Array CGH in patients with developmental delay or intellectual disability: are there phenotypic clues to pathogenic copy number variants?

Array comparative genomic hybridization (array CGH) is now widely adopted as a first-tier clinical diagnostic test in individuals with unexplained developmental delay/intellectual disability (DD/ID) and congenital anomalies. Our study aimed at enlarging the phenotypic spectrum associated with clinically relevant copy number variants (CNVs) as well as delineating clinical criteria, which may help separating patients with pathogenic CNVs from those without pathogenic CNVs. We performed a retrospective review of clinical and array CGH data of 342 children with unexplained DD/ID. The phenotypic features of patients with clinically significant CNV were compared with those without pathogenic CNVs. Array CGH detected pathogenic CNVs in 13.2% of the patients. Congenital anomalies, especially heart defects, as well as primary microcephaly, short stature and failure to thrive were clearly more frequent in children with pathogenic CNVs compared with children with normal array CGH results. Thus, we assume that in patients with unexplained DD/ID, array CGH will more probably detect a significant CNV if any of these features is part of the patient's phenotype.

Dramatically altered environmental lighting conditions in women with high-risk pregnancy during hospitalization.

The maternal circadian time structure is incredibly important in the entrainment and programing of the fetal and newborn circadian time structure. Natural sunlight is the primary environmental time cue for entrainment of circadian rhythms, but high-risk pregnant women spend most of their time indoors with artificial light sources and extremely low levels of natural light both during the day and night. Because the daily level, timing, duration of light exposure and its spectral properties are important in maintaining the normal circadian physiology in humans, we aimed to evaluate the environmental lighting conditions in high-risk pregnant women admitted to hospital for long-term stay. About 30 patients were included in the study. Exposed illuminance, color temperature and effective circadian radiation dose were measured and recorded every 10 s by light dosimeters attached to the patients' clothing. We documented the illuminance of 29 pregnant women on 235 inpatient days. Median (IQR) measured illuminance was 70 (28-173) lux in the morning, 124 (63-241) lux in the afternoon, 19 (6-53) lux in the evening and 0 (0-0) lux at the night. Median illuminance for the 235 inpatient days of assessment was below the recommended EU standard of 100 lux-60.5% of the mornings and 42.7% of the afternoons. The women confined to indoor locations rarely achieved an illuminances more than 300 lux in the morning and in the afternoon. Compared to women with outdoor mobility, those confined indoors have a significantly lower illuminance and color temperature, both in the morning and in the afternoon. Our study presents the first information about the dramatically altered environmental lighting conditions experienced by high-risk pregnant women during their hospital stay. Their exposure to light while in the hospital is significantly lower than exposure to natural daylight levels and below the recommended EU standard.

Alterations of human placental epidermal growth factor receptor in intrauterine growth retardation.

We studied human placental microvillous EGF receptor (EGFR) and its relationship with maternal and placental features in 14 cases of intrauterine growth retardation. Placental EGFR phosphorylation was significantly decreased or absent in 12 cases of small for gestational age neonates, as shown by SDS-PAGE, autoradiography, and scanning analysis. Specific [125I]EGF binding and Scatchard plots of the binding data showed a decreased number of EGFR in 6 of the 12 cases, with a mean maximal binding capacity of 1.09 +/- 0.32 pmol/mg for high affinity sites (mean control value = 2.30 +/- 0.23 pmol/mg). Most of the hypertensive women and smokers belonged to this subgroup. In three of the remaining six cases of small gestational age placentas with low EGFR phosphorylation, there was no maternal pathology or significant parenchymatous placental lesions. Five showed a 175-kD EGFR species when probed by [125I]EGF cross-linking and Western blotting with RK2 and C-Term, two polyclonal anti-EGFR antibodies, suggesting abnormal transduction of the EGF-induced signal. The sixth placenta yielded a single 145-kD EGFR band consistent with an abnormal EGFR structure; Western blot analysis showed no immunoreactive band. In conclusion, maternal and placental pathologies in intrauterine growth retardation are associated with various alterations of placental EGFR, pointing out the importance of EGFR ligands in the regulatory pathway of placental and fetal growth.

