A retrospective study on the efficacy of prenatal diagnosis for pregnancies at risk of mitochondrial DNA disorders.

PURPOSE: 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. METHODS: We report on a retrospective review of 20 years of prenatal diagnosis of pathogenic mtDNA variants in 80 pregnant women and 120 fetuses. RESULTS: 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. CONCLUSION: 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.

OTC deficiency in females: Phenotype-genotype correlation based on a 130-family cohort.

OTC deficiency, an inherited urea cycle disorder, is caused by mutations in the X-linked OTC gene. Phenotype-genotype correlations are well understood in males but still poorly known in females. Taking advantage of a cohort of 130 families (289 females), we assessed the relative contribution of OTC enzyme activity, X chromosome inactivation, and OTC gene sequencing to genetic counseling in heterozygous females. Twenty two percent of the heterozygous females were clinically affected, with episodic (11%), chronic (7.5%), or neonatal forms of the disease (3.5%). Overall mortality rate was 4%. OTC activity, ranging from 0% to 60%, did not correlate with phenotype at the individual level. Analysis of multiple samples from 4 mutant livers showed intra-hepatic variability of OTC activity and X inactivation profile (range of variability: 30% and 20%, respectively) without correlation between both parameters for 3 of the 4 livers. Ninety disease-causing variants were found, 27 of which were novel. Mutations were classified as "mild" or "severe," based on male phenotypes and/or in silico prediction. In our cohort, a serious disease occurred in 32% of females with a severe mutation, compared to 4% in females with a mild mutation (odds ratio = 1.365; P = 1.6e-06). These data should help prenatal diagnosis for heterozygous females and genetic counseling after fortuitous findings of OTC variants in pangenomic sequencing.

Segregation of mitochondrial DNA mutations in the human placenta: implication for prenatal diagnosis of mtDNA disorders.

BACKGROUND: Mitochondrial DNA (mtDNA) disorders have a high clinical variability, mainly explained by variation of the mutant load across tissues. The high recurrence risk of these serious diseases commonly results in requests from at-risk couples for prenatal diagnosis (PND), based on determination of the mutant load on a chorionic villous sample (CVS). Such procedures are hampered by the lack of data regarding mtDNA segregation in the placenta.The objectives of this report were to determine whether mutant loads (1) are homogeneously distributed across the whole placentas, (2) correlate with those in amniocytes and cord blood cells and (3) correlate with the mtDNA copy number. METHODS: We collected 11 whole placentas carrying various mtDNA mutations (m.3243A>G, m.8344A>G, m.8993T>G, m.9185T>C and m.10197G>A) and, when possible, corresponding amniotic fluid samples (AFSs) and cord blood samples. We measured mutant loads in multiple samples from each placenta (n= 6-37), amniocytes and cord blood cells, as well as total mtDNA content in placenta samples. RESULTS: Load distribution was homogeneous at the sample level when average mutant load was low (<20%) or high (>80%) at the whole placenta level. By contrast, a marked heterogeneity was observed (up to 43%) in the intermediate range (20%-80%), the closer it was to 40%-50% the mutant load, the wider the distribution. Mutant loads were found to be similar in amniocytes and cord blood cells, at variance with placenta samples. mtDNA content correlated to mutant load in m.3243A>G placentas only. CONCLUSION: These data indicate that (1) mutant load determined from CVS has to be interpreted with caution for PND of some mtDNA disorders and should be associated with/substituted by a mutant load measurement on amniocytes; (2) the m.3243A>G mutation behaves differently from other mtDNA mutations with respect to the impact on mtDNA copy number, as previously shown in human preimplantation embryogenesis.

Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis.

Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans.

Poor correlations in the levels of pathogenic mitochondrial DNA mutations in polar bodies versus oocytes and blastomeres in humans.

