X-chromosome inactivation in female patients with Fabry disease.

Fabry disease (FD) is an X-linked genetic disorder caused by the deficient activity of lysosomal α-galactosidase (α-Gal). While males are usually severely affected, clinical presentation in female patients may be more variable ranging from asymptomatic to, occasionally, as severely affected as male patients. The aim of this study was to evaluate the existence of skewed X-chromosome inactivation (XCI) in females with FD, its concordance between tissues, and its contribution to the phenotype. Fifty-six females with FD were enrolled. Clinical and biological work-up included two global scores [Mainz Severity Score Index (MSSI) and DS3], cardiac magnetic resonance imaging, measured glomerular filtration rate, and measurement of α-Gal activity. XCI was analyzed in four tissues using DNA methylation studies. Skewed XCI was found in 29% of the study population. A correlation was found in XCI patterns between blood and the other analyzed tissues although some punctual variability was detected. Significant differences in residual α-Gal levels, severity scores, progression of cardiomyopathy and deterioration of kidney function, depending on the direction and degree of skewing of XCI were evidenced. XCI significantly impacts the phenotype and natural history of FD in females.

Inhibition of human placental endothelial cell proliferation and angiogenesis by netrin-4.

INTRODUCTION: Netrin-4 is a secreted member of the laminin-related protein family, known to be involved in axonal guidance and endothelial cell survival, proliferation, and migration. We have recently reported the cellular localization of netrin-4 and its receptor neogenin in human first trimester and term placenta. A strong expression of netrin-4 was observed in trophoblast and in endothelial cells, suggesting a potential role of this protein in placental angiogenesis. In relation to human pregnancy, it has been reported that circulating netrin-4 were increased in fetal umbilical cord blood of intrauterine growth restriction IUGR compared to normal pregnancy suggesting an adverse effect of this protein on placental and fetal development. The aim of this study was to determine the role of netrin-4 in placental angiogenesis. METHODS: The effects of netrin-4 on proliferation, migration, tube-like organization, and spheroid sprouting of human placental microvascular endothelial cells (HPEC) were studied. RESULTS: We demonstrated that netrin-4 inhibits HPEC proliferation, tube-like formation, migration and spheroid sprouting, suggesting a direct role of netrin-4 in the regulation of intra-villus angiogenesis. DISCUSSION: This is the first report of an anti-angiogenic activity of netrin-4 in human placenta. This study brings new insights into netrin-4 roles in placental angiogenesis and suggests possible involvements of netrin-4 in angiogenesis-related pathologies such as IUGR.

EG-VEGF controls placental growth and survival in normal and pathological pregnancies: case of fetal growth restriction (FGR).

Identifiable causes of fetal growth restriction (FGR) account for 30 % of cases, but the remainders are idiopathic and are frequently associated with placental dysfunction. We have shown that the angiogenic factor endocrine gland-derived VEGF (EG-VEGF) and its receptors, prokineticin receptor 1 (PROKR1) and 2, (1) are abundantly expressed in human placenta, (2) are up-regulated by hypoxia, (3) control trophoblast invasion, and that EG-VEGF circulating levels are the highest during the first trimester of pregnancy, the period of important placental growth. These findings suggest that EG-VEGF/PROKR1 and 2 might be involved in normal and FGR placental development. To test this hypothesis, we used placental explants, primary trophoblast cultures, and placental and serum samples collected from FGR and age-matched control women. Our results show that (1) EG-VEGF increases trophoblast proliferation ([(3)H]-thymidine incorporation and Ki67-staining) via the homeobox-gene, HLX (2) the proliferative effect involves PROKR1 but not PROKR2, (3) EG-VEGF does not affect syncytium formation (measurement of syncytin 1 and 2 and ß hCG production) (4) EG-VEGF increases the vascularization of the placental villi and insures their survival, (5) EG-VEGF, PROKR1, and PROKR2 mRNA and protein levels are significantly elevated in FGR placentas, and (6) EG-VEGF circulating levels are significantly higher in FGR patients. Altogether, our results identify EG-VEGF as a new placental growth factor acting during the first trimester of pregnancy, established its mechanism of action, and provide evidence for its deregulation in FGR. We propose that EG-VEGF/PROKR1 and 2 increases occur in FGR as a compensatory mechanism to insure proper pregnancy progress.

