Genome-wide paternal uniparental disomy as a cause of Beckwith-Wiedemann syndrome associated with recurrent virilizing adrenocortical tumors.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by fetal macrosomia, macroglossia, and abdominal wall defects. BWS patients are at risk to develop Wilms tumor, neuroblastoma, hepatoblastoma, and adrenal tumors. A young woman with BWS features, but with inconclusive genetic evidence for the disease, came to clinical observation for signs of virilization at the age of 16 years. An adrenocortical tumor was diagnosed and surgically resected. The tumor underwent 2 local relapses that were also surgically treated. The patient was also operated to remove a breast fibroadenoma. SNP arrays were used to analyze chromosome abnormalities in normal and tumor samples from the patient and her parents. The patient presented genome-wide mosaic paternal uniparental disomy (patUPD) both in the adrenocortical and the breast tumors, with different degrees of loss of heterozygosity (LOH). The more recent relapses of the adrenocortical tumor showed a loss of part of chromosome 17p that was absent in the first tumor. Analysis of a skin biopsy sample also showed mosaic patUPD with partial LOH, while no LOH was detected in leukocyte DNA. This case shows that virilizing adrenocortical tumors may be a clinical feature of patients with BWS. The SNP array technology is useful to diagnose genome-wide patUPD mosaicism in BWS patients with an inconclusive molecular diagnosis and underlines the tumorigenic potential of the absence of the maternal genome combined with an excess of the paternal genome.

Split hand/foot malformation with long-bone deficiency and BHLHA9 duplication: report of 13 new families.

Split hand/foot malformation (SHFM) with long-bone deficiency (SHFLD, MIM#119100) is a rare condition characterized by SHFM associated with long-bone malformation usually involving the tibia. Previous published data reported several unrelated patients with 17p13.3 duplication and SHFLD. Recently, the minimal critical region had been reduced, suggesting that BHLHA9 copy number gains are associated with this limb defect. Here, we report on 13 new families presenting with ectrodactyly and harboring a BHLHA9 duplication.

[Mild cystic fibrosis: genetics - extending follow-up is necessary]. / Les formes atténuées de la mucoviscidose : génétique - suivi prolongé nécessaire.

The diagnosis of mild cystic fibrosis is first suspected on mild lung disease or absence of pancreatic insufficiency and is assessed by biological analysis. The sweat test is not always conclusive. The nasal potential difference and molecular analysis of CFTR gene allow confirming diagnosis. A regular follow-up in cystic fibrosis clinical centre is essential all life long. The genotype, especially during neonatal period, cannot be used to predict individually the course of the disease. Genetic counselling must be recommended to the parents in order to propose an analysis of CFTR gene to give the appropriate genetic counselling and to consider with them which family members could be concerned, especially in the event of parental project. The research of heterozygote status in related for prenatal diagnosis is not recommended for all mutations.

Molecular heterogeneity in fetal forms of type II lissencephaly.

Type II lissencephaly (type II LIS) is a group of autosomal recessive congenital muscular dystrophies (CMD) associated with defects in alpha-DG O-glycosylation, which comprises Walker-Warburg syndrome, Fukuyama cerebral and muscular dystrophy, or muscle-eye-brain disease. The most severe forms of these diseases often have a fetal presentation and lead to a pregnancy termination. We report here the first molecular study on fetal type II LIS in a series of 47 fetuses from 41 unrelated families. Sequencing of the different genes known to be involved in alpha-DG O-glycosylation allowed the molecular diagnosis in 22 families: involvement of POMT1 was demonstrated in 32% of cases, whereas POMGNT1 and POMT2 were incriminated in 15% and in 7% of cases, respectively. We found 30 different mutations in these three genes, 25 were described herein for the first time, 15 in POMT1, and five in POMT2 and POMGNT1. Despite sequencing of FKRP, FCMD, and LARGE, no definitive molecular diagnosis could be made for the other half of our cases. Preliminary results concerning genotype-phenotype correlations show that the choice of the first gene sequenced should depend on the clinical severity of the type II LIS; POMT1 and POMT2 for severest clinical picture and POMGNT1 for milder disease. The other genes, FKRP, FCMD, and LARGE, seem not to be implicated in the fetal form of CMD.