Because the mtDNA amount remains stable in the early embryo until uterine implantation, early human development is completely dependent on the mtDNA pool of the mature oocyte. Both quantitative and qualitative mtDNA defects therefore may negatively impact oocyte competence or early embryonic development. However, nothing is known about segregation of mutant and wild-type mtDNA molecules during human meiosis. To investigate this point, we compared the mutant levels in 51 first polar bodies (PBs) and their counterpart (oocytes, blastomeres, or whole embryos), at risk of having (1) the "MELAS" m.3243A>G mutation in MT-TL1 (n = 30), (2) the "MERRF" m.8344A>G mutation in MT-TK (n = 15), and (3) the m.9185T>G mutation located in MT-ATP6 (n = 6). Seven out of 51 of the PBs were mutation free and had homoplasmic wild-type counterparts. In the heteroplasmic PBs, measurement of the mutant load was a rough estimate of the counterpart mutation level (R(2) = 0.52), and high mutant-load differentials between the two populations were occasionally observed (ranging from -34% to +34%). The mutant-load differentials between the PB and its counterpart were higher in highly mutated PBs, suggestive of a selection process acting against highly mutated cells during gametogenesis or early embryonic development. Finally, individual discrepancies in mutant loads between PBs and their counterparts make PB-based preconception diagnosis unreliable for the prevention of mtDNA disorder transmission. Such differences were not observed in animal models, and they emphasize the need to conduct thorough studies on mtDNA segregation in humans.

Segregation of mtDNA throughout human embryofetal development: m.3243A>G as a model system.

Mitochondrial DNA (mtDNA) mutations cause a wide range of serious diseases with high transmission risk and maternal inheritance. Tissue heterogeneity of the heteroplasmy rate ("mutant load") accounts for the wide phenotypic spectrum observed in carriers. Owing to the absence of therapy, couples at risk to transmit such disorders commonly ask for prenatal (PND) or preimplantation diagnosis (PGD). The lack of data regarding heteroplasmy distribution throughout intrauterine development, however, hampers the implementation of such procedures. We tracked the segregation of the m.3243A>G mutation (MT-TL1 gene) responsible for the MELAS syndrome in the developing embryo/fetus, using tissues and cells from eight carrier females, their 38 embryos and 12 fetuses. Mutant mtDNA segregation was found to be governed by random genetic drift, during oogenesis and somatic tissue development. The size of the bottleneck operating for m.3243A>G during oogenesis was shown to be individual-dependent. Comparison with data we achieved for the m.8993T>G mutation (MT-ATP6 gene), responsible for the NARP/Leigh syndrome, indicates that these mutations differentially influence mtDNA segregation during oogenesis, while their impact is similar in developing somatic tissues. These data have major consequences for PND and PGD procedures in mtDNA inherited disorders.

[Extending preimplantation genetic diagnosis to HLA typing: the French exception]. / Le diagnostic pré-implantatoire couplé au typage HLA: l'expérience française.

Umut-Talha, a "sibling savior", was born on 26 January 2011 at Beclère Hospital after embryo selection at the Paris preimplantation genetic diagnosis (PGD) center. His birth revived the controversy over "double PGD". This procedure, authorized in France since 2006, allows couples who already have a child with a serious, incurable genetic disease, to opt for PGD in order to select a healthy embryo that is HLA-matched to the affected sibling and who may thus serve as an ombilical cord blood donor. The procedure is particularly complex and the baby take-home rate is still very low. Double PGD is strictly regulated in France, and candidate couples must first receive individual authorization from the Biomedicine Agency. In our experience, these couples have a strong desire to have children, as reflected by the large number of prior spontaneous pregnancies (25% of couples). Likewise, most of these couples request embryo transfer even when there is no HLA-matched embryo, which accounts for more than half of embryo transfers. The controversy surrounding this practice has flared up again in recent weeks, over the concepts of "designer babies" and "double savior siblings" (the baby is selected to be free of the hereditary disease, and may also serve as a stem cell donor for the affected sibling).

[Ten years' experience of preimplantation genetic diagnosis in Paris: remaining obstacles and potential solutions]. / Diagnostic pré-implantatoire: dix ans d'expérience en région parisienne: impasse actuelle et solutions à venir.

Preimplantation genetic diagnosis (PGD) has been authorized in France since 1999. Encouraging results have been obtained during the past 10 years in our Paris center, where 832 patients have undergone 1056 IVF-PGD procedures. With the advent of new techniques for the identification of genetic disease markers, our center can now offer PGD procedures for aneuploidy and 75 single-gene diseases. New indications for PGD have also been developed, such as mitochondrial DNA diseases, amyloid neuropathy, pulmonary arterial hypertension, and HLA typing The implantation rate is currently 29,6% and, by 31 December 2009, 151 healthy babies had been born. Unfortunately, demand for PGD procedures far outstrips available technical capacity, and the waiting period is longer than 18 months. Increased funding is urgently needed

Mitochondrial DNA mutations do not impact early human embryonic development.

