- Email: [email protected]
Polar body mutation load analysis in a patient with A3243G tRNALeu(UUR) point mutation
Mitochondrion 11 (2011) 626–629
Contents lists available at ScienceDirect
Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o
Short communication
Polar body mutation load analysis in a patient with A3243G tRNALeu(UUR) point mutation Mado Vandewoestyne a,1, Björn Heindryckx b,1, Trees Lepez a, Rudy Van Coster c, Jan Gerris b, Petra De Sutter b, Dieter Deforce a,⁎ a b c
Laboratory for Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium Department of Pediatric Neurology & Metabolism, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
a r t i c l e
i n f o
Article history: Received 30 December 2010 received in revised form 18 March 2011 accepted 29 March 2011 Available online 6 April 2011 Keywords: mtDNA heteroplasmy PCR-RFLP Mutation load Polar body analysis
a b s t r a c t Diseases associated with point mutations in the mitochondrial DNA (mtDNA) are maternally inherited. We evaluated whether pre-implantation genetic diagnosis, based on polar body mutation load detection could be used to distinguish healthy from affected oocytes. Restriction Fragment Length Polymorphism (RFLP) analysis was used and validated, to determine A3243G tRNALeu(UUR) mutation load in metaphase II oocytes and their respective first polar bodies. The results of this study show for the first time that the mutation load measured in the polar bodies correlates well with the mutation load in the respective oocytes. Therefore, human polar body analysis can be used as diagnostic tool to prevent transmission of mitochondrial disorders. © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction The mutation rate in mammalian mitochondrial DNA (mtDNA) is much higher than in nuclear DNA (Ballard and Whitlock, 2004). Pathogenic mutations in mtDNA may affect all copies of the mitochondrial genome, a state termed “homoplasmy”. However, in most cases only a proportion of the mtDNA is affected. This state is termed “heteroplasmy” (Lightowlers et al., 1997) and the ratio of wild type versus mutant mtDNA is indicated by the mutation load. In most heteroplasmic disorders, a minimal critical percentage of mutated mtDNA copies has to be present in cells or tissues before dysfunction and clinical symptoms become apparent (DiMauro and Schon, 2001), but in others the mutant load and disease phenotype correlate poorly (Poulton and Bredenoord, 2010). Because of the maternal inheritance of these mitochondrial disorders, couples at-risk look for preimplantation genetic diagnosis (PGD) (Poulton et al., 2009) aiming to select mutant-free embryos or, when these are unavailable, selecting embryos with acceptable mutation load,
⁎ Corresponding author at: Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium. Tel.: + 32 92648052; fax: + 32 92206688. E-mail addresses: [email protected] (M. Vandewoestyne), [email protected] (B. Heindryckx), [email protected] (T. Lepez), [email protected] (R. Van Coster), [email protected] (J. Gerris), [email protected] (P. De Sutter), [email protected] (D. Deforce). 1 Equal contribution.
likely to be sub-symptomatic. For the latter, the threshold mutation load depends on the specific mutation (Poulton and Bredenoord, 2010). Three possible strategies can be followed for PGD to prevent mitochondrial disorder transmission. After fertilisation is achieved, blastomere or blastocyst biopsy can be performed to determine the mutation load in the embryo. Using a heteroplasmic mouse model, it was shown that mutation load of individual blastomeres is similar, with a maximal difference of 6% between individual blastomeres (Dean et al., 2003). Nevertheless, heteroplasmic mouse models might differ from humans with single point mutations and could give rise to a different segregation pattern during early embryo development. In this respect, Monnot et al. recently showed that the mutation load is comparable between blastomeres of a single human embryo, also with a maximal difference of 6% between blastomeres in patients with an A3243G mutation (Monnot et al., 2011). One embryo was cultured up to the blastocyst stage followed by PGD for trophectoderm cells. The mutation load of the trophectoderm (day 5) was equivalent to the mutation load in day 3 blastomeres. PGD by blastomere biopsy has also been successfully used to prevent transmission of mtDNA disease in patients with NARP (T8993G) mutation on a limited number of analysed embryos, resulting in the birth of an unaffected child (Steffann et al., 2006). The latter was recently successfully reproduced for the same mutation by Thorburn et al. (2009). For PGD on embryonic level, both the blastomere and blastocyst approach necessitate the creation of embryos which implies ethical concerns. Furthermore, two blastomeres are commonly analysed when PGD for mtDNA mutations is performed, reducing the viability of the
1567-7249/$ – see front matter © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2011.03.123
M. Vandewoestyne et al. / Mitochondrion 11 (2011) 626–629
embryo (Marchington et al., 2009) and its embryonic developmental potential up to the blastocyst stage (Goossens et al., 2008). The third strategy, analysis of the first polar body, can be performed before fertilisation without damaging the oocyte and without the need of fertilisation of unsuitable oocytes when these oocytes would be cryopreserved. Recent studies have shown that vitrification of human oocytes does not impede pregnancy rates (Nagy et al., 2009). In addition, PGD on the first polar body could be combined with PGD on the second polar body and/or on one blastomere to increase the reliability of PGD. Heteroplasmic mouse models have indicated a positive correlation of mtDNA heteroplasmy level between oocytes and polar bodies (Dean et al., 2003). This study aims to prove that this also holds true in human and that analysis of the first polar body can be used for PGD. A protocol for A3243G mutation load quantification was developed. After validation, this protocol was used to assess the mutation load in single peripheral blood mononuclear cells (PBMCs) isolated from five patients. Finally, the correlation between the mutation load in the ooplasm of mature oocytes and their respective first polar bodies was analysed in one patient with known A3243G mutation. 2. Materials and methods 2.1. Patients Blood samples from five patients with a known A3243G mutation were collected by venous puncture in EDTA sample tubes. Additionally, seven mature oocytes and their respective first polar body and one immature oocyte were collected from one patient (patient 2). All samples were obtained after approval of the Ethical Committee of the Ghent University Hospital after written informed consent of the patients. 