A fertilin-derived peptide improves in vitro maturation and ploidy of human oocytes.

OBJECTIVE: To analyze the effect of a cyclic fertilin-derived peptide (cFEE) on in vitro maturation of human oocytes. DESIGN: Randomized study. SETTING: Fertility center in an academic hospital. PATIENT(S): Not applicable. INTERVENTION(S): Human immature germinal vesicle-stage oocytes (n = 1,629) donated for research according to French bioethics laws were randomly allocated to groups treated with 1 or 100 µM of cFEE or to a control group. They were incubated at 37 °C in 6% CO2 and 5% O2, and their maturation was assessed using time-lapse microscopy over 24 hours. In vitro maturated metaphase II oocytes were analyzed for chromosomal content using microarray comparative genomic hybridization, and their transcriptomes were analyzed using Affymetrix Clariom D microarrays. MAIN OUTCOME MEASURE(S): The percentage of oocytes undergoing maturation in vitro was observed. Aneuploidy and euploidy were assessed for all chromosomes, and differential gene expression was analyzed in oocytes treated with cFEE compared with the control to obtain insights into its mechanism of action. RESULT(S): cFEE significantly increased the percentage of oocytes that matured in vitro and improved euploidy in meiosis II oocytes by the up-regulation of FMN1 and FLNA genes, both of which encode proteins involved in spindle structure. CONCLUSION(S): cFEE improves human oocyte maturation in vitro and reduces aneuploidy. It may prove useful for treating oocytes before fertilization in assisted reproductive technology and for in vitro maturation in fertility preservation programs to improve oocyte quality and the chances for infertile couples to conceive.

Cyclic fertilin-derived peptide stimulates in vitro human embryo development.

OBJECTIVE: To study the cyclic fertilin peptide effects on preimplantation human embryogenesis. Cyclic fertilin peptide reproduces the structure of the binding site of the sperm Fertilin ß (also named A Disintegrin and Metalloprotease 2: ADAM2) disintegrin domain. It binds to the oocyte membrane and increases sperm-oocyte fusion index in human and fertilization rate in mouse, providing healthy pups. It also improves human oocyte maturation and chromosome segregation in meiosis I and binds to human embryo blastomeres, suggesting that it has a membrane receptor. DESIGN: Thawed human embryos at the 3 to 4 cells stage were randomly included in a dose-response study with cyclic fertilin peptide. Inner cell mass (ICM), trophectoderm (TE), and total cell numbers were evaluated in top- and good-quality blastocysts. SETTING: The study was performed in an academic hospital and research laboratory. PATIENT(S): Human embryos donated for research. This project was approved by the French "Agence de la Biomédecine." INTERVENTION(S): Immunofluorescence and tissue-specific gene expression analysis, using Clariom D microarrays, were performed to study its mechanism of action. MAIN OUTCOME MEASURE(S): Cyclic fertilin peptide improves blastocyst formation by almost 20%, the concentration of 1 µM being the lowest most efficient concentration. It significantly increases twice the TE cell number, without modifying the ICM. It increases the in vitro hatching rate from 14% to 45%. RESULT(S): Cyclic fertilin peptide stimulates TE growth. In the ICM, it induces transcriptional activation of intracellular protein and vesicle-mediated transport. CONCLUSION(S): Cyclic fertilin peptide dramatically improves human embryo development potential. It could be used to supplement culture medium and improve the in vitro human embryo development. Starting supplementation immediately after fertilization, instead of day 2, could significantly upgrade assisted reproductive technology outcome.

Small-molecule flunarizine increases SMN protein in nuclear Cajal bodies and motor function in a mouse model of spinal muscular atrophy.

