Xq25 duplication: the crucial role of the STAG2 gene in this novel human cohesinopathy.

The Xq25 duplications syndrome has recently emerged as a distinct clinical entity. We report here on six new patients belonging to two unrelated families and harbouring an Xq25 microduplication detected by array CGH. Similarly to previously reported cases, the phenotype of our patients is characterized by delayed milestones, speech disturbance, intellectual disability, abnormal behaviours and a characteristic facial dysmorphism. The common duplicated interval allowed further refinement of the shortest region of overlap to 173 kb, including only one gene, STAG2, which encodes a component of the cohesin complex. We suggest that increased STAG2 gene copy number and dysregulation of its downstream target genes may be responsible for the specific clinical findings of this syndrome. Therefore, the Xq25 microduplication could be considered as a novel cohesinopathy, thus increasing the group of these disorders.

Simplified gyral pattern in severe developmental microcephalies? New insights from allometric modeling for spatial and spectral analysis of gyrification.

The strong positive-allometric relationship between brain size, cortical extension and gyrification complexity, recently highlighted in the general population, could be modified by brain developmental disorders. Indeed, in case of brain growth insufficiency, the pathophysiological relevance of the "simplified gyral pattern" phenotype is strongly disputed since almost no genotype-phenotype correlations have been found in primary microcephalies. Using surface scaling analysis and newly-developed spectral analysis of gyrification (Spangy), we tested whether the gyral simplification in groups of severe microcephalies related to ASPM, PQBP1 or fetal-alcohol-syndrome could be fully explained by brain size reduction according to the allometric scaling law established in typically-developing control groups, or whether an additional disease effect was to be suspected. We found the surface area reductions to be fully explained by scaling effect, leading to predictable folding intensities measured by gyrification indices. As for folding pattern assessed by spectral analysis, scaling effect also accounted for the majority of the variations, but an additional negative or positive disease effect was found in the case of ASPM and PQBP1-linked microcephalies, respectively. Our results point out the necessity of taking allometric scaling into account when studying the gyrification variability in pathological conditions. They also show that the quantitative analysis of gyrification complexity through spectral analysis can enable distinguishing between even (predictable, non-specific) and uneven (unpredictable, maybe disease-specific) gyral simplifications.

A finite-element analysis of Kienbock's disease.

A finite-element analysis model of the lunate was established using geometrical data obtained from cadaveric bones. The lunate cortex was modelled with triangular and quadrilateral elements and its intraosseous structure was represented either as a homogenous elastic structure or as an anisotropic network of cortical bone beams (trabeculae) with different orientations and thicknesses. Compressive loads applied to the metacarpus were distributed in the carpus against the fixed radius and ulna. The ulnar variance had a strong influence on the ratios radiolunate/ulnolunate total load and peak pressures. The distribution of internal stresses was markedly affected by the lunate uncovering index. The evolution of a simulated incomplete fracture was dramatically influenced by morphological parameters: with positive ulnar variance, the fracture did not progress, but in the presence of three associated conditions, negative ulnar variance, a high lunate uncovering index and angulated trabeculae, the fracture progressed and the proximal part of the lunate collapsed. This study supports the concept that some lunates are predisposed to Kienböck's disease because their anatomy induces abnormal internal stresses, which allow an incomplete fracture to progress, under heavy loading conditions, and cause progressive collapse and localised trabecular osteonecrosis.

[Clinical study and prevalence of fetal alcohol syndrome in medico-social institutions of the Reunion Island]. / Etude clinique et prévalence du syndrome d'alcoolisation foetale pris en charge dans les établissements médicosociaux de l'île de la Réunion.

BACKGROUND: Fetal alcohol syndrome (FAS) is a major problem in the Reunion Island and the Public Health Authorities decided to determine its prevalence in their medico-social centers on 31 December 1996. MATERIAL AND METHODS: A questionnaire was established to identify affected patients in the 20 medico-social centers in charge of 1320 children. Eighty-eight children were selected and 87 could be analyzed. RESULTS: Sixty-four of 87 (76.3%) were FAS and 23 of 87 (23.7%) had closely alcohol-related diseases. The prevalence was between 7.1 and 14.1% and lower than expected from available data. CONCLUSION: The study allowed to precise the social and familial factors predisposing to alcohol addiction during pregnancy. A TV prevention message will be broadcasted after this study.

Modifications to the mechanical behavior of the wrist after fracture of the scaphoid. Modeling by finite element analysis.