2.2. Preparation of the dilution series for validation of the developed protocol A dilution series (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%) was prepared using DNA extracted from PBMCs from a healthy volunteer (mutation load 0%) and from transmitochondrial cybrid cells (Jahangir Tafrechi et al., 2007) with a mutation load of nearly 100% (kindly supplied by Prof. A. K. Raap, Department of Molecular Cell Biology, Leiden University, The Netherlands). These dilutions with known mutation load were used to optimise and validate the protocol. All samples were run in triplicate and the mean mutation load was calculated from two measurements for each of these analyses (the ratios for 6-FAM (6-carboxyfluorescein) labelled fragments and for NED (7′,8′-benzo-5′-fluoro-2′,4,7-trichloro-5-carboxyfluorescein) labelled fragments, see polymerase chain reaction). To exclude the possibility of contamination, negative controls (containing no DNA) were used. 2.3. Isolation of single peripheral blood mononuclear cells (PBMCs) PBMCs were isolated from the patient's EDTA blood samples by density gradient centrifugation on Ficoll-Paque Plus (GE Healthcare, Diegem, Belgium) according to manufacturer's instructions. From each patient sample, 200,000 PBMCs were cytospun on a silanized slide (Dako, Glostrup, Denmark) by centrifugation (5 min at 400 ×g, Rotofix 32A, Hettich, Tuttlingen, Germany). The slides were air dried for 10 min and fixed for one minute in a 70% ethanol solution (absolute ethanol, Merck BV, Schiphol-Rijk, The Netherlands). Single cells were isolated by laser pressure catapulting (LPC) using a PALM MicroBeam laser microdissection system (PALM/Zeiss, Munich, Germany) as described earlier (Vandewoestyne et al., 2009a,b). Briefly, the high energy generated by the focused laser light was used to catapult the single cells into the cap of a standard 0.2 ml microfuge tube (Westburg, Leusden, The Netherlands) containing 10 μl of PicoPure DNA extraction buffer (PicoPure DNA extraction kit, Arcturus, Mountain View, CA, USA). DNA was extracted
627
from the catapulted cells, using the PicoPure DNA extraction kit (Arcturus). The samples were incubated at 65 °C for 3 h, centrifuged briefly and heated to 95 °C for 10 min to inactivate proteinase K. 2.4. Isolation of oocytes and polar bodies Stimulation was performed with the short gonadotrophin-releasing hormone agonist protocol for 7 days (Decapeptyl®, Ipsen, Paris, France) and recombinant follicle-stimulating hormone (Gonal-F®, MerckSerono, Geneva, Switzerland) until human chorionic gonadotrophin administration (Pregnyl®, Organon, Oss, The Netherlands) when at least half of the follicles were 18–20 mm in diameter. Cumulus-oocyte complexes were collected by ultrasound-guided transvaginal aspiration 35 h post-hCG. About 3 h after retrieval, oocytes were denuded by hyaluronidase and mechanical pipetting and assessed for nuclear maturation: (i) GV stage with an intact germinal vesicle (GV); (ii) metaphase I (MI) stage showing absence of a germinal vesicle or 1st polar body (PB) and (iii) MII stage with 1st PB. Denuded oocytes were maintained in Cook Fertilisation Medium (Cook Ireland Ltd, Ireland) at 37 °C in 6% CO2 and air atmosphere until further processed. For PB biopsy, an opening was made in the zona pellucida by laser pulses (Zilos-tk, Alere, Tilburg, The Netherlands) with the 1st PB located at 12 o'clock. The PB was gently aspirated into a 20 μm biopsy pipette and put separately from the oocyte. Subsequently oocytes and polar bodies were aspirated into a fine glass needle and transferred into a standard 0.2 ml microfuge tube (Westburg) containing 10 μl of PicoPure DNA extraction buffer (PicoPure DNA extraction kit). DNA extraction was performed as described for the single PBMCs. 2.5. Polymerase chain reaction (PCR) The mtDNA was amplified using a 6-FAM labelled forward primer (5′-CCCACACCCACCCAAGAACA-3′) which anneals from location 3204 to 3223 on the mtDNA and a NED labelled backward primer (5′-TGGCCATGGGTATGTTGTTA-3′) which anneals from location 3300 to 3319 (Applied Biosystems, Foster City, CA, USA). PCR was performed in an end volume of 20 μl containing 10 μl of DNA extract and 10 μl of reaction mix. The reaction mix consisted of 0.5 μM forward primer, 0.5 μM backward primer, 200 μM GeneAmp 10 mM dNTP mix with dTTP (Applied Biosystems), Gene Amp 10× PCR buffer (Applied Biosystems), 0.3 μg/μl Bovine Serum Albumin (BSA) (Sigma Aldrich, Bornem, Belgium), 0.5 mM MgCl2 (Qiagen, Germantown, MD, USA) and 1.3 U Hotstar Taq DNA polymerase (Qiagen). The conditions used to amplify the 116-bp fragment from the single cells were as follows: a 15 min hot-start at 95 °C, followed by a varying amount of (see further) PCR cycles at 95 °C for 1 min, 63 °C for 1 min, and 72 °C for 80 s, followed by one final elongation step at 72 °C for 10 min. As heteroduplex formation may lead to an underestimation of the mutation load (Jahangir Tafrechi et al., 2007), we checked at how many PCR cycles this phenomenon starts to occur and made sure to use less PCR cycles to measure the mutation load. For the dilution series, 24 to 32 cycles were used to amplify the DNA. For the PBMCs, oocytes and polar bodies 32, 20 and 30 PCR cycles were used respectively. 2.6. Restriction enzyme digestion and capillary electrophoresis 5 μl of ApaI restriction enzyme mix was added to the PCR product. The restriction mix was composed of 0.05 μl ApaI (80 U/μl, Promega Corporation, Madison, WI, USA), 0.5 μl BSA (Promega), 0.5 μl 10× buffer A (Promega) and 4 μl water. Digestion was performed at 37 °C for 4 h. To ensure proper digestion of all amplicons, another 5 μl of ApaI restriction enzyme mix was added to the PCR product and digestion was performed for another 4 h. The amplified fragments were separated and analysed by capillary electrophoresis using an ABI 3100 Genetic Analyzer (Applied Biosystems).
628
M. Vandewoestyne et al. / Mitochondrion 11 (2011) 626–629
2.7. Mutation load calculation
Table 2 Detection of mutation load in PBMCs of 5 patients with known A3243G mutation.
After PCR, a double stranded 116-bp fragment labelled with 6-FAM as well as with NED was generated. Digestion with the restriction enzyme ApaI cleaved the mutant amplicon into a 6-FAM labelled 41-bp fragment and a NED labelled 75-bp fragment, whilst the wild-type amplicon remained undigested. After capillary electrophoresis, the mutation load was calculated by dividing the peak area of the 6-FAM 41-bp fragment or the NED 75-bp fragment by the sum of this peak area and the peak area of the corresponding 116-bp fragment.
