Anne-Lise Delezoide

Fibroblast Growth Factor Receptor 3 Mutations Promote Apoptosis but Do Not Alter Chondrocyte Proliferation in Thanatophoric Dysplasia

Thanatophoric dysplasia (TD) is a lethal skeletal disorder caused by recurrent mutations in the fibroblast growth factor receptor 3 (FGFR 3) gene. The mitogenic response of fetal TD I chondrocytes in primary cultures upon stimulation by either FGF 2 or FGF 9 did not significantly differ from controls. Although the levels of FGFR 3 mRNAs in cultured TD chondrocytes were similar to controls, an abundant immunoreactive material was observed at the perinuclear level using an anti-FGFR 3 antibody in TD cells. Transduction signaling via the mitogen-activated protein kinase pathway was assessed by measuring extracellular signal-regulated kinase activity (ERK 1 and ERK 2). Early ERKs activation following FGF 9 supplementation was observed in TD chondrocytes (2 min) as compared with controls (5 min) but no signal was detected in the absence of ligand. By contrast ligand-independent activation of the STAT signaling pathway was demonstrated in cultured TD cells and confirmed by immunodetection of Stat 1 in the nuclei of hypertrophic TD chondrocytes. Moreover, the presence of an increased number of apoptotic chondrocytes in TD fetuses was associated with a higher expression of Bax and the simultaneous decrease of Bcl-2 levels. Taken together, these results indicate that FGFR 3 mutations in TD I fetuses do not hamper chondrocyte proliferation but rather alter their differentiation by triggering premature apoptosis through activation of the STAT signaling pathway.

Spatio-temporal expression of FGFR 1, 2 and 3 genes during human embryo-fetal ossification

Mutations in FGFR 1-3 genes account for various human craniosynostosis syndromes, while dwarfism syndromes have been ascribed exclusively to FGFR 3 mutations. However, the exact role of FGFR 1-3 genes in human skeletal development is not understood. Here we describe the expression pattern of FGFR 1-3 genes during human embryonic and fetal endochondral and membranous ossification. In the limb bud, FGFR 1 and FGFR 2 are initially expressed in the mesenchyme and in epidermal cells, respectively, but FGFR 3 is undetectable. At later stages, FGFR 2 appears as the first marker of prechondrogenic condensations. In the growing long bones, FGFR 1 and FGFR 2 transcripts are restricted to the perichondrium and periosteum, while FGFR 3 is mainly expressed in mature chondrocytes of the cartilage growth plate. Marked FGFR 2 expression is also observed in the periarticular cartilage. Finally, membranous ossification of the skull vault is characterized by co-expression of the FGFR 1-3 genes in preosteoblasts and osteoblasts. In summary, the simultaneous expression of FGFR 1-3 genes in cranial sutures might explain their involvement in craniosynostosis syndromes, whereas the specific expression of FGFR 3 in chondrocytes does correlate with the involvement of FGFR 3 mutations in inherited defective growth of human long bones.

Undersulfation of Proteoglycans Synthesized by Chondrocytes from a Patient with Achondrogenesis Type 1B Homozygous for an L483P Substitution in the Diastrophic Dysplasia Sulfate Transporter

Achondrogenesis type 1B is an autosomal recessive, lethal chondrodysplasia caused by mutations in the gene encoding a sulfate/chloride antiporter of the cell membrane (Superti-Furga, A., Hästbacka, J., Wilcox, W. R., Cohn, D. H., van der Harten, J. J., Rossi, A., Blau, N., Rimoin, D. L., Steinmann, B., Lander, E. S., and Gitzelmann, R. (1996) Nat. Genet. 12, 100-102). To ascertain the consequences of the sulfate transport defect on proteoglycan synthesis, we studied the structure and sulfation of proteoglycans in cartilage tissue and in fibroblast and chondrocyte cultures from a fetus with achondrogenesis 1B. Proteoglycans extracted from epiphyseal cartilage and separated on agarose gels migrated more slowly than controls and stained poorly with alcian blue. The patients cultured cells showed reduced incorporation of [35S]sulfate relative to [3H]glucosamine, impaired uptake of sulfate, and higher resistance to chromate toxicity compared to control cells. Epiphyseal chondrocytes cultured in alginate beads synthesized proteoglycans of normal molecular size as judged by gel filtration chromatography, but undersulfated as judged by ion exchange chromatography and by the amount of nonsulfated disaccharide. High performance liquid chromatography analysis of chondroitinase-digested proteoglycans showed that sulfated disaccharides were present, although in reduced amounts, indicating that at least in vitro, other sources of sulfate can partially compensate for sulfate deficiency. A t1475c transition causing a L483P substitution in the eleventh transmembrane domain of the sulfate/chloride antiporter was present on both alleles in the patient who was the product of a consanguineous marriage. The results indicate that the defect of sulfate transport is expressed in both chondrocytes and fibroblasts and results in the synthesis of proteoglycans bearing glycosaminoglycan chains which are poorly sulfated but of normal length.