Promoting effect of glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically defined medium.

Stromal-vascular cells obtained from adult human subcutaneous adipose tissue were cultured in a chemically defined serum-free medium. In the presence of 0.2 nM triiodothyronine and 0.5 microM insulin, up to 25% of the cells were able to undergo terminal adipose differentiation within 18 d, as assessed by lipid accumulation and the expression of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities. Addition of cortisol resulted in a potent dose-dependent stimulation of the adipose differentiation process. Cortisol could be replaced by dexamethasone and partly by aldosterone, but not by sex steroids. The proportion of differentiated cells was dependent upon the age of the donor; when isolated from young adults, up to 70% of the stromal-vascular cells expressed the adipocyte phenotype as compared with 5-10% when the cells were isolated from the oldest subjects. An inverse relationship was observed between the age of the 27 normal-weight donors and the extent of GPDH expression after maintenance of the cells for 18 d in chemically defined medium supplemented with insulin, triiodothyronine, and cortisol (r = -0.787, P less than 0.001). It is concluded that adult human adipose tissue still contains precursor cells that are able to undergo adipose differentiation in vitro. This improved culture system may offer the opportunity to characterize other adipogenic factors as well as antiadipogenic factors involved in the control of adipose tissue growth.

Do we taste fat?

Sense of taste informs the body about the quality of ingested foods. Five sub-modalities allowing the perception of sweet, salty, sour, bitter, and umami stimuli are classically depicted. However, the inborn attraction of mammals for fatty foods raises the possibility of an additional orosensory modality devoted to fat perception. For a long time, dietary lipids were thought to be detected only by trigeminal (texture perception), retronasal olfactory, and post-ingestive cues. This minireview analyses recent findings showing that gustation also plays a significant role in dietary lipid perception.

[Psychometric properties of the Euroqol measure in patients with muscular dystrophy]. / Qualités métrologiques de l'outil de mesure Euroqol sur un échantillon de personnes myopathes.

OBJECTIVE: To analyse patient-reported outcome measures and to assess acceptability, test-retest reliability and responsiveness of the Euroqol measure (EQ), a conceptual health-related quality-of-life measure (HRQoL), for patients with muscular dystrophy. MATERIALS AND METHOD: One hundred and four consecutive outpatients completed the EQ in Reims university hospital between April 2002 and February 2005, and 60 patients were followed over 1 year. The acceptability of the EQ-5D and EQ-EVA measures was assessed by using the completion rate per dimension as an indicator; test-retest reliability was assessed with kappa and Spearman coefficients for qualitative data and the intraclass coefficient correlation (ICC) for quantitative data. Over the year, EQ-EVA score responsiveness was calculated according to the standardised response of the mean (SRM). RESULTS: Participation rate (96.3%) and EQ-5D completion rates were excellent, between 95.2 and 100%. Test-retest reliability after 15+/-7 days was excellent for the autonomy domain (kappa coefficient=0.81) and moderate for the other dimensions. EQ-EVA score stability was satisfactory (ICC=0.72). Global perceived health (EQ-EVA) was not associated with level of dependency but was associated with pain domain scores. EQ-EVA responsiveness was moderate (effect size=0.6) in the patients with a change in health status over 1 year and in reference to the relevant SF-36 item. CONCLUSION: EQ is a well-accepted tool for measuring HRQoL in this group of patients with muscular dystrophy. The prognostic interest of these subjective measures has yet to be demonstrated; however, these measures provide interesting additional information.

Fetuin modulates growth and differentiation of Ob17 preadipose cells in serum-free hormone-supplemented medium.

A serum-free hormone-supplemented medium able to support the growth of rodent adipose precursor cells has been used to characterize additional components from serum required for the differentiation of preadipose Ob17 cells into adipose-like cells. Fetuin is shown to behave as a growth-promoting agent for these cells. In addition to growth hormone, triiodothyronine and a low-molecular weight component(s) also purified from serum, fetuin is required for the full expression of the differentiation program. Other serum proteins as well as other mitogenic factors are unable to substitute for fetuin. A possible role of fetuin in the development of adipose tissue is discussed.