Although based on their long-term clinical evolution there appeared to be no doubt that fractures of the scaphoid modify the mechanical behavior of the carpus, the mechanisms of these modifications have not yet been investigated. This study based on finite element analysis provides insight into the sequence behind the onset of arthritis of the wrist, highlighting the existence of pressure peaks at the nonunion and at the midcarpal interface (scaphoid-capitate and lunate-capitate). This evidence explains the clinical evolution of nonunions of the scaphoid. Our study indirectly demonstrates the role played by the scaphoid within the wrist as a force transmission column. The use of finite element analysis for the modeling of simple or complex osteoarticular systems may prove to be a highly useful tool for the understanding of these mechanisms.

Lineage-dependent collagen expression and assembly during osteogenic or chondrogenic differentiation of a mesoblastic cell line.

The mesoblastic clone, C1, behaves as a tripotential progenitor able to self-renew and to differentiate toward osteogenesis, chondrogenesis, or adipogenesis in response to specific inducers. In this study, expression and deposition by the C1 cells of essential components of the extracellular matrix, collagens type I, II, III, V, XI, VI, IX, and X were followed along the osteogenic and chondrogenic pathways, through biochemical, immunochemical, and electron microscopy analyses. Implementation of each program involves profiles of collagen synthesis and matrix assembly close to those documented in vivo. Depending on the applied inducers, cells adopt a defined identity and, controls acting at transcriptional and posttranslational levels adapt the set of deposited collagens to one particular cell fate. Osteogenic C1 cells selectively build a type I collagen matrix also containing type III, V, and XI collagens but selectively exclude type II collagen. Chondrogenic C1 cells first elaborate a type II collagen network and then acquire hypertrophic chondrocyte properties while assembling a type X collagen matrix as in the growth plate. This study provides an example of how a mesoblastic cell line can develop, in vitro, each of its genetic programs up to terminal differentiation. Intrinsic factors and time-dependent cell-matrix interactions might, as in vivo, underline the implementation of an entire differentiation program.

Kidney cortex cells derived from SV40 transgenic mice retain intrinsic properties of polarized proximal tubule cells.

BACKGROUND: We have developed a nontransformed immortalized mice kidney cortex epithelial cell (MKCC) culture from a mouse transgenic for a recombinant plasmid adeno-SV40 (PK4). Methods and Results. After 12 months in culture, the immortalized cells had a stable homogeneous epithelial-like phenotype, expressed simian virus 40 (SV40) T-antigen, but failed to induce tumors after injection in nude mice. Epithelium exhibited polarity with an apical domain bearing many microvilli separated from lateral domains by junctional complexes with ZO1 protein. The transepithelial resistance was low. A Na-dependent glucose uptake sensitive to phlorizin and a Na-dependent phosphate uptake sensitive to arsenate were present. Western blot analysis of membrane fractions showed that anti-Na-Pi antiserum reacted with a 87 kD protein. The Na/H antiporters NHE-1, NHE-2, and NHE-3 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). The corresponding proteins with molecular weights of 111, 81, and 75 kD, respectively, could be detected by Western blot and were shown to be functional. Parathyroid hormone (PTH) induced a tenfold increase in cAMP and reduced the Na-dependent phosphate uptake and NHE-3 activity, as observed in proximal tubule cells. Isoforms alpha, delta, epsilon, and zeta of protein kinase C (PKC) were present in the cells. Angiotensin II (Ang II) elicited a translocation of the PKC-alpha toward the basolateral and apical domains. CONCLUSION: Thus, the MKCC culture retains the structural and functional properties of proximal tubular cells. To our knowledge, it is the first cell culture obtained from transgenic mice that exhibits the NHE-3 antiporter and type II Na-Pi cotransporter. MKCCs also display functional receptors for PTH and Ang II. Thus, MKCCs offer a powerful in vitro system to study the cellular mechanisms of ion transport regulation in proximal epithelium.

[MELAS syndrome in a five year-old child: clinical, biological and genetic characteristics]. / Syndrome MELAS chez un enfant de 5 ans: caractéristiques cliniques, biologiques et génétiques.

BACKGROUND: MELAS syndrome is a rare mitochondrial cytopathy; its diagnosis can be difficult. CASE REPORT: A 6-month-old boy presented with febrile seizures, possibly due to viral meningitis. At 7 months, he developed myoclonia and "brain attacks" and, subsequently, myoclonical attacks, regression of psychomotor and mental acquisitions, and progressive visual loss. The ratio of lactatorachia/lactacidemia was increased. The molecular genetic analysis showed an heteroplasmic point mutation with A-to-G mutation at nucleotide 3243 of the mitochondrial tRNA(leu) (UUR) gene. He was the second child of a mother having frequent headaches. His great aunt, a sister of his maternal grandmother, was mentally retarded and had frequent epileptic seizures and hemiparesy since her childhood. CONCLUSION: Any unusual neurological symptom, particularly when combined with "illegitimate" symptoms, should lead to search for a mitochondrial cytopathy.

