Limitations of Preimplantation Genetic Diagnosis for Mitochondrial DNA Diseases

Steffann et al., 2014Steffann J. Gigarel N. Samuels D.C. Monnot S. Borghese R. Hesters L. Frydman N. Burlet P. Frydman R. Benachi A. et al.Cell Rep. 2014; 7 (this issue): 933-934Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar highlight significant differences in predicting the transmission of mitochondrial DNA (mtDNA) mutations by preimplantation genetic diagnosis (PGD) in human and rhesus macaque embryos. We previously demonstrated considerable segregation of mtDNA variants between daughter blastomeres within a monkey 8-cell embryo, implying that sampling and analyzing of one or two blastomeres may not be fully predictive of total mutation load in the remaining embryo. Moreover, monkey offspring produced from heteroplasmic embryos were nearly homoplasmic, suggesting that mtDNA mutation levels may increase drastically due to a genetic bottleneck (Lee et al., 2012Lee H.S. Ma H. Juanes R.C. Tachibana M. Sparman M. Woodward J. Ramsey C. Xu J. Kang E.J. Amato P. et al.Cell Rep. 2012; 1: 506-515Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The authors reviewed several studies describing mtDNA mutation distribution among blastomeres in preimplantation human embryos. Analysis of two biopsied blastomeres from each embryo demonstrated comparable mtDNA mutation levels. Thus, the authors argue that PGD is a highly reliable approach to selecting embryos with sufficiently low mtDNA mutation levels for transfer. As an explanation for differences seen between nonhuman primate and human embryos, the authors point out that our rhesus monkey study is based on an “artificial” mixture of two mtDNA haplotypes in unfertilized oocytes, whereas heteroplasmy in human embryos occurs “naturally.” As we discussed in our study, we cannot exclude that such an extreme case of heteroplasmy involving mitochondrial sequence polymorphisms between two distant genomes could have contributed to the segregation patterns seen in monkey. However, differences in the specific molecular techniques used to measure mtDNA heteroplasmy levels in human and monkey studies may also have affected the interpretation of results. In particular, our study used ARMS-qPCR, a highly sensitive quantitative assay that allows accurate assessment of heteroplasmy load at a single-cell level (Lee et al., 2012Lee H.S. Ma H. Juanes R.C. Tachibana M. Sparman M. Woodward J. Ramsey C. Xu J. Kang E.J. Amato P. et al.Cell Rep. 2012; 1: 506-515Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In contrast, the human studies were based on semiquantitative fluorescent PCR followed by restriction enzyme digestion, which is associated with higher technical errors in heteroplasmy assessment due to heteroduplex formation or incomplete digestion (Monnot et al., 2011Monnot S. Gigarel N. Samuels D.C. Burlet P. Hesters L. Frydman N. Frydman R. Kerbrat V. Funalot B. Martinovic J. et al.Hum. Mutat. 2011; 32: 116-125Crossref PubMed Scopus (89) Google Scholar). Regardless of the technical validity of mtDNA mutation assessment, the ultimate validation of PGD success is the birth of healthy children with very low or undetectable mtDNA mutation load. The authors cite live births of four children who were conceived by IVF with embryo selection post-PGD in families carrying mtDNA mutations (Monnot et al., 2011Monnot S. Gigarel N. Samuels D.C. Burlet P. Hesters L. Frydman N. Frydman R. Kerbrat V. Funalot B. Martinovic J. et al.Hum. Mutat. 2011; 32: 116-125Crossref PubMed Scopus (89) Google Scholar, Steffann et al., 2006Steffann J. Frydman N. Gigarel N. Burlet P. Ray P.F. Fanchin R. Feyereisen E. Kerbrat V. Tachdjian G. Bonnefont J.P. et al.J. Med. Genet. 2006; 43: 244-247Crossref PubMed Scopus (110) Google Scholar, Treff et al., 2012Treff N.R. Campos J. Tao X. Levy B. Ferry K.M. Scott Jr., R.T. Fertil. Steril. 2012; 98: 1236-1240Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Although very limited clinical follow-up was reported in these cases, it is on the basis of these studies that Steffann et al. conclude that PGD is a reliable technique for lowering or completely eliminating the maternal transmission of mtDNA mutations to children. One of these four reported PGD cases involved a family affected with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome due to the common m.3243A>G mutation in mtDNA (Treff et al., 2012Treff N.R. Campos J. Tao X. Levy B. Ferry K.M. Scott Jr., R.T. Fertil. Steril. 