Autocrine control of adipose cell differentiation by prostacyclin and PGF2 alpha.

The mitogenic-adipogenic effect exerted by arachidonic acid, which leads to terminal differentiation of Ob1771 mouse preadipocytes, has been shown to be (i) blocked by cyclooxygenase inhibitors, (ii) mimicked by a stable analogue of prostacyclin (carbaprostacyclin) and (iii) potentiated by PGF2 alpha. Since these prostanoids are known to be synthesized and secreted by preadipocytes, we have proposed that both prostacyclin as the key mediator and PGF2 alpha as a modulator control the expression of terminal events of adipose conversion by means of an autocrine mechanism (Gaillard, D. et al. and Negrel, R. et al. Biochem. J. (1989) 257, 389-397 and 399-405). In order to test this hypothesis, the release of prostacyclin, characterized under the form of its stable degradation product 6-keto-PGF1 alpha, and that of PGF2 alpha have been studied in the culture medium of Ob1771 cells. A striking increase in the release of 6-keto-PGF1 alpha and to a minor degree of PGF2 alpha was observed when cells were exposed to arachidonic acid as shown by using [3H]arachidonic acid prelabelled cells or by radio-immunoassays. Since antagonists of PGF2 alpha and PGI2 receptors were not available, specific antibodies directed against PGF2 alpha and 6 beta-PGI1, another stable analogue of prostacyclin, were added as neutralizing agents in the culture medium. These antibodies were able to counteract the mitogenic-adipogenic effect of arachidonic acid. Prostacyclin and PGF2 alpha thus appear as autocrine mediators in the process of adipose conversion.

Less HLA-G expression in Plasmodium falciparum-infected third trimester placentas is associated with more natural killer cells.

During pregnancy, maternal immune tolerance of the fetal semi-allogeneic graft is partly the consequence of extravillous trophoblast HLA-G expression and its interaction with natural killer (NK) cells. Plasmodium falciparum malaria is frequently associated with maternal and fetal complications. Local HLA-G expression and the number of NK cells were evaluated immunohistochemically in P. falciparum-infected and uninfected placentas (15 each) collected in a seasonal malaria-hypoendemic area. In control placentas, HLA-G was almost always expressed in extravillous trophoblast whereas, in infected placentas, it was significantly more weakly expressed in extravillous trophoblast but was also detected in intervillous space macrophages. NK cells were evaluated in intervillous and intravillous spaces and in basal plate. NK cells were always more abundant in basal plate than in intervillous and intravillous spaces in infected or control placentas. For each area, more NK cells were seen in infected than control placentas. These data suggest that HLA-G down-regulation and more NK cells in placentas may be among the mechanisms involved in poor birth outcome associated with P. falciparum infection.

[Cardiac surgery without transfusion in 2005]. / Chirurgie cardiaque sans transfusion en 2005. A propos d'une série de 200 témoins de Jehovah opérés.

PURPOSE: Between January 1991 and October 2003, 200 Jehovah Witnesses adult patients underwent elective cardiac surgery. To asses the impact on continuing progress of blood saving protocols and the increasing operative risk of patients proposed to surgery, we have re-assessed our results in this specific population. METHODOLOGY: Files of the first 100 patients operated upon between 1991 and 1998 were reviewed, and compared to the following 100 ones treated between 1998 to today. All patients were scored using the Euroscore model. RESULTS: In the latest series, patients are older (68 vs 51) and 13% underwent an iterative procedure, although there was none in the first series. Three deaths occurred after one month at the beginning of our experience, only one in the latest series. Operative risk factors had distinctly deteriorated, with more redux, and ejection fraction lower than 35%. Major progress to maintain morbi-mortality stability were multifactorial: preoperative erythropoietin in order to reach an haemoglobin minimal value of 14 g/dL, Cornell University protocol, mini-ECC, warm blood cardioplegia, ultra-early extubation. CONCLUSION: Cardiac surgery without transfusion can be realised with an equivalent risk to that of classical surgery, despite an operative risk aggravation, due to the association of recent conservative techniques.