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Mean mutation load in peripheral blood (%) Detected mutation load on single cells (%)
3. Results 3.1. Validation of the developed protocol The developed protocol was validated by determining the mutation load of artificial mixtures with known levels of heteroplasmy. All measurements were performed in triplicate. A mean mutation load was calculated from the two measurements for each of these analyses (the ratios for 6-FAM labelled fragments and for NED labelled fragments, see polymerase chain reaction). The mean and standard deviation of these 6 values are presented in Table 1. For mixtures containing less than 10% of mutated DNA fragments, the standard deviation is 0.59% or less. For mixtures containing 10% or more mutated DNA fragments, the standard deviation is higher, but does not exceed 2.52%, except for the 70% mixture where the standard deviation is 6.41% due to an outlier in the measurements. Omission of this outlier results in a standard deviation of 2.18%. Contamination was excluded by the use of negative controls, containing no template DNA. 3.2. Mutation load in single peripheral blood mononuclear cells (PBMCs) After validation of this procedure, we isolated single PBMCs from five patients with known A3243G point mutation in their mtDNA. From each patient 20 PBMCs were isolated by LPC. The detected mutation load ranged between 0 and 94.7%. The results are shown in Table 2. Patients one to four all possessed mutation positive as well as negative cells. For the fifth patient, an asymptomatic patient with a known mutation load of 3.6% in the blood, only one cell showed the presence of the A3243G point mutation. This single PBMC had a mutation load of 91.7%. All other cells were negative for the point mutation. Considering the enormous difference between the mutation load of this single cell and the mean mutation load in the blood, Table 1 Validation of the mutation load detection protocol. Input mutation load (%)
Mean measured mutation load (%)
Standard deviation on the mean measured mutation load (%)
0 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 ~ 100
0.00 0.70 1.97 2.94 4.29 5.40 6.90 7.86 9.08 9.61 12.39 22.15 37.38 46.94 55.63 63.51 71.53 82.80 87.98 99.08
0.00 0.15 0.43 0.12 0.49 0.09 0.53 0.34 0.59 0.40 1.45 2.52 2.10 1.31 2.17 0.84 6.41 1.86 2.38 1.32
Average mutation load of the 20 single cells (%)
12.7
50
50
44
3.6
0 83.0 68.6 1.61 0 0 0 No result 0 0 9.5 0 0 1.9 91.4 0 0 0.8 0.4 1.0 13.6
69.3 31.2 46.7 94.7 89.2 6.8 0 No result 0 No result 30.9 59.6 0 0 71.8 0 63.5 0 0 7.6 31.7
87.5 0 0 10.6 43.4 0 0 0 1.8 36.1 0 15.7 0 3.1 50.2 8.1 9.5 60.0 43.8 0 18.5
26.0 0 52.4 22.0 0 0 0 0 26.4 0 65.1 0 0 86.6 No result No result 72.7 46.5 0 18.5 23.1
0 0 No result 0 0 0 0 0 91.7 0 0 0 0 0 0 0 0 0 0 0 4.8
another 80 single PBMCs were isolated from this patient. In total, from the 100 single PBMCs that were isolated from this patient, 90 PBMCs were negative for the point mutation and one PBMC showed no result. The mutation load detected in the remaining nine PBMCs was 91.7%, 53.0%, 3.2%, 2.6%, 2.1%, 3.1%, 36.6%, 77.6% and 2.4% respectively. The average mutation load percentages, based on the isolation of 20 single PBMCs per patient, correlate to some extent with the mean mutation load determined using standard laboratory procedures (Meulemans et al., 2007) in the peripheral blood of these patients, as shown in Table 2. Nevertheless, the amount of single PBMCs is too small to draw conclusions on the correlation with this mean mutation load in the peripheral blood. Based on the isolation of 100 PBMC, the average mutation load for the fifth patient was 2.8% (+−13.5%) which correlates well with the mean mutation load determined in the peripheral blood of this patient (3.6%). 3.3. Mutation load in mature oocytes and their respective polar bodies In total, 8 oocytes were collected, of which 7 at the MII stage and 1 GV oocyte. First PB biopsy was successful in all 7 MII oocytes despite their rather small and flattened nature and their attachment to the ooplasm. Additionally, mutation load analysis was performed in the GV oocyte that progressed to the MI stage at time of DNA extraction. The distribution of heteroplasmy could successfully be detected in every sample. The results of the mutation load determination of these 15 samples are given in Table 3. These data show a good correlation of the mutation load between the ooplasm of a mature oocyte and its corresponding first polar body. The mutation load in the oocytes ranges from 12.1% to 74.2% and in the polar bodies from 8.9% to 71.1%. Using a Wilcoxon Signed Ranks test in SPSS (non-parametric 2-tailed test for paired samples), a p-value of 0.091 was obtained. Therefore we can state that there is no significant difference between the mutation load as determined from oocytes versus their polar bodies. 4. Discussion In this study a protocol was optimised for measuring the degree of mtDNA mutation load in single cells. Validation of the developed protocol showed high accuracy over the full heteroplasmy range and
M. Vandewoestyne et al. / Mitochondrion 11 (2011) 626–629 Table 3 Detection of mutation load in seven mature (metaphase II, MII) oocytes and their respective 1st polar body and in one immature (GV) oocyte of one patient with known A3243G mutation. Stage
Mutation load (%) oocyte
Mutation load (%) corresponding first polar body
MII MII MII MII MII MII MII GV
74.2 59.2 15.4 57.0 52.4 57.7 12.1 33.5
71.1 58.6 16.6 48.0 45.0 57.9 8.9 /
high sensitivity, even in the low (b10%) heteroplasmy range. Our results show a large variation in heteroplasmy between individual PBMCs of patients carrying the A3243G mutation. Similar results have been reported for fibroblasts (Cavelier et al., 2000; Matthews et al., 1995) and lymphocytes (Monnot et al., 2011; Saitoh et al., 1999). In contradiction to Saitoh et al., several PBMCs without the A3243G mutation were detected in all five of our patients. Our data are supported by Monnot et al. (2011) who also detected mutation free PBMCs. Experiments in heteroplasmic mice suggest equal distribution of mtDNA in the first or second polar body and the respective oocyte (Dean et al., 2003). To confirm this predictive capacity in a human setting, determination of the mutation load of seven mature oocytes and their respective first polar bodies was performed by the optimised protocol described here. Our data show, for the first time, an equal distribution of mutant and wild-type mtDNA between the ooplasm and its respective polar body. Unfortunately, no mutation free oocytes could be detected. Nevertheless, the data obtained on the PBMCs of this patient suggest that mutation free oocytes could possibly be found in case more oocytes would be analysed. This hypothesis is supported by the analyses of Brown et al. (2001), who reported undetectable levels of mutant mtDNA in 9.8% of 82 primary oocytes analysed in a patient with the A3243G mutation. Of notice, this patient showed a mean mutation load of 7.24% in blood, which is much lower than the mean mutation load in blood of our patient (50%). Interestingly, analysis of seven human oocytes from a patient with a known T8993G mutation with a mean mutation load of 50% in the blood, showed six oocytes with mutation load of N95%, whilst one oocyte was mutation free (Blok et al., 1997). If no mutation free polar bodies can be detected, the main question remains which threshold mutation load would be acceptable for fertilisation of the respective oocyte. For mutations such as the T8993G and T8993C, welldocumented and predictive percentages of mutation loads exist (Poulton and Bredenoord, 2010) in contrast to the A3243G mutation of our patient. For this mtDNA point mutation, some evidence suggests that a mutation load below 30% results in healthy children (Monnot et al., 2009). In conclusion, our data clearly show that the mutation load in the first polar body can efficiently and reliably be detected and is representative for the mutation load in the oocyte, making PGD by analysis of the first polar body possible for patients with an A3243G point mutation. Acknowledgements The authors would like to thank Prof A. K. Raap for the supply of the DNA extracted from the cybrid cell lines. For assistance in oocyte and polar body collection, the authors wish to thank assistance of V. Muyshond and S. Lierman from the IVF laboratory. The Research was funded by a PhD grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders
629
(IWT-Vlaanderen) awarded to Mado Vandewoestyne and to Trees Lepez and by a grant from the Fund for Scientific Research in Flanders (FWO-Vlaanderen) awarded to the Laboratory of Pharmaceutical Biotechnology. Both Petra De Sutter and Rudy Van Coster are holders of a fundamental clinical research mandate by the Fund for Scientific Research in Flanders (FWO-Vlaanderen).
