Coamplification of Nuclear Pseudogenes and Assessment of Heteroplasmy of Mitochondrial DNA Mutations

doi:10.1006/bbrc.1998.8666

Biochemical and Biophysical Research Communications

Volume 247, Issue 1, 9 June 1998, Pages 57-59

https://doi.org/10.1006/bbrc.1998.8666 Get rights and content

Abstract

The potential co-amplification of actual mtDNA and nucleus-embedded mtDNA sequences was studied for the mtDNA domains encompassing the major disease-causing mtDNA mutations. By using two different cell lines devoid of mtDNA (ρ° cell lines), it is shown that nucleus-embedded mtDNA sequences readily co-amplified with most of the mtDNA domains encompassing disease-causing mtDNA mutations. The selection of mtDNA primers for specificity on ρ° cells constitutes a simple procedure to avoid such co-amplification. It appears mandatorypriorto quantify mtDNA mutations, especially when delivering prenatal diagnosis or predictive genetic advise.

References (9)

T. Tsuzuki et al.
Gene
(1983)
P. Rustin et al.
Clin. Chim. Acta
(1994)
D.C. Wallace
Annu. Rev. Biochem.
(1992)
A. Rötig et al.
J. Clin. Invest.
(1990)

There are more references available in the full text version of this article.

Cited by (91)