[Increased nuchal translucency and cystic hygroma in the first trimester: prenatal diagnosis and neonatal outcome]. / Hyperclarté nucale et hygroma cervical au premier trimestre de la grossesse: diagnostic prénatal et devenir néonatal.

OBJECTIVE: A prospective study of pregnancy outcome in fetuses with increased nuchal translucency above the 95th centile (group NT) or cystic hygroma (group CH) at 10 to 14 weeks of gestation was performed. PATIENTS AND METHODS: Maternal and fetal data (nuchal translucency, caryotype, pregnancy outcome) and infant follow-up of 223 fetuses with first trimester nuchal translucency thickness (183 NT and 40 CH) were analysed. RESULTS: The measurement of nuchal translucency thickness shows a significant difference between group CH and NT (7.4+/-2.9 mm compared 3.7+/-0.8 mm). Chromosomal abnormalities were present in 55% (22/40) in group CH, with 9 cases/22 (40.9%) of Turner syndrome, compared with 14.2% (26/183) in group NT with trisomy 21 in 15 cases/26 (57.7%) (P<0.05). The rate of unfavourable outcome of pregnancy (spontaneous abortion, elective termination of pregnancy, serious structural anomalies) was 80% (32/40) in group CH compared with 18% (33/183) in group NT (P<0.05). In chromosomally normal pregnancies, the rate of fetus with no visible serious structural anomalies was 44.4% (8/18) in group CH compared with 93% (146/157) in group NT (P<0.05). DISCUSSION AND CONCLUSION: Our data show ultrasonographic evaluation of the fetal nuchal translucency thickness at the first trimester is actually indispensable. Neonatal outcome and malformation rate in fetuses with increased nuchal translucency or cystic hygroma are different, even with normal karyotype.

Expression of transforming growth factor alpha (TGF alpha), epidermal growth factor receptor (EGF-R) and cell proliferation during human palatogenesis: an immunohistochemical study.

Transforming growth factor alpha (TGF alpha) is not only a potent mitogen for several cell types, it interferes with cell differentiation. To investigate the possible role of TGF alpha in the fusion of the palatal processes in humans, the distribution of TGF alpha and its receptor (epidermal growth factor receptor = EGF-R) were studied using immunohistochemistry. 23 human palates were obtained from embryos and fetuses at 6 to 12 weeks of gestation and embedded in paraffin. In each case, the degree of cell proliferation was assessed using an antibody reacting with the nuclear antigen Ki-67. The epithelial and mesenchymal cell phenotypes were studied with anti-cytokeratin and anti-vimentin antibodies. TGF alpha and its receptor were detected in all the human palates, regardless of the stage of fusion. They were more highly expressed in the epithelial cells than in the mesenchymal cells of the palatal shelves. At first, proliferative activity was intense in both the mesenchyme and the epithelia and was later principally limited to the nasal or oral epithelia and also to the degenerating epithelial seam. At 10 weeks, when the midline palatal seam broke up into epithelial islands, the epithelial cells remained immunolabeled for TGF alpha, EGF-R and showed an increased number of proliferating cells. Programmed cell death (PCD) of medial edge epithelia (MEE) has been well documented, however other mechanisms must be considered during palatogenesis. Complex interactions between different growth factors have a probable role in epithelial mesenchymal transformation (EMT) and migration as well as in extracellular matrix synthesis.

The influence of tissue pretreatment on the immunohistochemical demonstration of type I and III collagens and tenascin in fetal human tooth germs.

The influence of tissue pretreatment on the PAP immunostaining for type I and III collagens and tenascin was studied in formalin-fixed and paraffin-embedded human tooth germs at the 24th and 25th weeks of fetal life. Three variables were considered: the type of buffer used (PBS or Tris), pepsin digestion and the use of normal serum as a blocking agent prior to immunostaining. All three proteins needed an enzymatic digestion to be intensely revealed. Pepsin promoted, even at low concentrations, an intracellular staining of type I collagen in the secretory odontoblasts and in the pulpal fibroblasts. Normal serum partially blocked unspecific immunoreaction when polyclonal rabbit antibodies were used. The Tris buffer increased the staining intensity of the three macromolecules and revealed an unusual tenascin-like immunoreactivity in the ameloblasts. This study demonstrated that pepsin digestion and the use of normal serum and different buffers may influence the immunoreactivity of ECM proteins.