References Ballard, J.W., Whitlock, M.C., 2004. The incomplete natural history of mitochondria. Mol. Ecol. 13 (4), 729–744. Blok, R.B., Gook, D.A., Thorburn, D.R., Dahl, H.H., 1997. Skewed segregation of the mtDNA nt 8993 (T–NG) mutation in human oocytes. Am. J. Hum. Genet. 60 (6), 1495–1501. Brown, D.T., Samuels, D.C., Michael, E.M., Turnbull, D.M., Chinnery, P.F., 2001. Random genetic drift determines the level of mutant mtDNA in human primary oocytes. Am. J. Hum. Genet. 68 (2), 533–536. Cavelier, L., Johannisson, A., Gyllensten, U., 2000. Analysis of mtDNA copy number and composition of single mitochondrial particles using flow cytometry and PCR. Exp. Cell Res. 259 (1), 79–85. Dean, N.L., Battersby, B.J., Ao, A., Gosden, R.G., Tan, S.L., Shoubridge, E.A., Molnar, M.J., 2003. Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases. Mol. Hum. Reprod. 9 (10), 631–638. DiMauro, S., Schon, E.A., 2001. Mitochondrial DNA mutations in human disease. Am. J. Med. Genet. 106 (1), 18–26. Goossens, V., De Rycke, M., De Vos, A., Staessen, C., Michiels, A., Verpoest, W., Van Steirteghem, A., Bertrand, C., Liebaers, I., Devroey, P., Sermon, K., 2008. Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis. Hum. Reprod. 23 (3), 481–492. Jahangir Tafrechi, R.S., van de Rijke, F.M., Allallou, A., Larsson, C., Sloos, W.C., van de Sande, M., Wahlby, C., Janssen, G.M., Raap, A.K., 2007. Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis. J. Histochem. Cytochem. 55 (11), 1159–1166. Lightowlers, R.N., Chinnery, P.F., Turnbull, D.M., Howell, N., 1997. Mammalian mitochondrial genetics: heredity, heteroplasmy and disease. Trends Genet. 13 (11), 450–455. Marchington, D., Malik, S., Banerjee, A., Turner, K., Samuels, D., Macaulay, V., Oakeshott, P., Fratter, C., Kennedy, S., Poulton, J., 2009. Information for genetic management of mtDNA disease: sampling pathogenic mtDNA mutants in the human germline and in placenta. J. Med. Genet. 47 (4), 257–261. Matthews, P.M., Brown, R.M., Morten, K., Marchington, D., Poulton, J., Brown, G., 1995. Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA. Hum. Genet. 96 (3), 261–268. Meulemans, A., Seneca, S., Smet, J., De Paepe, B., Lissens, W., Van Coster, R., Debeer, A., De Meirleir, L., Jaeken, J., 2007. A new family with the mitochondrial tRNAGLU gene mutation m.14709TNC presenting with hydrops fetalis. Eur. J. Paediatr. Neurol. 11 (1), 17–20. Monnot, S., Gigarel, N., Hesters, L., Burlet, P., Benachi, A., Dumez, Y., Tachdjian, G., Rötig, A., Frydman, R., Munnich, A., Frydman, N., Bonnefont, J.P., Steffann, J., 2009. The challenge of prenatal and preimplantation genetic diagnosis of mitochondrial DNA disorders. European society of human genetics annual meeting; Austria, pp. 23–26. Monnot, S., Gigarel, N., Samuels, D.C., Burlet, P., Hesters, L., Frydman, N., Frydman, R., Kerbrat, V., Funalot, B., Martinovic, J., Benachi, A., Feingold, J., Munnich, A., Bonnefont, J.P., Steffann, J., 2011. Segregation of mtDNA throughout human embryofetal development: m.3243ANG as a model system. Hum. Mutat. 32 (1), 116–125. Nagy, Z.P., Chang, C.C., Shapiro, D.B., Bernal, D.P., Kort, H.I., Vajta, G., 2009. The efficacy and safety of human oocyte vitrification. Semin. Reprod. Med. 27 (6), 450–455. Poulton, J., Bredenoord, A.L., 2010. 174th ENMC international workshop: applying preimplantation genetic diagnosis to mtDNA diseases: implications of scientific advances 19–21 March 2010, Naarden, The Netherlands. Neuromuscul. Disord. 20 (8), 559–563. Poulton, J., Kennedy, S., Oakeshott, P., Wells, D., 2009. Preventing transmission of maternally inherited mitochondrial DNA diseases. Bmj. 338, b94. Saitoh, S., Momoi, M.Y., Yamagata, T., Nakauchi, H., Nihei, K., Fujii, M., 1999. Single-cell analysis of mitochondrial DNA in patients and a carrier of the tRNA(Leu)(UUR) gene mutation. J. Inherit. Metab. Dis. 22 (5), 608–614. Steffann, J., Frydman, N., Gigarel, N., Burlet, P., Ray, P.F., Fanchin, R., Feyereisen, E., Kerbrat, V., Tachdjian, G., Bonnefont, J.P., Frydman, R., Munnich, A., 2006. Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis. J. Med. Genet. 43 (3), 244–247. Thorburn, D.R., Wilton, L., Stock-Myer, S., 2009. Healthy baby girl born following preimplantation genetic diagnosis for mitochondrial DNA m.8993tNg mutation. Mol. Genet. Metab. 98, 5–6. Vandewoestyne, M., Van Hoofstat, D., Van Nieuwerburgh, F., Deforce, D., 2009a. Automatic detection of spermatozoa for laser capture microdissection. Int. J. Legal Med. 123 (2), 169–175. Vandewoestyne, M., Van Hoofstat, D., Van Nieuwerburgh, F., Deforce, D., 2009b. Suspension fluorescence in situ hybridization (S-FISH) combined with automatic detection and laser microdissection for STR profiling of male cells in male/female mixtures. Int. J. Legal Med. 123 (5), 441–447.
