalainen et al., 1993Suomalainen A Majander A Pihko H Peltonen L Syvanen AC Quantification of tRNA3243(Leu) point mutation of mitochondrial DNA in MELAS patients and its effects on mitochondrial transcription.Hum Mol Genet. 1993; 2: 525-534Crossref PubMed Scopus (64) Google Scholar; Juvonen et al. Juvonen et al., 1994Juvonen V Huoponen K Syvanen AC Nikoskelainen E Savontaus ML Quantification of point mutations associated with Leber hereditary optic neuroretinopathy by solid-phase minisequencing.Hum Genet. 1994; 93: 16-20Crossref PubMed Scopus (30) Google Scholar). In the present study, we were able to detect heteroplasmy, at a level of 0.5% T (see fig. 1). In the initial screen, heteroplasmic nucleotide variations were observed at 6 of the 288 sites analyzed. The C309T variant was observed in three individuals, at levels of 80%, 3%, and 50%. C16093T was seen in one individual (at a level of 28%) and C16189T in another individual (at a level of 98%). Interestingly, one individual was heteroplasmic at both nt 309 (at a level of 3%) and nt 16189 (at a level of 97%) (data not shown), indicating that one mitochondrial genome contains the 309C and 16189C variants and that the other mitochondrial genome contains the 309T and 16189T variants. For three of the women with detected heteroplasmy in their leukocyte samples, we were able to retrieve several cervical-smear samples collected during 20–25 years. From a fourth woman, who was deceased and had been affected by spinocerebellar ataxia type 7, we obtained autopsy samples from 14 different tissues, in addition to three cervical-cell samples taken during 9 years. DNA was extracted from the papanicolaou-stained cell smears (Josefsson et al. Josefsson et al., 1999Josefsson A Livak K Gyllensten U Detection and quantitation of human papillomavirus by using the fluorescent 5′ exonuclease assay.J Clin Microbiol. 1999; 37: 490-496PubMed Google Scholar) from the four women and from ∼100 mg of the tissue samples of the deceased woman. mtDNA fragments spanning nt 309 and nt 16189 were amplified by PCR, from 200 ng of DNA. C309T is a deletion of a C residue in a C homopolymer stretch (Bendall and Sykes Bendall and Sykes, 1995Bendall KE Sykes BC Length heteroplasmy in the first hypervariable segment of the human mtDNA control region.Am J Hum Genet. 1995; 57: 248-256PubMed Google Scholar), and the first nucleotide (a T residue) following the homopolymer stretch is detected in the minisequencing reaction. The relative amounts of the T and C sequence variants at nt 309 and nt 16189 were determined accurately by minisequencing-quantification standard curves, analyzed in parallel (fig. 1a). The quantitative analysis demonstrated that the level of heteroplasmy of the C309T and C16189T variants remained unaltered in cervical cells in all four individuals during the time studied (fig. 1b). The autopsy tissue samples contained the C309T variant in heteroplasmic form, at levels of 84%–91% (fig. 2). Our longitudinal analysis, during 1 or 2 decades, of four individuals with different levels of heteroplasmy in the control region of mtDNA, combined with the data from autopsy samples from several tissues of one of the individuals, suggests that heteroplasmy in the mtDNA control region is inherited and remains stable throughout life, without selection for either of the variants. On the basis of our study, heteroplasmy in the mitochondrial control region is rare and does not seem to be the result of somatic accumulation of sequence variation, as has been proposed elsewhere (Michikawa et al. Michikawa et al., 1999Michikawa Y Mazzucchelli F Bresolin N Scarlato G Attardi G Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication.Science. 1999; 286: 774-779Crossref PubMed Scopus (621) Google Scholar; Calloway et al. Calloway et al., 2000Calloway CD Reynolds RL Herrin Jr, GL Anderson WW The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age.Am J Hum Genet. 2000; 66: 1384-1397Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Our findings are important and reassuring for forensic identity testing and human evolutionary studies, which, so far, have been extensively based on the analysis of polymorphic sequence variation in the control region of mtDNA. Informed consent to retrieve the cervical samples from pathology departments had been obtained from all individuals analyzed. We are grateful to Margit Gustavsson, Erik Willander, Tomas Höckenström, and the staff at the pathology departments in Uppsala and in Umeå, for assistance with the localization of the samples. We thank Jenni Jonasson for extraction of DNA from the tissue samples, and we thank Agneta Josefsson for advice on extraction of DNA from cervical smears. This study was approved by the Ethics Committee of the Medical Faculty, Uppsala University, and was financially supported by the Swedish Medical Research Council.