Expression and immunohistochemical localization of laminin and type IV collagen in developing human fetal tracheal glands.

The expression and distribution of laminin and type IV collagen, two major components of the basement membrane, were investigated at the epithelio-mesenchymal interface of the human developing tracheal glands from 10 to 37 weeks of gestation. The localization of these molecules was assessed by indirect immunoperoxidase and indirect immunofluorescence staining and correlated to morphogenesis and epithelial cell differentiation. Laminin and type IV collagen were detected as early as 10 weeks of gestation in a continuous, linear pattern in the basement membrane surrounding the epithelial tracheal tube. By 12 weeks of gestation the basement membrane developed large openings at the tips of the budding glands beneath poorly differentiated cells, concomitant with the onset of morphogenetic movements. The remodeling of the basement membrane led to branching epithelial morphogenesis. The maturation and the functional differentiation of the secretory cells appeared later in the epithelium, when the basement membrane was strongly labeled with both anti-laminin and anti-type IV collagen antibodies, after 24 weeks of gestation. At this time the basement membrane became regular and thick and the maturation of serous cells increased progressively. These results suggest that the remodeling of the basement membrane takes place very early during gestation and that the morphogenesis and the maturation of the tracheal glands are rapidly achieved in humans.

In vivo localization of the insulin-like growth factors I and II (IGF I and IGF II) gene expression during human lung development.

The insulin-like growth factors I and II (IGF I and IGF II) are synthesized in many organs during human development and are involved in the growth and differentiation of tissues. Correlations between lung growth and maturation and the local production of IGFs have been poorly explored in humans. Using in situ hybridization we localized the synthesis of IGFs in the human fetal respiratory tract over an extended period of the gestation and we demonstrated time dependent changes. IGF mRNAs were expressed throughout gestation with a clear predominance of IGF II and a decreasing expression of both IGFs after the 20th week of gestation. They were mainly detected in the mesodermal-derived components of the respiratory tract, especially in the undifferentiated mesenchyme of the lung buds up to 20 weeks of gestation. At this time the local production of collagen and the proliferation of adjacent epithelial cells were predominant features. Later, mesenchymal hybridization decreased. Weak epithelial hybridization was observed during the first stages of growth and progressively decreased when the epithelium underwent maturation: early in the trachea, later in the distal lung buds. A consistent expression of IGF II, but not IGF I, in the endothelium, throughout gestation, was also observed. The IGFs may act on the near epithelial cell proliferation in both autocrine and paracrine ways. They may also stimulate the maturation of the connective tissue. This endogenous production of growth factors may play a role in the somatic growth during prenatal life.

Gene transfer to human fetal pulmonary tissue developed in immunodeficient SCID mice.

Human fetal lung rudiments (8-12 weeks of development) undergo considerable growth upon microsurgical ectopic implantation in the xenograft-tolerant SCID mouse, and differentiate into a lung-like tissue that includes: (i) bronchial structures lined with pseudostratified, secretory, ciliated epithelium surrounded by smooth muscle and cartilage rings, (ii) submucosal glands, and (iii) alveolar sacs. Normal expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was detected by immunostaining in those grafts, and similar differentiation was observed from either normal or cystic fibrosis (CF) fetal lung rudiments. Upon microinjection into human CF or normal lung grafts in SCID mice, beta-galactosidase-adenovirus gene constructs were efficiently transduced into epithelial and glandular cells. Such an in vivo replica of the human respiratory tissue may be a useful experimental model to study normal and pathologic lung development, and to assay candidate therapeutic gene constructs preclinically.

Twelve novel mutations in the tissue-nonspecific alkaline phosphatase gene (ALPL) in patients with various forms of hypophosphatasia.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report here the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 11 families affected by various forms of hypophosphatasia. Nineteen distinct mutations were found, 7 of which were previously reported. Eleven of the 12 new mutations were missense mutations (Y11C, A34V, R54H, R135H, N194D, G203V, E218G, D277Y, F310G, A382S, V406A), the last one (998-1G>T) was a mutation affecting acceptor splice site.