Controlled conversion of an immortalized mesodermal progenitor cell towards osteogenic, chondrogenic, or adipogenic pathways.

The teratocarcinoma-derived C1 clone behaves as a mesodermal tripotential progenitor cell whose choice of fate, either osteoblast, chondroblast, or adipoblast, is strictly dependent on the spatial organization of the cells and the nature of the induction. In the absence of cell contact before the addition of inducers, the C1 cells maintain a stable undifferentiated phenotype while expressing potential regulators of embryonic mesodermal stem cell fate such a M-twist and Id1. Upon establishment of cell contacts before the induction of differentiation, the early genes characteristic of the three fates become expressed. In the presence of beta glycerophosphate and ascorbate, provided the cells have formed aggregates, 95% of the C1 cells mineralize with a kinetics of gene expression close to that of osteoblasts (Poliard, A., D. Lamblin, P. J. Marie, M. H. Buc, and O. Kellerman. 1993. J. Cell Sci. 106:503-512). With 10(-6)M dexamethasone, 80% of the same aggregates differentiate into foci of chondroblast-like cells. The kinetics of expression of the genes encoding type II, IX, X, and XI collagens, aggrecan and link protein during the conversion toward cartilage hypertrophy resembles that accompanying in vivo chondrogenesis. The synergistic action of dexamethasone and insulin convert most confluent C1 cells into functional adipocytes and induce a pattern of gene expression close to that reported for adipoblast cell lines. The C1 clone with its capacity to differentiate along three alternative pathways with high frequency, therefore appears as a valid in vitro model for deciphering the molecular basis of mesoblast ontogeny.

Commitment of the teratocarcinoma-derived mesodermal clone C1 towards terminal osteogenic differentiation.

The mesodermal clone C1 was derived from the multipotent embryonal carcinoma 1003 cell line transformed with the plasmid pK4 carrying SV40 oncogenes under the control of the adenovirus E1A promoter. We have shown that the C1 clone becomes committed to the osteogenic pathway when cultured in aggregates in the presence of mediators of the osteogenic differentiation. To further validate C1 as a model with which to study osteogenesis in vitro the kinetics of its differentiation was studied, focusing on the histology of the aggregates and on the expression of a set of genes corresponding to representative bone matrix proteins. The presence of ascorbic acid and beta- glycerophosphate specifically leads to mineralization in almost 100% of the aggregates. Transcription of the above genes, silent in exponentially growing cells, specifically occurred with the establishment of cell-cell contacts independently of the presence of ascorbic acid and inorganic phosphate. The latter, however, were absolutely required for matrix deposition and mineralization. In their presence, one observed an overall decline in type I collagen and alkaline phosphatase transcripts while osteocalcin and osteopontin transcripts preferentially accumulated in cells lining the mineralizing foci. Concomitantly, type I collagen and osteocalcin became extracellularly deposited. The osteogenic differentiation of C1 occurred while cells were still proliferating. The C1 clone thus behaves as a mesodermal stem cell, becoming committed to the osteogenic pathway upon: firstly, establishment of cellular contacts; and secondly, addition of ascorbate and beta-glycerophosphate. It therefore appears to be a promising in vitro system for deciphering the molecular basis of osteoblast ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of in vitro mineralization by an osteogenic clonal cell line (C1) derived from mouse teratocarcinoma.

We have previously reported the isolation of an osteogenic clonal cell line (C1) derived from mouse teratocarcinoma and immortalized by the SV 40 oncogenes. In this report we describe the kinetics of osteogenic differentiation of aggregated C1 cells by following the matrix deposition and mineralization and the expression of alkaline phosphatase. We show that after addition of beta-glycerophosphate and ascorbic acid, more than 95% of C1 aggregates synthesize a bone matrix which is deposited as early as 2 days and increases progressively with time in culture. Matrix calcification is evidenced by von Kossa staining and tetracycline incorporation into the mineral whereas no calcification appears in control cultures. Calcium is detectable in mineralizing aggregates at 2 days and calcium content increases linearly with time in culture, being 125-fold higher in mineralizing nodules than in control aggregates at 30 days. Aggregated C1 cells are characterized by a high activity of the bone type isoenzyme of alkaline phosphatase, a marker of osteoblast phenotype. Upon addition of inducers, alkaline phosphatase activity decreases by five-fold after the onset of mineralization and remains stable thereafter. The down-regulation of alkaline phosphatase activity is confirmed at the cellular level by histochemical staining. The mRNA levels for alkaline phosphatase decline during osteogenesis, following a pattern similar to the decrease in protein activity. Analysis of DNA synthesis by (3H)-thymidine incorporation and quantification of labelled nuclei on autoradiographs shows that C1 cells proliferation is not down-regulated during the time course of differentiation and that proliferating C1 cells still express alkaline phosphatase activity during osteogenic differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin uptake, storage, and synthesis in an immortalized committed cell line derived from mouse teratocarcinoma.

