Phenotype in Two Consanguineous Tunisian Families With non Syndromic Autosomic Recessive Retinitis Pigmentosa Caused by an USH2A Mutation

Purpose: To assess the clinical phenotype in two consanguineous Tunisian families with non syndromic autosomic recessive retinitis Pigmentosa (arRP) caused by an USH2A mutation.Methods: All accessible members of family A and B were included and underwent full ophthalmic examination with best corrected Snellen visual acuity, kinetic visual field testing, fundus photography, optical coherence tomography and full field electroretinography. Haplotype analyses were used to test linkage in the families to 20 arRP loci, including ABCA4, LRAT, USH2A, RP29, CERKL, CNGA1, CNGB1, CRB1, EYS, RP28, MERTK, NR2E3, PDE6A, PDE6B, RGR, RHO, RLBP1, TULP1. In addition, index patients were sent to AsperOphthalmics for arRP mutation screening.Results: Twenty three patients from the two families were ascertained for the study. Eight of the 23 members were clinically affected with arRP without hearing loss. Age range at baseline was 35 to 63 years (mean age was 46.5 years). For all affected members, night blindness appeared during the second decade. Visual acuity at baseline ranged from 20/50 to 20/32. Kinetic visual field was severely constricted. Fundus examination revealed typical RP changes with bone spicule-shaped pigment deposits in the mid periphery along with atrophy of the retina, narrowing of the vessels and waxy optic discs. Tomograms showed a thinning and even loss the outer nuclear layer of the fovea. ERG was unrecordable in scotopic conditions and the cone responses were markedly hypovolted. Haplotype analysis did not reveal any homozygosity. Screening at AsperOphthalmis showed a compound heterozygous [p.A1953G]+[p.I5126T] in family A and [p.G713R]+[p.W4149R] in family B.Conclusions: For these families, changes were typical of those that have been described in patients with moderate to severe forms of non syndromic recessive RP. Our findings support the need to consider possible involvement of USH2A not only in patients with Usher syndrome but also in patients with non syndromc arRP. Despite consanguinity, the presence of non-homozygous mutants illustrates the complexity of molecular analysis.

Botulinum toxin A in non-dystonic tremors.

The present pilot study evaluated the effect of botulinum toxin A on primarily non-dystonic tremors using accelerometry in a single-blind, placebo-controlled design. Resting, postural, intention, or head tremor were assessed before and approximately 1 month after intramuscular saline and botulinum toxin A (25-50 U) respectively. Half of the patients showed > or = 30% placebo effect. Tremor in 10 of 17 patients (60%) studied improved further after botulinum toxin A (range 30-95%), exceeding the placebo effect by > or = 30%. Nine patients demonstrated clinically significant focal weakness in the extensor muscles after botulinum toxin A which interfered with fine movements. Patients were subdivided into PD-like and ET-like tremor(s). Both groups experienced large placebo effects for resting tremor, with little or no further improvement after botulinum toxin A. The improvement in postural tremor after botulinum toxin A, of 40% in the PD-like and 57% in the ET-like groups, however, was approximately twice that of placebo. In conclusion, botulinum toxin A exerts a modest tremorlytic effect, however the dose, and its distribution over the sites injected, need to be optimised to minimise focal weakness.

Analysis of angiotensin II- and ACTH-driven mineralocorticoid functions and omental adiposity in a non-genetic, hyperadipose female rat phenotype

The hypothalamic damage induced by neonatal treatment with monosodium l-glutamate (MSG) induces several metabolic abnormalities, resulting in a rat hyperleptinemic-hyperadipose phenotype. This study was conducted to explore the impact of the neonatal MSG treatment, in the adult (120 days old) female rat on: (a) the in vivo and in vitro mineralocorticoid responses to ACTH and angiotensin II (AII); (b) the effect of leptin on ACTH- and AII-stimulated mineralocorticoid secretions by isolated corticoadrenal cells; and (c) abdominal adiposity characteristics. Our data indicate that, compared with age-matched controls, MSG rats displayed: (1) enhanced and reduced mineralocorticoid responses to ACTH and AII treatments, respectively, effects observed in both in vivo and in vitro conditions; (2) adrenal refractoriness to the inhibitory effect of exogenous leptin on ACTH-stimulated aldosterone output by isolated adrenocortical cells; and (3) distorted omental adiposity morphology and function. This study supports that the adult hyperleptinemic MSG female rat is characterized by enhanced ACTH-driven mineralocorticoid function, impaired adrenal leptin sensitivity, and disrupted abdominal adiposity function. MSG rats could counteract undesirable effects of glucocorticoid excess, by developing a reduced AII-driven mineralocorticoid function. Thus, chronic hyperleptinemia could play a protective role against ACTH-mediated allostatic loads in the adrenal leptin resistant, MSG female rat phenotype.