Estrogen-related receptor gamma modulates energy metabolism target genes in human trophoblast.

Placenta growth and functions depend on correct trophoblast migration, proliferation, and differentiation. The placenta has a critical role in gas and nutrient transport. To accomplish these numerous functions, the placenta depends on a highly efficient energy metabolism control. Recent studies showed that the orphan nuclear receptor Estrogen-Related Receptor gamma (ERRγ) is highly expressed in human placentas. As ERRγ has been described as a major energy metabolism regulator, we investigated ERRγ expression and putative roles on energy homeostasis in human trophoblast from first trimester placentas. First, we showed that ERRγ expression level increased during pregnancy and that ERRγ was more abundant in villous than in extravillous trophoblasts. We also observed that ERRγ expression increased during trophoblast differentiation. Second, we demonstrated that mitochondrial biogenesis and expression of some energy metabolism target genes decreased when ERRγ expression was impaired. Altogether, these results suggest that ERRγ could be implicated in the energy metabolism regulation of human trophoblasts.

Identification and localization of netrin-4 and neogenin in human first trimester and term placenta.

We describe here for the first time the characterization of family member of netrins, netrin-4 and its receptor neogenin, during the development of the placenta. By using western blots and RT-PCR, we demonstrated the presence of netrin-4 and its receptor neogenin protein as well as their transcripts. Using immunohistochemistry, we studied the distribution of netrin-4 and neogenin in both the first trimester and term placenta. We observed staining of netrin-4 in villous and extravillous cytotrophoblasts, syncytiotrophoblast, and endothelial cells whereas staining in stromal cells was faint. In decidua, we observed netrin-4 labelling in glandular epithelial cells, perivascular decidualized cells, and endothelial cells. However, neogenin was absent in villous and extravillous cytotrophoblasts and was expressed only on syncytiotrophoblast and placental stromal cells in the first trimester and at term placenta. The pattern of distribution suggests that a functional netrin-4-neogenin pathway might be restricted to syncytiotrophoblasts, mesenchymal cells, and villous endothelial cells. This pathway function might vary with its localization in the placenta. It is possibly involved in angiogenesis, morphogenesis, and differentiation.

Genome wide expression profile in human HTR-8/Svneo trophoblastic cells in response to overexpression of placental alkaline phosphatase gene.

During pregnancy, placental growth allows the adaptation of the feto-maternal unit to fetal requirements. Placental alkaline phosphatase (PLAP) is a phosphomonoesterase produced increasingly until term by the placenta and also ectopically in some tumors. To precise the role of this enzyme in the placenta, we analyzed the genome wide expression profile of HTR-8/Svneo trophoblastic cells after overexpression of the alkaline phosphatase gene (ALPP). We showed that ALPP overexpression mainly altered expression of genes implicated in cellular growth and proliferation. These results were confirmed by the study of cellular effects in HTR-8/Svneo cells overexpressing ALPP and in HTR-8/Svneo cells in which ALPP expression was suppressed by siRNA. We showed that PLAP exerts a positive effect on DNA replication and acts as a proliferative factor in trophoblastic cells.

Forensic and police identification of "X" bodies. A 6-years French experience.

The identification of X bodies is an everyday preoccupation in forensic pathology. This retrospective analysis studied all methods of identification and characteristics of unidentified bodies arrived in the Department of Forensic Medicine and Pathology (University Hospital R. Poincaré, Garches, France) during a 6-years period (2003-2009). The aim was to determine the identification methods used during all the forensic investigations, but also to study causes and manner of death in this sample of the population. A total of 9.1% of all autopsies were on X cadavers (217 cases out of 2384). On this total, only 134 of them have been included in our series after exclusion of archaeological and animal samples, but also of unidentified individuals or incomplete data available. Almost 28% of them have been identified with molecular biology (DNA), 23% with odontological examination, 7.5% with fingerprinting and 6.7% with autopsy data. Manner of death was mainly suicide (40.3%) especially by asphyxia following drowning, then accidental death (17.9%) especially consecutive to multiple trauma after traffic accident, acute carbon monoxide intoxication or carbonization in a fire. A total of 11.9% natural deaths were found (50% of them being of cardio-vascular origin) and 11.2% of homicides (with the use of firearm in a third of them). For 18.7% of X cadavers, the mode of death was undetermined. 46.4% of all unidentified bodies in our series were only identified by the police investigations, using physical recognition (direct or with photographs) or personal effects or identity documents in close relationship with the body. Our study highlights the fact that quite half of all unidentified bodies are inhumed with an identity not scientifically proved. Bodies which remained unidentified after all investigations represent 10.2% of X cadavers (if we consider a group of 176 cases composed of our study sample of 134 cases plus 24 subjects identified just before the autopsy and the 18 cases which remained unidentified) and 0.8% of all autopsies performed in the department.