Contents lists available at ScienceDirect
Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o
Short communication
Polar body mutation load analysis in a patient with A3243G tRNALeu(UUR) point mutation Mado Vandewoestyne a,1, Björn Heindryckx b,1, Trees Lepez a, Rudy Van Coster c, Jan Gerris b, Petra De Sutter b, Dieter Deforce a,⁎ a b c
Laboratory for Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium Department of Pediatric Neurology & Metabolism, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
a r t i c l e
i n f o
Article history: Received 30 December 2010 received in revised form 18 March 2011 accepted 29 March 2011 Available online 6 April 2011 Keywords: mtDNA heteroplasmy PCR-RFLP Mutation load Polar body analysis
a b s t r a c t Diseases associated with point mutations in the mitochondrial DNA (mtDNA) are maternally inherited. We evaluated whether pre-implantation genetic diagnosis, based on polar body mutation load detection could be used to distinguish healthy from affected oocytes. Restriction Fragment Length Polymorphism (RFLP) analysis was used and validated, to determine A3243G tRNALeu(UUR) mutation load in metaphase II oocytes and their respective first polar bodies. The results of this study show for the first time that the mutation load measured in the polar bodies correlates well with the mutation load in the respective oocytes. Therefore, human polar body analysis can be used as diagnostic tool to prevent transmission of mitochondrial disorders. © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction The mutation rate in mammalian mitochondrial DNA (mtDNA) is much higher than in nuclear DNA (Ballard and Whitlock, 2004). Pathogenic mutations in mtDNA may affect all copies of the mitochondrial genome, a state termed “homoplasmy”. However, in most cases only a proportion of the mtDNA is affected. This state is termed “heteroplasmy” (Lightowlers et al., 1997) and the ratio of wild type versus mutant mtDNA is indicated by the mutation load. In most heteroplasmic disorders, a minimal critical percentage of mutated mtDNA copies has to be present in cells or tissues before dysfunction and clinical symptoms become apparent (DiMauro and Schon, 2001), but in others the mutant load and disease phenotype correlate poorly (Poulton and Bredenoord, 2010). Because of the maternal inheritance of these mitochondrial disorders, couples at-risk look for preimplantation genetic diagnosis (PGD) (Poulton et al., 2009) aiming to select mutant-free embryos or, when these are unavailable, selecting embryos with acceptable mutation load,
⁎ Corresponding author at: Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium. Tel.: + 32 92648052; fax: + 32 92206688. E-mail addresses: [email protected] (M. Vandewoestyne), [email protected] (B. Heindryckx), [email protected] (T. Lepez), [email protected] (R. Van Coster), [email protected] (J. Gerris), [email protected] (P. De Sutter), [email protected] (D. Deforce). 1 Equal contribution.
likely to be sub-symptomatic. For the latter, the threshold mutation load depends on the specific mutation (Poulton and Bredenoord, 2010). Three possible strategies can be followed for PGD to prevent mitochondrial disorder transmission. After fertilisation is achieved, blastomere or blastocyst biopsy can be performed to determine the mutation load in the embryo. Using a heteroplasmic mouse model, it was shown that mutation load of individual blastomeres is similar, with a maximal difference of 6% between individual blastomeres (Dean et al., 2003). Nevertheless, heteroplasmic mouse models might differ from humans with single point mutations and could give rise to a different segregation pattern during early embryo development. In this respect, Monnot et al. recently showed that the mutation load is comparable between blastomeres of a single human embryo, also with a maximal difference of 6% between blastomeres in patients with an A3243G mutation (Monnot et al., 2011). One embryo was cultured up to the blastocyst stage followed by PGD for trophectoderm cells. The mutation load of the trophectoderm (day 5) was equivalent to the mutation load in day 3 blastomeres. PGD by blastomere biopsy has also been successfully used to prevent transmission of mtDNA disease in patients with NARP (T8993G) mutation on a limited number of analysed embryos, resulting in the birth of an unaffected child (Steffann et al., 2006). The latter was recently successfully reproduced for the same mutation by Thorburn et al. (2009). For PGD on embryonic level, both the blastomere and blastocyst approach necessitate the creation of embryos which implies ethical concerns. Furthermore, two blastomeres are commonly analysed when PGD for mtDNA mutations is performed, reducing the viability of the
1567-7249/$ – see front matter © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2011.03.123
M. Vandewoestyne et al. / Mitochondrion 11 (2011) 626–629
embryo (Marchington et al., 2009) and its embryonic developmental potential up to the blastocyst stage (Goossens et al., 2008). The third strategy, analysis of the first polar body, can be performed before fertilisation without damaging the oocyte and without the need of fertilisation of unsuitable oocytes when these oocytes would be cryopreserved. Recent studies have shown that vitrification of human oocytes does not impede pregnancy rates (Nagy et al., 2009). In addition, PGD on the first polar body could be combined with PGD on the second polar body and/or on one blastomere to increase the reliability of PGD. Heteroplasmic mouse models have indicated a positive correlation of mtDNA heteroplasmy level between oocytes and polar bodies (Dean et al., 2003). This study aims to prove that this also holds true in human and that analysis of the first polar body can be used for PGD. A protocol for A3243G mutation load quantification was developed. After validation, this protocol was used to assess the mutation load in single peripheral blood mononuclear cells (PBMCs) isolated from five patients. Finally, the correlation between the mutation load in the ooplasm of mature oocytes and their respective first polar bodies was analysed in one patient with known A3243G mutation. 2. Materials and methods 2.1. Patients Blood samples from five patients with a known A3243G mutation were collected by venous puncture in EDTA sample tubes. Additionally, seven mature oocytes and their respective first polar body and one immature oocyte were collected from one patient (patient 2). All samples were obtained after approval of the Ethical Committee of the Ghent University Hospital after written informed consent of the patients. 