Parathyroid hormone increases epidermal growth factor receptors in cultured human trophoblastic cells from early and term placenta.

The effect of PTH on the epidermal growth factor (EGF) receptor was analyzed during the in vitro differentiation of human cytotrophoblasts. The cytotrophoblasts were isolated by a trypsin-DNase method from first trimester and term placentas and purified on a Percoll gradient. In culture, these cells aggregated and fused together to form a syncytium. This in vitro differentiation was associated with a 2-fold increase in 125I-EGF binding after 48 h of culture. The addition of 0.1 microM PTH (PTH-treated cells) to the culture medium induced a significant 2- to 3-fold increase (P less than 0.005) in EGF binding. The effect was dose related with a maximum obtained at a 1 nM concentration. Scatchard analyses revealed that PTH-treated cells possess a 2-fold higher number of high affinity sites as compared to control cells from early placenta (0.71 +/- 0.06 pmol/mg protein and 0.34 +/- 0.04 pmol/mg protein, respectively) and from term placenta (1.24 +/- 0.10 pmol/mg protein and 0.61 +/- 0.07 pmol/mg protein, respectively). The apparent Kd values for high affinity sites (0.15 nM) and for low affinity sites (4 nM) were not altered either by the gestational age of the cells or by PTH treatment. With respect to the EGF-dependent phosphorylation in membranes of trophoblast cells in culture, it was found that the phosphorylation of two major proteins of 175 kilodaltons and 35 kilodaltons, is greatly increased in PTH-treated cell membranes in the presence of EGF. This PTH-induced effect on EGF receptors was associated with an augmented functional response of trophoblastic cells to EGF. PTH increased the EGF-stimulated secretion of hCG. These results demonstrate that PTH increases the number of biologically active EGF receptors during the in vitro differentiation of human trophoblast cells. This PTH-induced effect suggests a role for this hormone in the regulation of the growth and the endocrine functions of these cells.

Glucose inhibits human placental GH secretion, in vitro.

Human placenta specifically expresses the GH-V gene leading to the production of placental Growth Hormone (PGH). During pregnancy, PGH levels increase progressively in maternal blood, but its regulation remains unknown. In this study the effect of glucose on PGH secretion by human term placenta was tested, in vitro, by means of two different experimental models: organ culture of villous tissue and primary culture of isolated cytotrophoblasts. PGH was assayed in the culture medium by an immunoradiometric assay using a specific PGH monoclonal antibody. The presence of glucose (25 mmol/L) in the culture medium significantly inhibited (p < 0.001) the secretion of PGH by either placental villous explants or by cultured trophoblast cells. This inhibitory effect of glucose on PGH secretion was dose-dependent. More than 50% inhibition being observed with 5.5 mmol/L. In the same conditions, the daily production of hPL and hCG, were unmodified. Furthermore, the glucose-induced inhibition of PGH secretion was more effective when cultured trophoblast cells are differentiated into syncytiotrophoblast. This study demonstrates, for the first time, that among the gestational polypeptide hormones secreted by the human placenta, only PGH secretion is modulated by glucose, suggesting a key metabolic role for this hormone during pregnancy.

Retinoid receptors expression in human term placenta: involvement of RXR alpha in retinoid induced-hCG secretion.

To investigate the role of retinoids on human placental development and functions, we characterized the spatial distribution of retinoid receptors in human term chorionic villi. In situ hybridization with 35S labeled sense and antisense probes for the RARs, alpha, beta, gamma and RXRs, alpha, beta, gamma, specifically detected only RAR alpha and RXR alpha. Both RAR alpha and RXR alpha mRNA were preferentially expressed in the trophoblast cell layer. This syncytiotrophoblast expression was confirmed by immunohistochemical analyses using anti-RAR alpha and RXR alpha antibodies. Using trophoblast cells in culture, we then studied the effect on hCG secretion of 0.1 microM RA physiological forms and of selective RAR alpha and RXR alpha synthetic agonists. Only RXR alpha specific ligands such as physiological 9-cis RA and synthetic Ro 25-7386 stimulated hCG secretion (doubled). These results suggest an important role for RXR alpha in human placental development and function.