2012; 98: 1236-1240Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). After two IVF cycles and trophectoderm biopsy, a male embryo with the lowest mutation load (12%) was identified and transferred, resulting in viable pregnancy. Although the authors’ report implied that the baby was healthy at birth and unlikely to manifest symptoms of MELAS later in life, the actual clinical details of that case were significantly more complicated. Specifically, the infant was delivered pre-term at 34 3/7 weeks’ gestation, weighing 1,890 g due to severe preeclampsia with placental atresia. The child then presented to the Mitochondrial-Genetics Clinic at The Children’s Hospital of Philadelphia with multiple medical problems that included a cyanotic spell associated with hypoxemia at 1 month of age, recurrent hypoglycemia (53–64 mg/dl) that self-resolved over time, recurrent hospitalizations for infections with prolonged recovery and associated dehydration, a prolonged period of projectile vomiting associated with C. difficile infection, a prolonged febrile seizure, gastresophageal reflux disease, mild developmental delays, and behavioral problems including mild hyperactivity, difficulty calming, frequent temper tantrums, head tapping, head banging on the wall and floor, and spinning. Upon physical exams at 6 weeks and 18 months of age, he was found to have decreased pupil photoreactivity and several minor dysmorphic features, including relative macroscaphocephaly, mildly coarse facies, thickened superior ear helix, epicanthal folds, short sublingual frenulum, mild fifth finger clinodactyly, and small umbilical hernia. His brain MRI at age 15 months revealed prominence of the ventricular system and extra-axial cerebrospinal fluid; prominent Virchow-Robin spaces; pineal cyst; increased FLAIR signal involving the posterior periventricular white matter, suggestive of incomplete myelination; and multiple areas of scattered punctate signal abnormality, most extensively in the cerebellum, right temporal-occipital border, and right occipital lobe, that were indicative of prior intracranial and intraventricular hemorrhage. Metabolic studies of his blood and urine identified intermittent mild lactic acidemia and an elevated ratio of lactate to pyruvate—including an occurrence at the time of an intercurrent febrile illness (Table S1)—generalized aminoaciduria, and elevated triglycerides (245 mg/dl; normal, < 125). Multiple genetic studies did not reveal chromosomal copy-number aberrations or nuclear gene mutations. However, ARMS-qPCR analysis performed by a CLIA-certified mitochondrial genetics diagnostics laboratory on samples collected from the boy at the ages of 6 weeks and 18 months demonstrated mutant m.3243A>G heteroplasmy loads of 47% and 46% in blood and 52% and 42% in urine, respectively (Table S1). It is not clear whether this child’s complex neurologic, developmental, and multisystem problems relate to his m.3243A>G mutation, the biopsy itself, or other aspects of the IVF procedure. While some of the child’s features are neither specific nor typical for MELAS, they could potentially relate to problems experienced in the perinatal course, such as preeclampsia and intrauterine growth restriction, which are known complications of the m.3243A>G mutation. Studies on the correlation between m.3243A>G mutation and clinical manifestations suggest that a 50% heteroplasmy level is on the borderline of where phenotypic expression of MELAS is expected (Jeppesen et al., 2006Jeppesen T.D. Schwartz M. Frederiksen A.L. Wibrand F. Olsen D.B. Vissing J. Arch. Neurol. 2006; 63: 1701-1706Crossref PubMed Scopus (69) Google Scholar). Regardless, careful clinical follow-up throughout the patient’s life is indicated. This case highlights that important uncertainties remain in predicting “safe” mtDNA mutation levels sufficient to assure long-term health in children born after PGD. A phenomenon that might be relevant to his outcome is the existence of the mtDNA bottleneck that occurs during the early peri-implantation period, suggesting that mutation levels in liveborn children may change significantly compared to heteroplasmy levels determined in preimplantation embryos. As recognized above, another potential problem may relate to limitations of the technical accuracy of the specific molecular assays implemented for quantification of mtDNA mutation levels at a single-cell level in biopsied preimplantation embryos. Although PGD appeared more successful in predicting mtDNA mutation levels in the other cases of three liveborn children cited by Steffann et al., additional limitations of this method must be considered (Wallace and Chalkia, 2013Wallace D.C. Chalkia D. Cold Spring Harb. Perspect. Biol. 2013; 5: a021220Crossref Scopus (251) Google Scholar). A major concern is that PGD simply selects for the embryo having the lowest heteroplasmy level and may reduce, but is unlikely to eliminate, the risk of transmitting mtDNA mutations. Additionally, women may not produce oocytes, and hence embryos, with levels of mutant mtDNA that are low enough to be acceptable for transfer. No exact recommendations exist for the determination of an acceptable heteroplasmy level that may be “safe” to select, because this likely depends on the mutation type, specific disease manifestations, and family history (Poulton and Bredenoord, 2010Poulton, J., and Bredenoord, A.L. (2010). 