2.2. Preparation of the dilution series for validation of the developed protocol A dilution series (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%) was prepared using DNA extracted from PBMCs from a healthy volunteer (mutation load 0%) and from transmitochondrial cybrid cells (Jahangir Tafrechi et al., 2007) with a mutation load of nearly 100% (kindly supplied by Prof. A. K. Raap, Department of Molecular Cell Biology, Leiden University, The Netherlands). These dilutions with known mutation load were used to optimise and validate the protocol. All samples were run in triplicate and the mean mutation load was calculated from two measurements for each of these analyses (the ratios for 6-FAM (6-carboxyfluorescein) labelled fragments and for NED (7′,8′-benzo-5′-fluoro-2′,4,7-trichloro-5-carboxyfluorescein) labelled fragments, see polymerase chain reaction). To exclude the possibility of contamination, negative controls (containing no DNA) were used. 2.3. Isolation of single peripheral blood mononuclear cells (PBMCs) PBMCs were isolated from the patient's EDTA blood samples by density gradient centrifugation on Ficoll-Paque Plus (GE Healthcare, Diegem, Belgium) according to manufacturer's instructions. From each patient sample, 200,000 PBMCs were cytospun on a silanized slide (Dako, Glostrup, Denmark) by centrifugation (5 min at 400 ×g, Rotofix 32A, Hettich, Tuttlingen, Germany). The slides were air dried for 10 min and fixed for one minute in a 70% ethanol solution (absolute ethanol, Merck BV, Schiphol-Rijk, The Netherlands). Single cells were isolated by laser pressure catapulting (LPC) using a PALM MicroBeam laser microdissection system (PALM/Zeiss, Munich, Germany) as described earlier (Vandewoestyne et al., 2009a,b). Briefly, the high energy generated by the focused laser light was used to catapult the single cells into the cap of a standard 0.2 ml microfuge tube (Westburg, Leusden, The Netherlands) containing 10 μl of PicoPure DNA extraction buffer (PicoPure DNA extraction kit, Arcturus, Mountain View, CA, USA). DNA was extracted
627
from the catapulted cells, using the PicoPure DNA extraction kit (Arcturus). The samples were incubated at 65 °C for 3 h, centrifuged briefly and heated to 95 °C for 10 min to inactivate proteinase K. 2.4. Isolation of oocytes and polar bodies Stimulation was performed with the short gonadotrophin-releasing hormone agonist protocol for 7 days (Decapeptyl®, Ipsen, Paris, France) and recombinant follicle-stimulating hormone (Gonal-F®, MerckSerono, Geneva, Switzerland) until human chorionic gonadotrophin administration (Pregnyl®, Organon, Oss, The Netherlands) when at least half of the follicles were 18–20 mm in diameter. Cumulus-oocyte complexes were collected by ultrasound-guided transvaginal aspiration 35 h post-hCG. About 3 h after retrieval, oocytes were denuded by hyaluronidase and mechanical pipetting and assessed for nuclear maturation: (i) GV stage with an intact germinal vesicle (GV); (ii) metaphase I (MI) stage showing absence of a germinal vesicle or 1st polar body (PB) and (iii) MII stage with 1st PB. Denuded oocytes were maintained in Cook Fertilisation Medium (Cook Ireland Ltd, Ireland) at 37 °C in 6% CO2 and air atmosphere until further processed. For PB biopsy, an opening was made in the zona pellucida by laser pulses (Zilos-tk, Alere, Tilburg, The Netherlands) with the 1st PB located at 12 o'clock. The PB was gently aspirated into a 20 μm biopsy pipette and put separately from the oocyte. Subsequently oocytes and polar bodies were aspirated into a fine glass needle and transferred into a standard 0.2 ml microfuge tube (Westburg) containing 10 μl of PicoPure DNA extraction buffer (PicoPure DNA extraction kit). DNA extraction was performed as described for the single PBMCs. 2.5. Polymerase chain reaction (PCR) The mtDNA was amplified using a 6-FAM labelled forward primer (5′-CCCACACCCACCCAAGAACA-3′) which anneals from location 3204 to 3223 on the mtDNA and a NED labelled backward primer (5′-TGGCCATGGGTATGTTGTTA-3′) which anneals from location 3300 to 3319 (Applied Biosystems, Foster City, CA, USA). PCR was performed in an end volume of 20 μl containing 10 μl of DNA extract and 10 μl of reaction mix. The reaction mix consisted of 0.5 μM forward primer, 0.5 μM backward primer, 200 μM GeneAmp 10 mM dNTP mix with dTTP (Applied Biosystems), Gene Amp 10× PCR buffer (Applied Biosystems), 0.3 μg/μl Bovine Serum Albumin (BSA) (Sigma Aldrich, Bornem, Belgium), 0.5 mM MgCl2 (Qiagen, Germantown, MD, USA) and 1.3 U Hotstar Taq DNA polymerase (Qiagen). The conditions used to amplify the 116-bp fragment from the single cells were as follows: a 15 min hot-start at 95 °C, followed by a varying amount of (see further) PCR cycles at 95 °C for 1 min, 63 °C for 1 min, and 72 °C for 80 s, followed by one final elongation step at 72 °C for 10 min. As heteroduplex formation may lead to an underestimation of the mutation load (Jahangir Tafrechi et al., 2007), we checked at how many PCR cycles this phenomenon starts to occur and made sure to use less PCR cycles to measure the mutation load. For the dilution series, 24 to 32 cycles were used to amplify the DNA. For the PBMCs, oocytes and polar bodies 32, 20 and 30 PCR cycles were used respectively. 2.6. Restriction enzyme digestion and capillary electrophoresis 5 μl of ApaI restriction enzyme mix was added to the PCR product. The restriction mix was composed of 0.05 μl ApaI (80 U/μl, Promega Corporation, Madison, WI, USA), 0.5 μl BSA (Promega), 0.5 μl 10× buffer A (Promega) and 4 μl water. Digestion was performed at 37 °C for 4 h. To ensure proper digestion of all amplicons, another 5 μl of ApaI restriction enzyme mix was added to the PCR product and digestion was performed for another 4 h. The amplified fragments were separated and analysed by capillary electrophoresis using an ABI 3100 Genetic Analyzer (Applied Biosystems).
628
M. Vandewoestyne et al. / Mitochondrion 11 (2011) 626–629
2.7. Mutation load calculation
Table 2 Detection of mutation load in PBMCs of 5 patients with known A3243G mutation.
After PCR, a double stranded 116-bp fragment labelled with 6-FAM as well as with NED was generated. Digestion with the restriction enzyme ApaI cleaved the mutant amplicon into a 6-FAM labelled 41-bp fragment and a NED labelled 75-bp fragment, whilst the wild-type amplicon remained undigested. After capillary electrophoresis, the mutation load was calculated by dividing the peak area of the 6-FAM 41-bp fragment or the NED 75-bp fragment by the sum of this peak area and the peak area of the corresponding 116-bp fragment.