We report the isolation and characterization of a serotoninergic cell line, 1C11, derived from a mouse teratocarcinoma. The clone 1C11 was immortalized through the expression of the simian virus 40 oncogenes. 1C11 presents two states: an immature epithelial-like state (1C11 precursor) and a more differentiated state (1C11). After induction by dibutyryl cyclic AMP and cyclohexanecarboxylic acid, almost 100% of 1C11 cells continue to divide and have acquired a neural-like phenotype. 1C11* cells coexpress several neural markers, such as synaptophysin (the membrane constituent of synaptic vesicles), the neuropeptide [Met5]enkephalin, and the neurotransmitter serotonin. 1C11* cells store endogenous serotonin and are able to synthesize serotonin from L-tryptophan and to catabolize it by monoamine oxidase B. Moreover, the cells take up serotonin by a carrier-mediated mechanism very similar to that of serotoninergic neurons. The expression of the simian virus 40 oncogenes, which promoted immortalization, does not therefore prevent further differentiation. This inducible cell line constitutes a valuable model for cellular and molecular studies concerning the physiology and the pharmacological modulation of the serotoninergic phenotype.

An immortalized osteogenic cell line derived from mouse teratocarcinoma is able to mineralize in vivo and in vitro.

The hybrid plasmid pK4 containing the early genes of the simian virus SV-40, under the control of the adenovirus type 5 E1a promoter, was introduced into the multipotent embryonal carcinoma (EC) 1003. Expression of the SV-40 oncogenes was observed at the EC cell stage, and this allowed the derivation of immortalized cells corresponding to early stages of differentiation. Among the immortalized mesodermal derivatives obtained, one clone, C1, is committed to the osteogenic pathway. C1 cells have a stable phenotype, synthesize type I collagen, and express alkaline phosphatase activity. Although immortalized and expressing the SV-40 T antigen, the cells continue to be able to differentiate in vivo and in vitro. In vivo, after injection into syngeneic mice, they produce osteosarcomas. In vitro, the cells form nodules and deposit a collagenous matrix that mineralizes, going to hydroxyapatite crystal formation, in the presence of beta-glycerophosphate. This clonal cell line, which originates from an embryonal carcinoma, therefore differentiates into osteogenic cells in vivo and in vitro. This immortalized cell line will be useful in identifying specific molecular markers of the osteogenic pathway, to investigate gene regulation during osteogenesis and to study the ontogeny of osteoblasts.

F9 teratocarcinoma aggregates express villin upon differentiation into visceral endoderm-like cells.

The visceral endoderm of the mouse embryo is a polarized epithelium which has recently been shown to express villin, a major actin binding component of absorptive epitheliums. We report here that villin is induced during differentiation of aggregates of the mouse embryonal carcinoma F9, an in vitro system widely used to study extraembryonic endoderm differentiation. Identical results were obtained with a variant of F9 which carries an immortalizing vector. Villin is coexpressed with F-actin and with alpha-foetoprotein, in most of the visceral endoderm-like cells lining the aggregates. This system is potentially useful to study (i) the induction of villin expression and (ii) the establishment of polarity in the visceral endoderm epithelium.

5-Azacytidine is able to induce the conversion of teratocarcinoma-derived mesenchymal cells into epithelia cells.

The inhibitor of DNA-methylation, 5-azacytidine (5- AzaC ) induced the appearance of cytokeratin-containing cells in several mesenchymal cell lines such as teratocarcinoma-derived fibroblasts, preadipocytes and myoblasts, NIH-3T3 fibroblasts and human embryonic fibroblasts. At optimal 5- AzaC concentrations the proportion of such cells was in the range of 10(-1) compared with 10(-6) -10(-4) in non-treated cultures. Dose-response curves indicated that the induction of cytokeratin was the result of an interaction of the drug with few targets. Stable, mature, keratinocyte cell lines, as well as lines of myoblasts and astrocytes, could be isolated from a teratocarcinoma-derived preadipocyte line, showing that 5- AzaC is able to provoke a wide range of complete phenotypic conversions. In these cell lines, the intermediate filaments corresponded to the morphological phenotype. Altogether, the results suggest that 5- AzaC preferentially activates certain genes.