Detection of distinct receptors for native and acetylated low-density lipoproteins in human placental microvilli by ligand-immunoblotting.

Ligand-immunoblotting was used to detect distinct receptors for native low-density lipoprotein and for acetylated low-density lipoprotein on microvillous membranes from human term placentas. Antisera directed against native and modified low-density lipoproteins were prepared in rabbits and their specificities were assessed by immunodiffusion and immunoelectrophoresis. The receptor for low-density lipoprotein was detected as a 160 kDa protein and that for acetylated low-density lipoprotein as a 200 kDa protein. These receptors were compared with their counterparts in cultured human skin fibroblasts, bovine adrenal cortex and J774 macrophage-like cells. This is the first investigation that visualizes the presence of receptors for both native and modified low-density lipoproteins in a steroidogenic tissue.

Human placental microvilli as a source of antigen for the preparation of a polyclonal antibody directed against the LDL receptor.

Polyclonal antibodies were prepared by immunization of rabbits with partially purified LDL receptor obtained from human placental microvilli. The antiserum reacted with membranes from human placental microvilli and human fibroblasts, as assessed by immunobinding studies. It also reacted with purified LDL receptors of both origins. The antiserum markedly inhibited 125I-labeled LDL binding to cultured human fibroblasts.

A one-nucleotide difference in a cAMP and phorbol ester response element leads to differential regulation of the human chorionic somatomammotropin A and B gene transcription.

Human chorionic somatomammotropin (hCS) is encoded by two highly related genes, hCS-A and hCS-B, located in the hGH/hCS gene locus. Both genes are expressed in the syncytiotrophoblast layer of the placenta and hCS release from trophoblast cells is known to be increased by cAMP and phorbol esters. However, it remains unclear whether this regulation acts at the level of hCS gene expression or secretion and whether both genes are affected. We examined the effects of cAMP and phorbol 12-myristate 13-acetate (PMA) on the transcription of the hCS-A and hCS-B genes. Transient expression experiments revealed a 7 bp cAMP- and PMA-responsive element (CRElhCS-A) spanning nucleotides-1102 to -1096 upstream of the hCS-A gene. In contrast, the homologous sequence upstream of hCS-B (CRElhCS-B), differing from CRElhCS-A by a single substitution, shows little or no response to cAMP. In band-shift assays, the CRElhCS-A oligonucleotide was shown to bind two factors related to CREBP and AP-1, whereas CRElhCS-B only competes for one of these complexes. Finally, Southwestern analysis revealed that the CRElhCS-A element binds two ubiquitous proteins of 100 kDa and 47 kDa respectively, whereas CRElhCS-B interacts only with the 47 kDa protein. Taken together, these results suggest that a 47 kDa protein binding to the CRElhCS-A and CRElhCS-B elements is involved in the PMA response of the hCS-A and hCS-B genes, and a 100 kDa protein plays a crucial role in cAMP regulation of the hCS-A gene.

Acetylated low density lipoprotein endocytosis by human syncytiotrophoblast in culture.

Low density lipoproteins (LDL) were chemically modified (acetyl LDL) and then conjugated to colloïdal gold (gold acetyl LDL), firstly, to visualize the acetyl LDL binding sites, and secondly, to demonstrate a possible internalization by human syncytiotrophoblast in culture. Cells were obtained by a trypsin DNase method followed by a Percoll gradient centrifugation. After 3 days of culture the syncytiotrophoblast characterization was performed by using ultramicroscopy, immunohistochemistry, and by studying the secretion of gestational hormones during culture. Binding experiments showed gold acetyl LDL attached to the membrane with random distribution. After incubation at 37 degrees C, gold acetyl LDL was internalized by the syncytiotrophoblast following the classical receptor mediated endocytosis process and a non-specific internalization process. These results suggest the existence in the placenta of a 'scavenger pathway' concomittant of the classical LDL internalization. This phenomenon may be related to the high amount of cholesterol required by the human placenta for its cellular growth and intensive progesterone synthesis.