174th ENMC international workshop: Applying pre-implantation genetic diagnosis to mtDNA diseases: Implications of scientific advances. 19–21 March, 2010. Naarden, The Netherlands. Neuromuscul Disord. 20, 559–563.Google Scholar). Given the possibility of random and rapid changes in mtDNA heteroplasmy, any level of mutant mtDNA present in embryos could lead to clinical disease that presents either in childhood or later in life and is more likely to result in maternal transmission of disease in future generations. A recently reported model of mtDNA heteroplasmy inheritance predicts that transfer of an embryo having a mutation level above 5% will have a significant chance of disease recurrence (Samuels et al., 2013Samuels D.C. Wonnapinij P. Chinnery P.F. Hum. Reprod. 2013; 28: 554-559Crossref PubMed Scopus (28) Google Scholar). Therefore, many families now request transfers of embryos with a mutation threshold of 5% or less (Sallevelt et al., 2013Sallevelt S.C. Dreesen J.C. Drüsedau M. Spierts S. Coonen E. van Tienen F.H. van Golde R.J. de Coo I.F. Geraedts J.P. de Die-Smulders C.E. Smeets H.J. J. Med. Genet. 2013; 50: 125-132Crossref PubMed Scopus (66) Google Scholar), although these may not be present in a given IVF cycle. Another important PGD-related consideration is that at least two cells are removed from the 8-cell embryo, which significantly reduces its developmental potential and chance of developing into a viable pregnancy and a healthy child. For example, nine IVF/PGD cycles that were initiated in four families having mtDNA mutations resulted in just one viable pregnancy (Sallevelt et al., 2013Sallevelt S.C. Dreesen J.C. Drüsedau M. Spierts S. Coonen E. van Tienen F.H. van Golde R.J. de Coo I.F. Geraedts J.P. de Die-Smulders C.E. Smeets H.J. J. Med. Genet. 2013; 50: 125-132Crossref PubMed Scopus (66) Google Scholar). Families with known mtDNA mutations who are searching for reproductive options will likely be better served by emerging methodologies that do not just select, but actively eliminate, mutant mtDNA in oocytes. In particular, spindle transfer (Lee et al., 2012Lee H.S. Ma H. Juanes R.C. Tachibana M. Sparman M. Woodward J. Ramsey C. Xu J. Kang E.J. Amato P. et al.Cell Rep. 2012; 1: 506-515Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, Tachibana et al., 2013Tachibana M. Amato P. Sparman M. Woodward J. Sanchis D.M. Ma H. Gutierrez N.M. Tippner-Hedges R. Kang E. Lee H.S. et al.Nature. 2013; 493: 627-631Crossref PubMed Scopus (302) Google Scholar) results in < 1% mutant mtDNA carryover to the resulting embryo. This reproductive technique will likely provide a more reliable and effective medical alternative to classical PGD for mtDNA disease. Until then, the current ambiguities of PGD outcomes for families with mtDNA mutations will require careful evaluation. Further applications should be carried out as a part of clinical trials under appropriate regulatory oversight, to ensure that parents are fully informed about the experimental nature and high risk of mtDNA mutation transmission to offspring. Additionally, it is critical that all children born after PGD are carefully followed long-term to monitor any changes in mtDNA mutation levels and associated clinical symptoms. Given the unpredictable nature of changes in mtDNA heteroplasmy that occur during development, further translational research investigations in relevant animal models are warranted. Research in rhesus macaques has provided a particularly valuable preclinical experimental system in which to better understand mechanisms of mtDNA inheritance and to develop novel treatment options for the prevention of intergenerational transmission of mtDNA diseases (Lee et al., 2012Lee H.S. Ma H. Juanes R.C. Tachibana M. Sparman M. Woodward J. Ramsey C. Xu J. Kang E.J. Amato P. et al.Cell Rep. 2012; 1: 506-515Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, Tachibana et al., 2013Tachibana M. Amato P. Sparman M. Woodward J. Sanchis D.M. Ma H. Gutierrez N.M. Tippner-Hedges R. Kang E. Lee H.S. et al.Nature. 2013; 493: 627-631Crossref PubMed Scopus (302) Google Scholar). We also highlight a need for the development of additional animal models of naturally arising pathogenic mtDNA mutations that will be essential for a better understanding of human biology and disease. We hope that the work of Steffann et al. and our response will contribute to a clearer understanding of the complexities in predicting mtDNA disease transmission and serve as a useful platform to stimulate further discussions. This work was supported by National Institutes of Health grants R01-HD063276, R01-HD057121, R01-HD059946, R01-EY021214, P51-OD011092 (S.M.), and R03-DK082446 (M.J.F.), as well as a grant from the Leducq Foundation (S.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Download .pdf (.07 MB) Help with pdf files Table S1. Tissue-Specific m.3243A>G Mutation Load and Blood Lactate and Pyruvate Levels of a Boy Born Following PGD Selection to a Mother with MELAS