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.

Comprehensive survey of mutations in RP2 and RPGR in patients affected with distinct retinal dystrophies: genotype-phenotype correlations and impact on genetic counseling.

X-linked forms of retinitis pigmentosa (RP) (XLRP) account for 10 to 20% of families with RP and are mainly accounted for by mutations in the RP2 or RP GTPase regulator (RPGR) genes. We report the screening of these genes in a cohort of 127 French family comprising: 1) 93 familial cases of RP suggesting X-linked inheritance, including 48 out of 93 families with expression in females but no male to male transmission; 2) seven male sibships of RP; 3) 25 sporadic male cases of RP; and 4) two cone dystrophies (COD). A total of 5 out of the 93 RP families excluded linkage to the RP2 and RP3 loci and were removed form the cohort. A total of 14 RP2 mutations, 12 of which are novel, were identified in 14 out of 88 familial cases of RP and 1 out of 25 sporadic male case (4%). In 13 out of 14 of the familial cases, no expression of the disease was noted in females, while in 1 out of 14 families one woman developed RP in the third decade. A total of 42 RPGR mutations, 26 of which were novel, were identified in 80 families, including: 69 out of 88 familial cases (78.4%); 2 out of 7 male sibship (28.6%); 8 out of 25 sporadic male cases (32.0%); and 1 out of 2 COD. No expression of the disease was noted in females in 41 out of 69 familial cases (59.4%), while at least one severely affected woman was recognized in 28 out of 69 families (40.6%). The frequency of RP2 and RPGR mutations in familial cases of RP suggestive of X-linked transmission are in accordance to that reported elsewhere (RP2: 15.9% vs. 6-20%; RPGR: 78.4% vs. 55-90%). Interestingly, about 30% of male sporadic cases and 30% of male sibships of RP carried RP2 or RPGR mutations, confirming the pertinence of the genetic screening of XLRP genes in male patients affected with RP commencing in the first decade and leading to profound visual impairment before the age of 30 years.

NDP gene mutations in 14 French families with Norrie disease.

Norrie disease is a rare X-inked recessive condition characterized by congenital blindness and occasionally deafness and mental retardation in males. This disease has been ascribed to mutations in the NDP gene on chromosome Xp11.1. Previous investigations of the NDP gene have identified largely sixty disease-causing sequence variants. Here, we report on ten different NDP gene allelic variants in fourteen of a series of 21 families fulfilling inclusion criteria. Two alterations were intragenic deletions and eight were nucleotide substitutions or splicing variants, six of them being hitherto unreported, namely c.112C>T (p.Arg38Cys), c.129C>G (p.His43Gln), c.133G>A (p.Val45Met), c.268C>T (p.Arg90Cys), c.382T>C (p.Cys128Arg), c.23479-1G>C (unknown). No NDP gene sequence variant was found in seven of the 21 families. This observation raises the issue of misdiagnosis, phenocopies, or existence of other X-linked or autosomal genes, the mutations of which would mimic the Norrie disease phenotype.

Osteopathia striata cranial sclerosis: non-random X-inactivation suggestive of X-linked dominant inheritance.

Osteopathia striata with cranial sclerosis (OS-CS) is a rare syndrome comprising macrocephaly, minor anomalies, conductive hearing loss, and mild mental retardation. The diagnosis is based on radiological findings, including cranial sclerosis and longitudinal striations of metaphyses of long bones. Here we report on 10 new cases of OS-CS, including two sporadic cases and three families, with an excess of affected females (9F/1M). Phenotypic variability was observed in our patients as well as several unusual findings. Hirschsprung disease, Pierre Robin sequence, coronal craniostenosis, and laryngotracheomalacia were associated with a poor prognosis. The X-inactivation pattern of peripheral blood lymphocytes in a mildly affected mother and her severely affected boy demonstrated a non-random X-inactivation in the mother. This finding, in combination with a sex ratio in favor of females and an increased morbidity and mortality in males, is highly suggestive of X-linked dominant inheritance.

Single-sperm analysis for recurrence risk assessment of spinal muscular atrophy.