The hereditary neurodegenerative disorder spinal muscular atrophy (SMA) is characterized by the loss of spinal cord motor neurons and skeletal muscle atrophy. SMA is caused by mutations of the survival motor neuron (SMN) gene leading to a decrease in SMN protein levels. The SMN deficiency alters nuclear body formation and whether it can contribute to the disease remains unclear. Here we screen a series of small-molecules on SMA patient fibroblasts and identify flunarizine that accumulates SMN into Cajal bodies, the nuclear bodies important for the spliceosomal small nuclear RNA (snRNA)-ribonucleoprotein biogenesis. Using histochemistry, real-time RT-PCR and behavioural analyses in a mouse model of SMA, we show that along with the accumulation of SMN into Cajal bodies of spinal cord motor neurons, flunarizine treatment modulates the relative abundance of specific spliceosomal snRNAs in a tissue-dependent manner and can improve the synaptic connections and survival of spinal cord motor neurons. The treatment also protects skeletal muscles from cell death and atrophy, raises the neuromuscular junction maturation and prolongs life span by as much as 40 percent (p < 0.001). Our findings provide a functional link between flunarizine and SMA pathology, highlighting the potential benefits of flunarizine in a novel therapeutic perspective against neurodegenerative diseases.

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.

A role for protein phosphatase PP1γ in SMN complex formation and subnuclear localization to Cajal bodies.

The spinal muscular atrophy (SMA) gene product SMN forms with gem-associated protein 2-8 (Gemin2-8) and unrip (also known as STRAP) the ubiquitous survival motor neuron (SMN) complex, which is required for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), their nuclear import and their localization to subnuclear domain Cajal bodies (CBs). The concentration of the SMN complex and snRNPs in CBs is reduced upon SMN deficiency in SMA cells. Subcellular localization of the SMN complex is regulated in a phosphorylation-dependent manner and the precise mechanisms remain poorly understood. Using co-immunoprecipitation in HeLa cell extracts and in vitro protein binding assays, we show here that the SMN complex and its component Gemin8 interact directly with protein phosphatase PP1γ. Overexpression of Gemin8 in cells increases the number of CBs and results in targeting of PP1γ to CBs. Moreover, depletion of PP1γ by RNA interference enhances the localization of the SMN complex and snRNPs to CBs. Consequently, the interaction between SMN and Gemin8 increases in cytoplasmic and nuclear extracts of PP1γ-depleted cells. Two-dimensional protein gel electrophoresis revealed that SMN is hyperphosphorylated in nuclear extracts of PP1γ-depleted cells and expression of PP1γ restores these isoforms. Notably, SMN deficiency in SMA leads to the aberrant subcellular localization of Gemin8 and PP1γ in the atrophic skeletal muscles, suggesting that the function of PP1γ is likely to be affected in disease. Our findings reveal a role of PP1γ in the formation of the SMN complex and the maintenance of CB integrity. Finally, we propose Gemin8 interaction with PP1γ as a target for therapeutic intervention in SMA.

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.

[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

[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).

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.

The loss of the snoRNP chaperone Nopp140 from Cajal bodies of patient fibroblasts correlates with the severity of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common autosomal recessive neurodegenerative disease caused by reduced survival motor neuron (SMN) levels. The assembly machinery containing SMN is implicated in the biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN is present in both the cytoplasm and nucleus, where it transiently accumulates in subnuclear domains named Cajal bodies (CBs) and functions in the maturation of snRNPs and small nucleolar (sno)RNPs. The impact of lowering SMN levels on the composition of CBs in SMA cells is still not completely understood. Here, we analyse the CB composition in immortalized and primary fibroblasts from SMA patients. We show that the U snRNA export factors PHAX and chromosome region maintenance 1 and the box C/D snoRNP core protein fibrillarin concentrate in CBs from SMA cells, whereas the box H/ACA core proteins GAR1 and NAP57/dyskerin show reduced CB localization. Remarkably, the functional deficiency in SMA cells is associated with decreased localization of the snoRNP chaperone Nopp140 in CBs that correlates with disease severity. Indeed, RNA interference knockdown experiments in control fibroblasts demonstrate that SMN is required for accumulation of Nopp140 in CBs. Conversely, overexpression of SMN in SMA cells restores the CB localization of Nopp140, whereas SMN mutants found in SMA patients are defective in promoting the association of Nopp140 with CBs. Taken together, we demonstrate that only a subset of CB functions (as indicated by the association of representative factors) are impaired in SMA cells and, importantly, we identify the decrease of Nopp140 localization in CBs as a phenotypic marker for SMA.