A sequence variation in the promoter of the placental alkaline phosphatase gene (ALPP) is associated with allele-specific expression in human term placenta.

Placental alkaline phosphatase (PLAP), encoded by the ALPP gene, is produced by the fetal side of the placenta. This enzyme displays strong genetic variability. Some of the variants were reported to be associated with pathology of pregnancy. We show here that the two most common ALPP allelic variants, Pl(1) and Pl(2), differ in mRNA expression level. This differential expression was independent of the parental origin and probably results from linkage disequilibrium with the sequence variation rs2014683G>A in the ALPP gene promoter that was shown to have allele-specific binding patterns to placental nuclear proteins. The possible role of this allelic-specific expression in placenta-related pathology is discussed.

Thrombin generation in patients with factor XI deficiency and clinical bleeding risk.

SUMMARY: Factor XI (FXI) deficiency is a rare bleeding disorder. Most patients with FXI deficiency are mild bleeders but certain patients with similar FXI activity exhibit different bleeding phenotype. Routine laboratory assays do not help physicians to estimate the individual bleeding risk in these patients. Thrombin generation test (TGT) is a more comprehensive, global function test of the clotting system. We investigated whether or not the bleeding tendency of patients with FXI deficiency is correlated with features of the TGT. Twenty-four patients with FXI deficiency were divided in two groups: (i) severe bleeders (n = 9) and (ii) mild or non-bleeders (n = 15). All severe bleeders had a personal history of surgery-related severe bleeding. Thrombin generation (TG) was measured in platelet-rich plasma (PRP) using a low concentration of tissue factor 0.5 pm. In patients exhibiting severe bleeding tendency, independently of their FXI level, a dramatically impaired TG was observed. For example, despite a low plasma FXI = 1 IU dl(-1), a clinically non-bleeding individual exhibited normal TG results whereas another patient with severe bleeding history and FXI = 40 IU dl(-1) had a very low TG capacity. Low velocity and delayed TG were the main parameters suggesting a higher bleeding risk. DNA analysis of patients reported eight novel mutations of the FXI gene but neither mutation location nor secretion or not of the variant correlated with the bleeding tendency. The results of this study suggest that TG measurement in PRP may be a useful tool to predict bleeding risk in FXI deficiency and should be studied further in larger prospective clinical studies.

Effects of 17beta-estradiol on preadipocyte proliferation in human adipose tissue: Involvement of IGF1-R signaling.

Estrogens are known to stimulate the proliferation of human preadipocytes. However, the molecular mechanisms underlying the increased cell growth by these steroids are poorly understood. In the present study, we have demonstrated that the proliferative effect of 17beta-estradiol involves the induction of both cell cycle gene expressions, c-myc and cyclin D1. Moreover, the mitogenic effects of 17beta-estradiol are suppressed by the pure antagonist ICI 182780 suggesting that estradiol action is mediated by estrogen receptor (ER). We have also shown that 17beta-estradiol is able to inhibit human preadipocyte apoptosis capacity as reflected by DNA fragmentation experiments and the mRNA expression of the pro- and antiapoptotic genes. Finally, 17beta-estradiol significantly induces both mRNA and protein expression of RIGF1 in human preadipose cells via ER and thus reinforces the signaling pathway of the proliferative factor, IGF1. Taken together, these data reinforce the concept of cross-talk between IGF1- and ER-signaling pathways in preadipocytes and indicate that IGFI may be a critical regulator of estrogen-mediated preadipose growth.

The 'relics of Joan of Arc': a forensic multidisciplinary analysis.