Preimplantation genetic testing for mitochondrial DNA mutation: ovarian response to stimulation, outcomes and follow-up
2023, Reproductive BioMedicine Online
How do carriers of pathogenic mitochondrial DNA (mtDNA) respond to ovarian stimulation?
A single-centre, retrospective study conducted between January 2006 and July 2021 in France. Ovarian reserve markers and ovarian stimulation cycle outcomes were compared for couples undergoing preimplantation genetic testing (PGT) for maternally inherited mtDNA disease (n = 18) (mtDNA-PGT group) with a matched-control group of patients undergoing PGT for male indications (n = 96). The PGT outcomes for the mtDNA-PGT group and the follow-up of these patients in case of unsuccessful PGT was also reported.
For carriers of pathogenic mtDNA, parameters of ovarian response to FSH and ovarian stimulation cycle outcomes were not different from those of matched-control ovarian stimulation cycles. The carriers of pathogenic mtDNA needed a longer ovarian stimulation and higher dose of gonadotrophins. Three patients (16.7%) obtained a live birth after the PGT process, and eight patients (44.4%) achieved parenthood through alternative methods: oocyte donation (n = 4), natural conception with prenatal diagnosis (n = 2) and adoption (n = 2).
To the best of our knowledge, this is the first study of women carrying a mtDNA variant who have undergone a PGT for monogenic (single gene defects) procedure. It is one of the possible options to obtain a healthy baby without observing an impairment in ovarian response to stimulation.
Emerging methods for and novel insights gained by absolute quantification of mitochondrial DNA copy number and its clinical applications
2022, Pharmacology and Therapeutics
The past thirty years have seen a surge in interest in pathophysiological roles of mitochondria, and the accurate quantification of mitochondrial DNA copy number (mCN) in cells and tissue samples is a fundamental aspect of assessing changes in mitochondrial health and biogenesis. Quantification of mCN between studies is surprisingly variable due to a combination of physiological variability and diverse protocols being used to measure this endpoint. The advent of novel methods to quantify nucleic acids like digital polymerase chain reaction (dPCR) and high throughput sequencing offer the ability to measure absolute values of mCN. We conducted an in-depth survey of articles published between 1969 -- 2020 to create an overview of mCN values, to assess consensus values of tissue-specific mCN, and to evaluate consistency between methods of assessing mCN. We identify best practices for methods used to assess mCN, and we address the impact of using specific loci on the mitochondrial genome to determine mCN. Current data suggest that clinical measurement of mCN can provide diagnostic and prognostic value in a range of diseases and health conditions, with emphasis on cancer and cardiovascular disease, and the advent of means to measure absolute mCN should improve future clinical applications of mCN measurements.
Cyclic fertilin-derived peptide stimulates in vitro human embryo development
2022, F and S Science
To study the cyclic fertilin peptide effects on preimplantation human embryogenesis. Cyclic fertilin peptide reproduces the structure of the binding site of the sperm Fertilin β (also named A Disintegrin and Metalloprotease 2: ADAM2) disintegrin domain. It binds to the oocyte membrane and increases sperm-oocyte fusion index in human and fertilization rate in mouse, providing healthy pups. It also improves human oocyte maturation and chromosome segregation in meiosis I and binds to human embryo blastomeres, suggesting that it has a membrane receptor.
Thawed human embryos at the 3 to 4 cells stage were randomly included in a dose-response study with cyclic fertilin peptide. Inner cell mass (ICM), trophectoderm (TE), and total cell numbers were evaluated in top- and good-quality blastocysts.
The study was performed in an academic hospital and research laboratory.
Human embryos donated for research. This project was approved by the French “Agence de la Biomédecine.”
Immunofluorescence and tissue-specific gene expression analysis, using Clariom D microarrays, were performed to study its mechanism of action.
Cyclic fertilin peptide improves blastocyst formation by almost 20%, the concentration of 1 μM being the lowest most efficient concentration. It significantly increases twice the TE cell number, without modifying the ICM. It increases the in vitro hatching rate from 14% to 45%.
Cyclic fertilin peptide stimulates TE growth. In the ICM, it induces transcriptional activation of intracellular protein and vesicle-mediated transport.
Cyclic fertilin peptide dramatically improves human embryo development potential. It could be used to supplement culture medium and improve the in vitro human embryo development. Starting supplementation immediately after fertilization, instead of day 2, could significantly upgrade assisted reproductive technology outcome.
Mitochondrial DNA mutations do not impact early human embryonic development
2021, Mitochondrion
Citation Excerpt :
Values used for the standard curve quantification ranged from 1,22 × 107 up to 1,22 copies/µl. Cross hybridization of oligonucleotide primers to genomic DNA was ruled out by PCR amplification on mtDNA-less Rho0 cells (Parfait et al., 1998). Real-time PCR reaction was performed on 5 µl of purified PCR or standard product (ranging from 6.1 × 107 copies to 6.1 copies), 10 µl Power SYBR Green PCR Master Mix (2X), 0.25 mM of each primer (12S-F:5′-TAGAGGAGCCTGTTCTGTAATCGA-3′ and 12S-R:5′-TGCGCTTACTTTGTAGCCTTCAT-3′) and 4 µl of nuclease free H2O for a final volume of 20 µl.
Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited disorders, although mechanisms regulating mother-to-offspring transmission have not yet been elucidated. To investigate if mtDNA mutations affect embryonic development, we compared morphology, viability and mtDNA content in control (n = 165) and mitochondrial (n = 16) human embryos at the cleavage-stage. mtDNA copy number (CN) was assessed in one or two embryonic cells, by real-time PCR. The presence of a maternal or embryonic mtDNA mutation did not impact on either embryonic quality or viability. mtDNA CN was not altered by mtDNA mutations, suggesting that mtDNA defects do not modify mtDNA metabolism at this early stage.
A retrospective study on the efficacy of prenatal diagnosis for pregnancies at risk of mitochondrial DNA disorders
2021, Genetics in Medicine
Prenatal diagnosis of mitochondrial DNA (mtDNA) disorders is challenging due to potential instability of fetal mutant loads and paucity of data connecting prenatal mutant loads to postnatal observations. Retrospective study of our prenatal cohort aims to examine the efficacy of prenatal diagnosis to improve counseling and reproductive options for those with pregnancies at risk of mtDNA disorders.
We report on a retrospective review of 20 years of prenatal diagnosis of pathogenic mtDNA variants in 80 pregnant women and 120 fetuses.
Patients with undetectable pathogenic variants (n = 29) consistently had fetuses free of variants, while heteroplasmic women (n = 51) were very likely to transmit their variant (57/78 fetuses, 73%). In the latter case, 26 pregnancies were terminated because fetal mutant loads were >40%. Of the 84 children born, 27 were heteroplasmic (mutant load <65%). To date, no medical problems related to mitochondrial dysfunction have been reported.
Placental heterogeneity of mutant loads questioned the reliability of chorionic villous testing. Fetal mutant load stability, however, suggests the reliability of a single analysis of amniotic fluid at any stage of pregnancy for prenatal diagnosis of mtDNA disorders. Mutant loads under 40% reliably predict lack of symptoms in the progeny of heteroplasmic women.
Are ATPase6 polymorphisms associated with osteosarcoma?
2014, Experimental and Molecular Pathology

View all citing articles on Scopus

^☆: Abbreviations: COX, cytochrome oxidase; FBSN, familial bilateral striatal necrosis; LHON, Leber's hereditary optic neuropathy; MELAS, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; MERRF, myoclonus epilepsy with ragged red fibers; MICM, maternally-inherited cardiomyopathy; MILS, maternally-inherited Leigh syndrome; mtDNA, mitochondrial DNA; NARP, neuropathy, ataxia and retinitis pigmentosa; PCR, polymerase chain reaction; PEO, progressive external ophthalmoplegia

¹: To whom correspondence should be addressed at INSERM U393, Tour Lavoisier, Hôpital des Enfants-Malades, 149 rue de Sèvres, 75743 Paris Cedex 15, France. FAX: (33) 1 47 34 85 14. E-mail:[email protected].

View full text

Regular ArticleCoamplification of Nuclear Pseudogenes and Assessment of Heteroplasmy of Mitochondrial DNA Mutations☆

Abstract

Gene

Clin. Chim. Acta

Annu. Rev. Biochem.

J. Clin. Invest.

Regular Article
Coamplification of Nuclear Pseudogenes and Assessment of Heteroplasmy of Mitochondrial DNA Mutations☆