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 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.

Molecular analysis of SMA patients without homozygous SMN1 deletions using a new strategy for identification of SMN1 subtle mutations.

Spinal muscular atrophy (SMA) is a common autosomal recessive disease. SMA is linked to the 5q13 locus in 95% of patients, and in at least 98% of them, the SMN1 homozygous deletion is found. Compound heterozygous patients, who have an SMN1 deletion associated with a subtle mutation, appear undeleted with the common molecular diagnostic test that detects only the homozygous absence of SMN1. In these patients, mutation screening in SMN1 is hampered by the presence of several copies of the highly homologous SMN2 gene. Here, we present a rapid and reliable strategy for detecting SMN mutations using long-range PCR, which avoids cloning and cDNA analysis. Using this method, we found 10 mutations, including five mutations never reported previously and five recurrent mutations; some of them are probably population-specific. Marker analysis of the 5q13 locus in these mutations showed common haplotypes, supporting the hypothesis of a common ancestor rather than a hot spot sequence. We also evaluate the suitability of automated SSCA and DHPLC for mutation scanning.

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.

Mapping of autosomal recessive chronic distal spinal muscular atrophy to chromosome 11q13.

Distal spinal muscular atrophy is a heterogeneous group of neuromuscular disorders caused by progressive anterior horn cell degeneration and characterized by progressive motor weakness and muscular atrophy, predominantly in the distal parts of the limbs. Here we report on chronic autosomal recessive distal spinal muscular atrophy in a large, inbred family with onset at various ages. Because this condition had some of the same clinical features as spinal muscular atrophy with respiratory distress, we tested the disease gene for linkage to chromosome 11q and mapped the disease locus to chromosome 11q13 in the genetic interval that included the spinal muscular atrophy with respiratory distress gene (D11S1889-D11S1321, Z(max) = 4.59 at theta = 0 at locus D11S4136). The sequencing of IGHMBP2, the human homologue of the mouse neuromuscular degeneration gene (nmd) that accounts for spinal muscular atrophy with respiratory distress, failed to detect any mutation in our chronic distal spinal muscular atrophy patients, suggesting that spinal muscular atrophy with respiratory distress and chronic distal spinal muscular atrophy are caused by distinct genes located in the same chromosomal region. In addition, the high intrafamilial variability in age at onset raises the question of whether nonallelic modifying genes could be involved in chronic distal spinal muscular atrophy.

A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by the loss of functional survival motor neuron 1 (SMN1) protein. This ubiquitously expressed protein is a component of a novel complex immunodetected in both the cytoplasm and the nucleus, which is associated with complexes involved in mRNA splicing, ribosome biogenesis and transcription. Here, we study a mutant protein corresponding to the N-terminal half of the protein that is encoded by the SMA frameshift mutation SMN 472del5. We show by confocal microscopy that the resulting mutant protein exhibits various distribution patterns in different transiently transfected COS cells. The mutant distributes into the nucleoplasm and/or the nucleolus, whereas the normal SMN protein accumulates at discrete nucleocytoplasmic dot-like structures previously named gems/Cajal bodies. The cell population with the nucleolar distribution is enriched upon treatment with mimosine, a synchronizing drug in late G(1) phase. Co-immunoprecipitation studies carried out on nuclear extracts reveal that both the endogenous SMN and mutant proteins are associated with complexes containing two major non-ribosomal nucleolar proteins, namely nucleolin and protein B23, and that the association is mediated, by among other things, RNA moieties. Both the association of the SMN protein with nucleolin-containing complexes and the nucleolin/B23 complex are disrupted in fibroblasts derived from a type I SMA patient harboring a homozygous SMN1 gene deletion. These findings suggest that altered assembly and/or stability of ribonucleoprotein complexes may contribute to the pathophysiological processes in SMA.