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Mean mutation load in peripheral blood (%) Detected mutation load on single cells (%)
3. Results 3.1. Validation of the developed protocol The developed protocol was validated by determining the mutation load of artificial mixtures with known levels of heteroplasmy. All measurements were performed in triplicate. A mean mutation load was calculated from the two measurements for each of these analyses (the ratios for 6-FAM labelled fragments and for NED labelled fragments, see polymerase chain reaction). The mean and standard deviation of these 6 values are presented in Table 1. For mixtures containing less than 10% of mutated DNA fragments, the standard deviation is 0.59% or less. For mixtures containing 10% or more mutated DNA fragments, the standard deviation is higher, but does not exceed 2.52%, except for the 70% mixture where the standard deviation is 6.41% due to an outlier in the measurements. Omission of this outlier results in a standard deviation of 2.18%. Contamination was excluded by the use of negative controls, containing no template DNA. 3.2. Mutation load in single peripheral blood mononuclear cells (PBMCs) After validation of this procedure, we isolated single PBMCs from five patients with known A3243G point mutation in their mtDNA. From each patient 20 PBMCs were isolated by LPC. The detected mutation load ranged between 0 and 94.7%. The results are shown in Table 2. Patients one to four all possessed mutation positive as well as negative cells. For the fifth patient, an asymptomatic patient with a known mutation load of 3.6% in the blood, only one cell showed the presence of the A3243G point mutation. This single PBMC had a mutation load of 91.7%. All other cells were negative for the point mutation. Considering the enormous difference between the mutation load of this single cell and the mean mutation load in the blood, Table 1 Validation of the mutation load detection protocol. Input mutation load (%)
Mean measured mutation load (%)
Standard deviation on the mean measured mutation load (%)
0 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 ~ 100
0.00 0.70 1.97 2.94 4.29 5.40 6.90 7.86 9.08 9.61 12.39 22.15 37.38 46.94 55.63 63.51 71.53 82.80 87.98 99.08
0.00 0.15 0.43 0.12 0.49 0.09 0.53 0.34 0.59 0.40 1.45 2.52 2.10 1.31 2.17 0.84 6.41 1.86 2.38 1.32
Average mutation load of the 20 single cells (%)
12.7
50
50
44
3.6
0 83.0 68.6 1.61 0 0 0 No result 0 0 9.5 0 0 1.9 91.4 0 0 0.8 0.4 1.0 13.6
69.3 31.2 46.7 94.7 89.2 6.8 0 No result 0 No result 30.9 59.6 0 0 71.8 0 63.5 0 0 7.6 31.7
87.5 0 0 10.6 43.4 0 0 0 1.8 36.1 0 15.7 0 3.1 50.2 8.1 9.5 60.0 43.8 0 18.5
26.0 0 52.4 22.0 0 0 0 0 26.4 0 65.1 0 0 86.6 No result No result 72.7 46.5 0 18.5 23.1
0 0 No result 0 0 0 0 0 91.7 0 0 0 0 0 0 0 0 0 0 0 4.8
another 80 single PBMCs were isolated from this patient. In total, from the 100 single PBMCs that were isolated from this patient, 90 PBMCs were negative for the point mutation and one PBMC showed no result. The mutation load detected in the remaining nine PBMCs was 91.7%, 53.0%, 3.2%, 2.6%, 2.1%, 3.1%, 36.6%, 77.6% and 2.4% respectively. The average mutation load percentages, based on the isolation of 20 single PBMCs per patient, correlate to some extent with the mean mutation load determined using standard laboratory procedures (Meulemans et al., 2007) in the peripheral blood of these patients, as shown in Table 2. Nevertheless, the amount of single PBMCs is too small to draw conclusions on the correlation with this mean mutation load in the peripheral blood. Based on the isolation of 100 PBMC, the average mutation load for the fifth patient was 2.8% (+−13.5%) which correlates well with the mean mutation load determined in the peripheral blood of this patient (3.6%). 3.3. Mutation load in mature oocytes and their respective polar bodies In total, 8 oocytes were collected, of which 7 at the MII stage and 1 GV oocyte. First PB biopsy was successful in all 7 MII oocytes despite their rather small and flattened nature and their attachment to the ooplasm. Additionally, mutation load analysis was performed in the GV oocyte that progressed to the MI stage at time of DNA extraction. The distribution of heteroplasmy could successfully be detected in every sample. The results of the mutation load determination of these 15 samples are given in Table 3. These data show a good correlation of the mutation load between the ooplasm of a mature oocyte and its corresponding first polar body. The mutation load in the oocytes ranges from 12.1% to 74.2% and in the polar bodies from 8.9% to 71.1%. Using a Wilcoxon Signed Ranks test in SPSS (non-parametric 2-tailed test for paired samples), a p-value of 0.091 was obtained. Therefore we can state that there is no significant difference between the mutation load as determined from oocytes versus their polar bodies. 4. Discussion In this study a protocol was optimised for measuring the degree of mtDNA mutation load in single cells. Validation of the developed protocol showed high accuracy over the full heteroplasmy range and
M. Vandewoestyne et al. / Mitochondrion 11 (2011) 626–629 Table 3 Detection of mutation load in seven mature (metaphase II, MII) oocytes and their respective 1st polar body and in one immature (GV) oocyte of one patient with known A3243G mutation. Stage
Mutation load (%) oocyte
Mutation load (%) corresponding first polar body
MII MII MII MII MII MII MII GV
74.2 59.2 15.4 57.0 52.4 57.7 12.1 33.5
71.1 58.6 16.6 48.0 45.0 57.9 8.9 /
high sensitivity, even in the low (b10%) heteroplasmy range. Our results show a large variation in heteroplasmy between individual PBMCs of patients carrying the A3243G mutation. Similar results have been reported for fibroblasts (Cavelier et al., 2000; Matthews et al., 1995) and lymphocytes (Monnot et al., 2011; Saitoh et al., 1999). In contradiction to Saitoh et al., several PBMCs without the A3243G mutation were detected in all five of our patients. Our data are supported by Monnot et al. (2011) who also detected mutation free PBMCs. Experiments in heteroplasmic mice suggest equal distribution of mtDNA in the first or second polar body and the respective oocyte (Dean et al., 2003). To confirm this predictive capacity in a human setting, determination of the mutation load of seven mature oocytes and their respective first polar bodies was performed by the optimised protocol described here. Our data show, for the first time, an equal distribution of mutant and wild-type mtDNA between the ooplasm and its respective polar body. Unfortunately, no mutation free oocytes could be detected. Nevertheless, the data obtained on the PBMCs of this patient suggest that mutation free oocytes could possibly be found in case more oocytes would be analysed. This hypothesis is supported by the analyses of Brown et al. (2001), who reported undetectable levels of mutant mtDNA in 9.8% of 82 primary oocytes analysed in a patient with the A3243G mutation. Of notice, this patient showed a mean mutation load of 7.24% in blood, which is much lower than the mean mutation load in blood of our patient (50%). Interestingly, analysis of seven human oocytes from a patient with a known T8993G mutation with a mean mutation load of 50% in the blood, showed six oocytes with mutation load of N95%, whilst one oocyte was mutation free (Blok et al., 1997). If no mutation free polar bodies can be detected, the main question remains which threshold mutation load would be acceptable for fertilisation of the respective oocyte. For mutations such as the T8993G and T8993C, welldocumented and predictive percentages of mutation loads exist (Poulton and Bredenoord, 2010) in contrast to the A3243G mutation of our patient. For this mtDNA point mutation, some evidence suggests that a mutation load below 30% results in healthy children (Monnot et al., 2009). In conclusion, our data clearly show that the mutation load in the first polar body can efficiently and reliably be detected and is representative for the mutation load in the oocyte, making PGD by analysis of the first polar body possible for patients with an A3243G point mutation. Acknowledgements The authors would like to thank Prof A. K. Raap for the supply of the DNA extracted from the cybrid cell lines. For assistance in oocyte and polar body collection, the authors wish to thank assistance of V. Muyshond and S. Lierman from the IVF laboratory. The Research was funded by a PhD grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders
629
(IWT-Vlaanderen) awarded to Mado Vandewoestyne and to Trees Lepez and by a grant from the Fund for Scientific Research in Flanders (FWO-Vlaanderen) awarded to the Laboratory of Pharmaceutical Biotechnology. Both Petra De Sutter and Rudy Van Coster are holders of a fundamental clinical research mandate by the Fund for Scientific Research in Flanders (FWO-Vlaanderen).