Archaeological remains can provide concrete cases, making it possible to develop, refine or validate medico-legal techniques. In the case of the so-called 'Joan of Arc's relics' (a group of bone and archaeological remains known as the 'Bottle of Chinon'), 14 specialists analysed the samples such as a cadaver X of carbonised aspect: forensic anthropologist, medical examiners, pathologists, geneticists, radiologist, biochemists, palynologists, zoologist and archaeologist. Materials, methods and results of this study are presented here. This study aims to offer an exploitable methodology for the modern medico-legal cases of small quantities of human bones of carbonised aspect.

Array comparative genomic hybridization in prenatal diagnosis: another experience.

OBJECTIVES: Etiologic diagnosis of multiple congenital abnormalities (MCAs) is often lacking. Large chromosome abnormalities can be detected by conventional cytogenetic methods, but more subtle chromosome micro-rearrangements and/or de novo abnormalities require multi-FISH analysis, which is hampered by the amount of material available in prenatal testing. METHODS: We used the comparative genomic hybridization (CGH) array, Genosensor Array 300, to screen for classic microdeletion syndromes and subtelomeric rearrangements in 39 consecutive fetuses with MCAs, after termination of pregnancy, in a prospective study. Thirty-seven of them had a normal karyotype, and two had a de novo unbalanced karyotype that could not be characterized with conventional cytogenetic methods. RESULTS: Two de novo unbalanced karyotypes were characterized by array CGH, and four additional abnormalities were diagnosed: an unbalanced inherited cryptic translocation, a deletion in band 22q11.2, a 1p36 deletion, and a 6p12.1-21.2 duplication. CONCLUSION: Chromosomal imbalances were therefore detected and/or characterized in 6 of 39 (15.4%) fetuses, indicating the value of routine array CGH in cases of MCAs and in uncharacterized chromosome rearrangements. Extension to all prenatal diagnoses may be warranted when copy number variation is identified and all FISH probes are commercially available.

Partial atlE sequencing of Staphylococcus epidermidis strains from prosthetic joint infections.

Partial atlE sequencing (atlE nucleotides 2782 to 3114 [atlE(2782-3114)]) was performed in 41 Staphylococcus epidermidis isolates from prosthetic joint infections (PJIs) and 44 isolates from skin as controls. The atlE(2782-3114) allele 1 (type strain sequence) was significantly more frequent in PJI strains (38/41 versus 29/44 in controls; P = 0.0023). Most PJI strains were positive for mecA, icaA/icaD, and IS256, and most belonged to the sequence type 27 subgroup, suggesting the involvement of few related clones.

Case report: Meiotic segregation in spermatozoa of a 46,X,t(Y;10)(q11.2;p15.2) fertile translocation carrier.

Translocations involving gonosomes are frequent in azoospermic patients and sometimes in oligozoospermic ones, conditions that lead to request for assisted reproduction treatment. This study reports an unexpectedly fertile 49-year-old man bearing a de-novo translocation 46,X,t(Y;10)(q11.2;q15.2) associated with a high chromosomal risk for offspring, and referred for familial investigations after the diagnosis of an unbalanced translocation 46,XX,der(10)t(Y;10)(q11.2;p15.2) in his naturally conceived and mentally retarded daughter. Chromosome molecular investigation confirmed Y long-arm inheritance in the daughter and absence of the Yq deletion in the father. Semen analysis showed a normal sperm count associated with moderate asthenospermia and severe teratospermia. A total of 984 spermatozoa were analysed using fluorescence in-situ hybridization (FISH). Alternate segregation pattern was found in 50.31% of the spermatozoa studied. The frequencies of adjacent I, adjacent II, 3:1 segregation, and diploidy (or 4:0 segregation) were respectively 39.62, 1.63, 7.83, and 0.61%. No interchromosomal effect was observed. This patient is the first fertile man in whom the meiotic segregation pattern of a Y-autosome translocation has been analysed. The imbalance risk was close to those observed for reciprocal translocations, and emphasizes the value of FISH studies in males with a chromosomal translocation in order to provide them a personalized risk evaluation.

Gamete cytogenetic study in couples with implantation failure: aneuploidy rate is increased in both couple members.