Increase in expression and activity of thrombomodulin in term human syncytiotrophoblast microvilli.

A comparative study of thrombomodulin (TM), a potent natural anticoagulant, was performed in first trimester and term human placentae. Immunoreactive TM was observed on fetal vascular endothelium and syncytiotrophoblast at both gestational ages. Staining was stronger in term than in early placentae, particularly along the microvillous apical membrane of the syncytiotrophoblast. Similarly, a higher level of TM mRNA was detected by RT-PCR (P<0.02) and Northern blot analysis in extracts of whole term placentae. The localization of TM on syncytial microvilli was confirmed by electron microscopy after immunogold labelling. When isolated microvilli were compared at both gestational ages; a significant 2.3-fold increase in TM protein was observed in term microvilli as compared to first trimester microvilli by Western blot analysis (P<0.005) and ELISA (P<0.05). This higher level of TM in term microvilli was associated with an increase in its ability to activate protein C, from 3.7 +/- 1.2 to 8.7 +/- 4.2 mOD/min/microg protein +/- s.d. (P<0.01) in first trimester and term microvilli, respectively. The modulation of biologically active TM at the syncytial membrane exposed to maternal blood according to the length of gestation suggests that TM may be involved both in maternal haemostasis within the intervillous spaces, and also in the trophoblast differentiation process.

Developmental expression of Glut1 glucose transporter and c-fos genes in human placental cells.

Glut1, the brain/erythrocyte glucose transporter is one major isoform of the human placenta and displays an age-specific pattern of expression with mRNA levels five-fold higher in first trimester than in term placenta. By contrast, the mRNA level of the insulin-regulatable glucose transporter Glut4 remains at the limit of detection throughout pregnancy indicating a very low expression of this isoform in the placenta. The nuclear proto-oncogenes c-fos and c-myc were also detectable in the human placenta, but c-fos only exhibited an age-specific pattern of expression with levels higher in third trimester than in term placenta. Primary cultures of human trophoblast cells from term placenta were used to further study the expression and regulation of Glut1 and c-fos genes. Fetal calf serum rapidly and transiently (15 to 60 min) stimulated c-fos and Glut1 gene expression suggesting that both genes share similar growth factor-controlled pathways. Glucose inhibited Glut1, but not c-fos expression. An eight-fold decrease in Glut1 mRNA was observed when glucose concentration in the medium was increased from 0 to 25 mM, whereas c-fos mRNA levels remained very low. These results suggest that in the human placenta, the expression of Glut1 is specifically regulated by glucose concentration. These data demonstrate that (1) Glut1 and c-fos mRNA transcripts are expressed in the human placenta exhibiting an age-specific pattern of expression, (2) In cultured trophoblast cells, both genes are stimulatable by fetal calf serum and in contrast to c-fos, Glut1 is negatively regulated by glucose. This differential regulation of Glut1 and c-fos genes could be relevant to specific metabolic and mitogenic pathways implicated in placental growth and differentiation.

High density lipoprotein interaction with human placenta: biochemical and ultrastructural characterization of binding to microvillous receptor and lack of internalization.

Specific receptor and internalization process for low density lipoprotein (LDL) and modified LDL (acetyl-LDL) have been well characterized in placental microvilli and in trophoblastic cells in culture. The aim of this study was to investigate high density lipoprotein (HDL3) binding and its eventual subsequent internalization in both these purified placental preparations. Isolated term placental microvilli were used for binding of [125I]HDL3 (devoid of apoprotein E). HDL3 were conjugated to colloidal gold for ultrastructural visualization of binding and internalization in syncytiotrophoblast in culture. Saturable binding of HDL3 was identified. Scatchard analysis revealed a Kd value of 24.2 +/- 8.0 micrograms HDL3 protein/ml and a maximum binding capacity at 4 degrees C of 128.2 +/- 54.5 micrograms HDL3 protein/mg of membrane protein. These sites have broad specificity: both LDL and acetyl-LDL were able to partially inhibit the HDL3 binding. Ultrastructural study confirms that gold-HDL3 bind specifically to syncytiotrophoblast membrane. However, after incubation at 37 degrees C, an internalization process similar to those described for gold-LDL and gold-acetyl-LDL was not observed for gold-HDL3. These results demonstrate specific HDL3 binding without internalization. The physiological significance of an HDL3 membranous interaction and the placental steroidogenesis remains to be established.