Clinical homogeneity of the Stüve-Wiedemann syndrome and overlap with the Schwartz-Jampel syndrome type 2

The Stüve-Wiedemann syndrome (SWS) is a rare disorder characterized by respiratory distress, hyperthermic episodes, and early lethality and radiologically by bowing of the long bones with internal cortical thickening and large metaphyses. We report findings in 8 new patients suggesting that this syndrome is clinically homogeneous. All patients had feeding and swallowing difficulties, respiratory insufficiency, abnormal appearance, muscle hypotonia, and postnatal short stature. Recurrent episodes of unexplained fever occurred in all and were the cause of death in 6 of 8 cases. Parental consanguinity and sib recurrence suggest autosomal recessive inheritance. The clinical, radiological, and histological similarities between our patients with SWS and those with the recently delineated neonatal Schwartz-Jampel syndrome (SJS type 2) lead us to suggest that SWS and SJS type 2 may be a single entity.

Clinical, radiological, and chondro-osseous findings in opsismodysplasia: survey of a series of 12 unreported cases

Opsismodysplasia ( opsismos in Greek = late) is a rare chondrodysplasia, first described in 1977 by Zonana et al 1 as a unique chondrodysplasia and designated “opsismodysplasia” only in 1984.2 The disorder is characterised clinically by micromelia with extremely short hands and feet and respiratory distress responsible for death in the first few years of life.2 The main radiological features include severe platyspondyly, major delay in skeletal ossification, and metaphyseal cupping. To date, 13 cases have been reported and recurrence in sibs and/or consanguinity have suggested an autosomal recessive mode of inheritance.1–6 Here, we describe the clinical, radiological and chondro-osseous findings of 12 previously unreported cases in nine families. We show that opsismodysplasia is not a consistently lethal condition and we identify the severity of the delayed bone ossification as an important feature, distinct from other forms of spondylo(epi)metaphyseal dysplasias.nnSeven male and five female cases originating from nine families of French (4/9), German, (1/9), and Algerian origin (4/9) were included in the study. All affected cases presented with the following inclusion criteria: (1) major delay in epiphyseal ossification, (2) platyspondyly, (3) metaphyseal cupping, and (4) very short metacarpals and phalanges. Half of the cases were prenatally diagnosed during ultrasound follow up of the pregnancies and half of them were diagnosed postnatally. Pregnancies were terminated between 15 and 29 weeks’ of gestation. For chondro-osseous studies, the samples were embedded in paraffin and HES routinely stained after fixation in 4% formaldehyde and decalcification with EDTA.nn### Key points

Safety study of Ciprofloxacin in newborn mice.

Ciprofloxacin, a broad-spectrum antimicrobial agent belonging to the fluoroquinolone family, is prescribed off-label in infants less than one year of age. Ciprofloxacin is included in the European Medicines Agency priority list of off-patent medicinal products requiring evaluation in neonates. This evaluation is undergoing within the TINN (Treat Infections in Neonates) FP7 EU project. As part of the TINN project, the present preclinical study was designed to assess the potential adverse effects of Ciprofloxacin on neurodevelopment, liver and joints in mice. Newborn mice received subcutaneous Ciprofloxacin at 10, 30 and 100xa0mg/kg/day from 2 to 12 postnatal days. Peak plasma levels of Ciprofloxacin were in the range of levels measured in human neonates. We examined vital functions inxa0vivo, including cardiorespiratory parameters and temperature, psychomotor development, exploratory behavior, arthro-, nephro- and hepato-toxic effects. We found no effect of Ciprofloxacin at 10 and 30xa0mg/kg/day. In contrast, administration at 100xa0mg/kg/day delayed weight gain, impaired cardiorespiratory and psychomotor development, caused inflammatory infiltrates in the connective tissues surrounding the knee joint, and moderately increased extramedullary hematopoiesis. The present study pleads for careful watching of cardiorespiratory and motor development in neonates treated with Ciprofloxacin, in addition to the standard surveillance of arthrotoxicity.

A new lethal syndrome of exomphalos, short limbs, and macrogonadism

We report a new lethal multiple congenital abnormality (MCA) syndrome of exomphalos, short limbs, nuchal web, macrogonadism, and facial dysmorphism in seven fetuses (six males and one female) belonging to three unrelated families. Xrays showed enlarged and irregular metaphyses with a heterogeneous pattern of mineralisation of the long bones. Pathological examination showed adrenal cytomegaly, hyperplasia of Leydig cells, ovarian stroma cells, and Langherans cells, and renal microcysts. We suggest that this condition is a new autosomal recessive MCA syndrome different from Beckwith-Wiedemann syndrome, especially as no infracytogenetic deletion or uniparental disomy of chromosome 11 was found.