With the detection of a homozygous deletion of the survival motor neuron 1 gene (SMN1), prenatal and preimplantation genetic diagnosis (PGD) for spinal muscular atrophy has become feasible and widely applied. The finding of a de novo rearrangement, resulting in the loss of the SMN1 gene, reduces the recurrence risk from 25% to a lower percentage, the residual risk arising from recurrent de novo mutation or germline mosaicism. In a couple referred to our PGD center because their first child was affected with SMA, the male partner was shown to carry two SMN1 copies. An analysis of the SMN1 gene and two flanking markers was performed on 12 single spermatozoa, to determine whether the father carried a CIS duplication of the SMN1 gene on one chromosome and was a carrier, or if the deletion has occurred de novo. We showed that all spermatozoa that were carriers of the 'at-risk haplotype' were deleted for the SMN1 gene, confirming the carrier status of the father. We provide an original application of single germ cell studies to recessive disorders using coamplification of the gene and its linked markers. This efficient and easy procedure might be useful to elucidate complex genetic situations when samples from other family members are not available.

Single cell quantification of the 8993T>G NARP mitochondrial DNA mutation by fluorescent PCR.

When a mitochondrial DNA (mtDNA) mutation is identified, the reliable and sensitive quantification of the mutation load is a prerequisite for evaluating the feasibility of prenatal/pregestational diagnosis of the disease. We have developed a quantification assay of the 8993T>G NARP mutation using semi-quantitative fluorescent PCR. The test was reproducible and the experimental values were linear even at extremely low concentrations of mutant mtDNA molecules, making quantification of the mutant load in individual cells feasible (including blastomeres). Studying single circulating lymphocytes from a single NARP 8993T>G patient, we found a broad distribution of the disease causing mutation (0-44%) supporting the remarkable variability of heteroplasmy at the cellular level. This observation and the experimental approach reported here should be relevant to either prenatal or preimplantation diagnosis.

Improved single-cell protocol for preimplantation genetic diagnosis of spinal muscular atrophy.

OBJECTIVE: To develop and validate a simple and reliable single-cell analysis protocol for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA). DESIGN: Molecular tests based on specific enzymatic digestion have already been described for SMA diagnosis. We modified the amplified DNA fragments so as to introduce a novel restriction site that provides an internal control for the completeness of the digestion. SETTING: The genetics and reproduction departments of two teaching hospitals. PATIENT(S): Six informed couples at risk of transmitting SMA. INTERVENTION(S): All patients underwent standard procedures associated with intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Improvement of SMA diagnostic efficiency and accuracy on single cell. RESULT(S): One hundred fifty lymphocytes were analyzed with our protocol. One hundred percent diagnostic accuracy was achieved from both homozygous normal and SMN1-deleted leukocytes. Successful molecular analysis was achieved for 36 of 42 biopsied embryos (86%). Twenty-five normal embryos were transferred, but no pregnancy was achieved. CONCLUSION(S): We developed an improved protocol for PGD of SMA that is simple, robust, and accurate; unfortunately, no pregnancies were achieved for any of the six patients who have undergone PGD in the program thus far.

Single cell co-amplification of polymorphic markers for the indirect preimplantation genetic diagnosis of hemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti loci on Xq28.

Preimplantation genetic diagnosis (PGD) first consisted of the selection of female embryos for patients at risk of transmitting X-linked recessive diseases. Advances in molecular biology now allow the specific diagnosis of almost any Mendelian disease. For families with an identified X-linked recessive disease-causing mutation, non-specific diagnosis by sex identification can be considered as a sub-standard method, since it involves the unnecessary disposal of healthy male embryos and reduces success rate by diminishing the pool of embryos eligible for transfer. The most telomeric part of the X-chromosome long arm is a highly gene-rich region encompassing disease genes such as haemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti. We developed five single-cell triplex amplification protocols with microsatellite markers DXS1073, DXS9901 (BGN), G6PD, DXS1108, DXS8087 and F8C-IVS13 located in this Xq terminal region. These tests allow the diagnosis of all diseases previously mentioned providing that the genetic material allowing the identification of the morbid allele can be obtained. The choice of the microsatellite set to use depends on the localisation of the gene responsible for the diagnosed pathology and on the informativity of the markers in particular families. Single-cell amplification efficiency was assessed on single lymphocytes. Amplification rate of the different markers ranged from 89-97% with an allele drop out rate of 2-19%. So far PGD has been carried out for three carrier females at risk of transmitting X-linked adrenoleukodystrophy, X-linked hydrocephalus and hemophilia A. The latter one is now pregnant.