Physiological role of human placental growth hormone.

Placental growth hormone (PGH) is the product of the GH-V gene specifically expressed in the syncytiotrophoblast layer of the human placenta. PGH differs from pituitary growth hormone by 13 amino acids. It has high somatogenic and low lactogenic activities. Assays of PGH by specific monoclonal antibodies reveal that in the maternal circulation from 15-20 weeks up to term, PGH gradually replaces pituitary growth hormone which becomes undetectable. It is secreted by the placenta in a non-pulsatile manner. This continuous secretion appears to have important implications for physiological adjustment to gestation and especially in the control of maternal IGF1 levels. PGH secretion is inhibited by glucose in vitro and in vivo, and is significantly decreased in the maternal circulation in cases of pregnancies with intrauterine growth retardation. PGH does not appear to have a direct effect on fetal growth, as this hormone is not detectable in the fetal circulation. However the physiological role of PGH might also include a direct influence on placental development via an autocrine or paracrine mechanism as suggested by the presence of specific GH receptors in this tissue.

Low-density lipoprotein binding sites in the microvillous membranes of human placenta at different stages of gestation.

Microvillous membranes isolated from early gestation placentas (8-12 weeks of amenorrhoea) and from mid-term placentas (20-22 weeks of amenorrhoea) were used to study the specific binding of low-density lipoprotein (LDL) to the trophoblast. The purity of the microvillous preparations has been assessed by electron microscopy and by their enrichment in two membrane markers, 5'-nucleotidase and alkaline phosphatase. Evidence was presented demonstrating the existence of saturable binding sites for [125I]LDL in placental microvilli as early as 6 weeks of pregnancy. The apparent KD values for these binding sites have been determined by Scatchard analyses to be 6.98 +/- 0.83 and 6.57 +/- 0.81 micrograms protein LDL/ml, for early gestation and mid-term preparations, respectively. This apparent KD value was unaffected by a pretreatment of the membranes by heparin, as indicated by the mean values of 7.13 +/- 0.89 and 6.97 +/- 0.75 micrograms protein LDL/ml obtained for immature microvilli preincubated with or without heparin, respectively. Large variations of binding capacity were observed in each gestational age group and no significant difference was found between them. These results indicate that the LDL binding sites of the human placenta, located on the microvillous membranes, (i) are present as early as the 6th week of pregnancy, and (ii) display the same high affinity and specificity for LDL as those of the term trophoblast.

Characterization of specific low-density lipoprotein binding sites in human term placental microvillous membranes.

Purified microvillous membranes prepared from normal term human placenta were studied for their ability to bind specifically low-density lipoprotein (LDL). Electron microscopic examination of the membrane preparations revealed essentially microvilli-like structures, and the enzyme analyses a 14-17-fold enrichment in the membrane markers 5'-nucleotidase and alkaline phosphatase. The binding of [125I]LDL was dependent on time, temperature, pH and protein concentration; it was saturable with a low capacity (130.4 +/- 22.2 ng/mg of membrane protein) and presented a high affinity (apparent Ka 6.12 +/- 1.32 micrograms protein per ml). These high-affinity binding sites were specific for LDL (high-density lipoprotein induced less competition than unlabelled LDL) and were sensitive to pronase digestion. Unlike the binding of LDL to other tissues, the [125I]LDL binding to microvillous membranes did not require divalent cations. The presence of specific LDL receptors on the placental microvillous membranes, located at the effective site of exchange between the maternal blood and the placental tissue, supports the concept that human placenta utilizes LDL-cholesterol for its progesterone synthesis.