References Ballard, J.W., Whitlock, M.C., 2004. The incomplete natural history of mitochondria. Mol. Ecol. 13 (4), 729–744. Blok, R.B., Gook, D.A., Thorburn, D.R., Dahl, H.H., 1997. Skewed segregation of the mtDNA nt 8993 (T–NG) mutation in human oocytes. Am. J. Hum. Genet. 60 (6), 1495–1501. Brown, D.T., Samuels, D.C., Michael, E.M., Turnbull, D.M., Chinnery, P.F., 2001. Random genetic drift determines the level of mutant mtDNA in human primary oocytes. Am. J. Hum. Genet. 68 (2), 533–536. Cavelier, L., Johannisson, A., Gyllensten, U., 2000. Analysis of mtDNA copy number and composition of single mitochondrial particles using flow cytometry and PCR. Exp. Cell Res. 259 (1), 79–85. Dean, N.L., Battersby, B.J., Ao, A., Gosden, R.G., Tan, S.L., Shoubridge, E.A., Molnar, M.J., 2003. Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases. Mol. Hum. Reprod. 9 (10), 631–638. DiMauro, S., Schon, E.A., 2001. Mitochondrial DNA mutations in human disease. Am. J. Med. Genet. 106 (1), 18–26. Goossens, V., De Rycke, M., De Vos, A., Staessen, C., Michiels, A., Verpoest, W., Van Steirteghem, A., Bertrand, C., Liebaers, I., Devroey, P., Sermon, K., 2008. Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis. Hum. Reprod. 23 (3), 481–492. Jahangir Tafrechi, R.S., van de Rijke, F.M., Allallou, A., Larsson, C., Sloos, W.C., van de Sande, M., Wahlby, C., Janssen, G.M., Raap, A.K., 2007. Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis. J. Histochem. Cytochem. 55 (11), 1159–1166. Lightowlers, R.N., Chinnery, P.F., Turnbull, D.M., Howell, N., 1997. Mammalian mitochondrial genetics: heredity, heteroplasmy and disease. Trends Genet. 13 (11), 450–455. Marchington, D., Malik, S., Banerjee, A., Turner, K., Samuels, D., Macaulay, V., Oakeshott, P., Fratter, C., Kennedy, S., Poulton, J., 2009. Information for genetic management of mtDNA disease: sampling pathogenic mtDNA mutants in the human germline and in placenta. J. Med. Genet. 47 (4), 257–261. Matthews, P.M., Brown, R.M., Morten, K., Marchington, D., Poulton, J., Brown, G., 1995. Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA. Hum. Genet. 96 (3), 261–268. Meulemans, A., Seneca, S., Smet, J., De Paepe, B., Lissens, W., Van Coster, R., Debeer, A., De Meirleir, L., Jaeken, J., 2007. A new family with the mitochondrial tRNAGLU gene mutation m.14709TNC presenting with hydrops fetalis. Eur. J. Paediatr. Neurol. 11 (1), 17–20. Monnot, S., Gigarel, N., Hesters, L., Burlet, P., Benachi, A., Dumez, Y., Tachdjian, G., Rötig, A., Frydman, R., Munnich, A., Frydman, N., Bonnefont, J.P., Steffann, J., 2009. The challenge of prenatal and preimplantation genetic diagnosis of mitochondrial DNA disorders. European society of human genetics annual meeting; Austria, pp. 23–26. Monnot, S., Gigarel, N., Samuels, D.C., Burlet, P., Hesters, L., Frydman, N., Frydman, R., Kerbrat, V., Funalot, B., Martinovic, J., Benachi, A., Feingold, J., Munnich, A., Bonnefont, J.P., Steffann, J., 2011. Segregation of mtDNA throughout human embryofetal development: m.3243ANG as a model system. Hum. Mutat. 32 (1), 116–125. Nagy, Z.P., Chang, C.C., Shapiro, D.B., Bernal, D.P., Kort, H.I., Vajta, G., 2009. The efficacy and safety of human oocyte vitrification. Semin. Reprod. Med. 27 (6), 450–455. Poulton, J., Bredenoord, A.L., 2010. 174th ENMC international workshop: applying preimplantation genetic diagnosis to mtDNA diseases: implications of scientific advances 19–21 March 2010, Naarden, The Netherlands. Neuromuscul. Disord. 20 (8), 559–563. Poulton, J., Kennedy, S., Oakeshott, P., Wells, D., 2009. Preventing transmission of maternally inherited mitochondrial DNA diseases. Bmj. 338, b94. Saitoh, S., Momoi, M.Y., Yamagata, T., Nakauchi, H., Nihei, K., Fujii, M., 1999. Single-cell analysis of mitochondrial DNA in patients and a carrier of the tRNA(Leu)(UUR) gene mutation. J. Inherit. Metab. Dis. 22 (5), 608–614. Steffann, J., Frydman, N., Gigarel, N., Burlet, P., Ray, P.F., Fanchin, R., Feyereisen, E., Kerbrat, V., Tachdjian, G., Bonnefont, J.P., Frydman, R., Munnich, A., 2006. Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis. J. Med. Genet. 43 (3), 244–247. Thorburn, D.R., Wilton, L., Stock-Myer, S., 2009. Healthy baby girl born following preimplantation genetic diagnosis for mitochondrial DNA m.8993tNg mutation. Mol. Genet. Metab. 98, 5–6. Vandewoestyne, M., Van Hoofstat, D., Van Nieuwerburgh, F., Deforce, D., 2009a. Automatic detection of spermatozoa for laser capture microdissection. Int. J. Legal Med. 123 (2), 169–175. Vandewoestyne, M., Van Hoofstat, D., Van Nieuwerburgh, F., Deforce, D., 2009b. Suspension fluorescence in situ hybridization (S-FISH) combined with automatic detection and laser microdissection for STR profiling of male cells in male/female mixtures. Int. J. Legal Med. 123 (5), 441–447.