PURPOSE: Implantation failure is known to be associated with an increased risk of aneuploidy in embryos, a situation leading to a pre-implantation genetic screening, not allowed in different countries like France. Our aim was to evaluate the gamete aneuploidy incidence in this context, using first polar body and spermatozoa aneuploidy screening. METHODS: Three groups were considered: 11 couples with pregnancy obtained after IVF for female infertility (group 1); 20 couples with pregnancy obtained after IVF for male infertility (group 2); and 35 couples with implantation failure (group 3). In group 3, 28 couples treated by ICSI volunteered for first polar body analysis (PB1). RESULTS: Spermatozoa aneuploidy rate was increased in groups 2 (1.6%) and 3 (2.1%) in comparison to group 1 (0.6%). PB1 aneuploidy rate was 35.4% in group 3. Finally, eight couples (32%) had no particular chromosomal risk in gametes, 15/25 (60%) presented an increased spermatic (>2%) or oocyte (>1/3) aneuploidy rate, and 2/25 (8%) had both. CONCLUSION: Those results confirm that implantation failure has a heterogeneous origin, that gamete chromosome abnormality rate is one of the major contributing factors, and that 1st Polar body and spermatozoa aneuploidy screening or pre-implantation genetics screening may be indicated for these couples.

A case of lethal hypophosphatasia providing new insights into the perinatal benign form of hypophosphatasia and expression of the ALPL gene.

Hypophosphatasia is a rare inherited bone disease caused by mutations in the alkaline phosphatase liver-type gene (ALPL) gene, with extensive allelic heterogeneity leading to a range of clinical phenotypes. We report here a patient who died from severe lethal hypophosphatasia, who was compound heterozygous for the mutation c.1133A>T (D361V) and the newly detected missense mutation c791A>G, and whose parents were both healthy. Because the c.1133A>T (D361V) mutation was previously reported to have a dominant-negative effect and to be responsible for the uncommon perinatal benign form of the disease, we studied the expression of the ALPL gene in this family. Analysis at the messenger RNA (mRNA) level, both quantitative and qualitative, showed that the paternal c.1133A>T (D361V) mutation was associated with over-expression of the ALPL gene and that the maternal c.791A>G mutation lead to complete skipping of exon 7. The results provide an explanation of the lethal phenotype in the patient where the two ALPL alleles are non-functional and in the asymptomatic father where over-expression of the normal allele could counteract the effect of the c.1133A>T (D361V) mutation by providing an increased level of normal mRNA. This may also explain the variable expression of hypophosphatasia observed in parents of patients with the perinatal benign form.

Fibrinogen angers with a new deletion (gamma GVYYQ 346-350) causes hypofibrinogenemia with hepatic storage.

INTRODUCTION: This study reports a family with chronically abnormal blood liver function tests (LFT) and congenital hypofibrinogenemia. The proposita had cirrhosis initially related to alcohol abuse and chronic viral hepatitis C (HCV), but abnormal LFT persisted even when alcohol intake was stopped and despite HCV treatment was efficient based on serum RNA negative testing. RESULTS: Needle biopsy specimens of the proposita and her brother showed eosinophilic intra-cytoplasmic inclusions that reacted strongly with fibrinogen antisera on direct immunofluorescence. Electron microscopic examination showed that the rough endoplasmic reticulum was filled with inclusions that consisted of densely packed, curved tubular structures arranged in a fingerprint-like pattern. Coagulation studies revealed low functional and antigenic fibrinogen concentrations suggestive of hypofibrinogenemia. Amplification and DNA sequencing showed a heterozygous deletion of the a7690 to g7704 nucleotides of the gamma chain gene in the 3'end of exon 8 (g 7690_7704del14; Genbank access M10014); this deletion encompassed the splicing site at position 7703 and predicts in a new putative consensus splicing sequence (AATGgtatgtt). RNA was extracted from a liver specimen from the proposita's brother. The cDNA obtained by reverse transcription polymerase chain reaction confirmed the usage of a newly generated donor site at position 7688 of the genomic sequence resulting in an in-frame heterozygous 5 amino acid deletion (GVYYQ 346-350; p.G372_Q376del) and that this mutation is responsible for a new splicing site at position 7688 of the genomic sequence. CONCLUSION: we suggest that the molecular defect in fibrinogen Angers results in an impaired assembly and causes defective secretion and hepatic storage of fibrinogen.