EGF increases retinoid X receptor-alpha expression in human trophoblastic cells in culture: relationship with retinoic acid induced human chorionic gonadotropin secretion.

In the present study, the effect of retinoic acid (RA) and epidermal growth factor (EGF) on the functions of human trophoblastic cells in culture were analysed. In these cells, RA potentiated the hCG secretion increase induced by EGF. To gain a better understanding of such a synergistic effect, the expression of retinoic acid receptors (RAR alpha and beta) and retinoid X receptor (RXR alpha) was studied by immunoblotting in RA- and EGF-treated cells. EGF treatment specifically increased the level of RXR alpha protein and RXR alpha transcripts. In parallel, we demonstrated that the choriocarcinoma cells JEG 3, which respond to RA by an increase in hCG secretion, express constitutively high levels of RXR alpha protein. Furthermore, RXR alpha-transfected trophoblastic cells also become RA responsive for hCG secretion. All these data suggest that RXR alpha expression is modulated by EGF, and may be involved in the effect of RA on hCG secretion.

Identification of specific binding sites for acetylated low density lipoprotein in microvillous membranes from human placenta.

The ability of microvillous membranes isolated from human placenta to specifically bind human low density lipoprotein (LDL) modified by acetic anhydride has been investigated. The presence of saturable high affinity binding sites specific for [125I]acetyl-LDL was demonstrated. Scatchard analysis of the binding data, obtained at 4 degrees C, revealed a single class of sites with a mean KD value of 3.63 +/- 1.16 micrograms acetyl-LDL protein/ml, and a maximal binding capacity of 335.1 +/- 148.8 ng acetyl-LDL protein/mg of membrane protein. At 37 degrees C, the binding capacity was increased, while the KD value was not modified. The specificity of these binding sites was assessed by competition studies: unlabelled acetyl-LDL were effective competitors, whereas native LDL, VLDL and HDL3 were ineffective. Conversely, unlabelled acetyl-LDL failed to prevent the binding of native [125I]LDL to placental microvilli. The [125I]acetyl-LDL binding was partially inhibited (about 35%) by dextran sulfate and fucoidin, and was abolished by a pretreatment of the microvillous membranes with pronase. The binding sites specific for acetyl-LDL are present during all the gestation and are distinctly different from the binding sites for native LDL, previously characterized in placental microvilli. These 2 types of binding sites may be related to the high amount of cholesterol required by the human placenta for progesterone synthesis and trophoblastic growth.

The enhancers of the human placental lactogen B, A, and L genes: progressive activation during in vitro trophoblast differentiation and importance of the DF-3 element in determining their respective activities.

The hCS-A and hCS-B genes encoding human chorionic somatomammotropin and the related hCS-L gene are very similar in their coding and flanking sequences. For each of these genes, downstream enhancers, varying in strength, have been identified with the help of cytotrophoblast-derived JEG-3 cells, which do not express the hCS genes. Here we study the activity of the hCS enhancers in human syncytiotrophoblast in primary culture, which naturally expresses the hCS genes. We show that the activity of the hCS-B gene enhancer is mediated by two elements, DF-3 and DF-4, whereas the hCS-L and hCS-A gene enhancers display weaker activity due to mutations in their respective DF-3 sites. Replacement of the hCS-B DF-3 site with the homologous hCS-A sequence causes hCS-B enhancer activity to decrease. Primary cytotrophoblasts differentiate in culture to form the syncytiotrophoblast. We show that during this process the production of hCS progressively increases and that concomitantly all three hCS enhancers are progressively activated. A targeted mutation in the 3' part of the DF-4 element abolishes the binding of a protein present only in syncytiotrophoblast extracts and inactivates the DF-4 element. Thus, a direct correlation exists between the appearance of this syncytiotrophoblast-specific protein and hCS enhancer activity. This primary culture model proves useful in studying the regulation of the hCS genes.