Early Carboniferous age of the Versoyen ophiolites and consequences: non-existence of a "Valais ocean'' (Lower Penninic, western Alps)

Ophiolites occur at several places in the Lower Penninic of the W and Central Alps. They are generally ascribed to oceanic crust of a so-called ``Valais ocean'' of Cretaceous age which plays a fundamental role in many models of Alpine paleogeography and geodynamics. The type locality and only observational base for the definition of a ``Valais ocean'' in the W Alps is the Versoyen ophiolitic complex, on the French-Italian boundary W of the Petit St-Bernard col. The idea of a "Valais ocean'' is based on two propositions that are since 40 years the basis for most reconstructions of the Lower Penninic: (1) The Versoyen forms the (overturned) stratigraphic base of the Cretaceous-Tertiary Valais-Tarentaise series; and (2) it has a Cretaceous age. We present new field and isotopic data that severely challenge both propositions. (1) The base of the Versoyen ophiolite is a thrust. It overlies a wildflysch with blocks of Versoyen rocks, named the Mechandeur Formation. This ``supra-Tarentaise'' wildflysch has been confused with an (overturned) stratigraphic transition from the Versoyen to the Valais-Tarentaise series. Thus the contact Versoyen/Tarentaise is not stratigraphic but tectonic, and the Versoyen ophiolite has no link with the Valais basin. This thrust corresponds to an inverse metamorphic discontinuity and to an abrupt change in tectonic style. (2) The contact of the Versoyen complex with the overlying Triassic-Jurassic Petit St-Bernard (PSB) series is stratigraphic (and not tectonic as admitted by all authors since 50 years). Several types of sedimentary structures polarize it and show that the PSB series is younger than the Versoyen. Consequently the Versoyen ophiolitic complex is Paleozoic and forms the basement of the PSB Mesozoic sediments. They both belong to a single tectonic unit, named the Versoyen-Petit St-Bernard nappe. (3) Ion microprobe U-Pb isotopic data on zircons from the main gabbroic intrusion in the Versoyen complex give a crystallization age of 337.0 +/- 4.1 Ma (Visean, Early Carboniferous). These zircons show typical oscillatory zoning and no overgrowth or corrosion. and are interpreted to date the Versoyen magmatism. These U-Pb data are in excellent agreement with our field observations and confirm the Paleozoic age of the Versoyen ophiolite. The existence of a ``Valais ocean'' of Cretaceous age in the W Alps becomes very improbable. The eclogite facies metamorphism of the Versoyen-Petit St-Bernard nappe results from an Alpine intra-continental subduction, guided by a Paleozoic oceanic suture. This is an example of the lone term influence of inherited deep-seated structures on a Much younger orogeny. This might well be a major cause of of the inherent complexity of the Alps.

Multiple adenosine 3',5'-cyclic [corrected] monophosphate response element DNA-binding proteins generated by gene diversification and alternative exon splicing.

The protein sequence deduced from the open reading frame of a human placental cDNA encoding a cAMP-responsive enhancer (CRE)-binding protein (CREB-327) has structural features characteristic of several other transcriptional transactivator proteins including jun, fos, C/EBP, myc, and CRE-BP1. Results of Southwestern analysis of nuclear extracts from several different cell lines show that there are multiple CRE-binding proteins, which vary in size in cell lines derived from different tissues and animal species. To examine the molecular diversity of CREB-327 and related proteins at the nucleic acid level, we used labeled cDNAs from human placenta that encode two different CRE-binding proteins (CREB-327 and CRE-BP1) to probe Northern and Southern blots. Both probes hybridized to multiple fragments on Southern blots of genomic DNA from various species. Alternatively, when a human placental c-jun probe was hybridized to the same blot, a single fragment was detected in most cases, consistent with the intronless nature of the human c-jun gene. The CREB-327 probe hybridized to multiple mRNAs, derived from human placenta, ranging in size from 2-9 kilobases. In contrast, the CRE-BP1 probe identified a single 4-kilobase mRNA. Sequence analyses of several overlapping human genomic cosmid clones containing CREB-327 sequences in conjunction with polymerase chain reaction indicates that the CREB-327/341 cDNAs are composed of at least eight or nine exons, and analyses of human placental cDNAs provide direct evidence for at least one alternatively spliced exon. Analyses of mouse/hamster-human hybridoma DNAs by Southern blotting and polymerase chain reaction localizes the CREB-327/341 gene to human chromosome 2. The results indicate that there is a dichotomy of CREB-like proteins, those that are related by overall structure and DNA-binding specificity as well as those that are related by close similarities of primary sequences.

Unusual astrocyte reactivity caused by the food mycotoxin ochratoxin A in aggregating rat brain cell cultures.

Ochratoxin A (OTA), a mycotoxin and widespread food contaminant, is known for its patent nephrotoxicity and potential neurotoxicity. Previous observations in vitro showed that in the CNS, glial cells were particularly sensitive to OTA. In the search for the molecular mechanisms underlying OTA neurotoxicity, we investigated the relationship between OTA toxicity and glial reactivity, in serum-free aggregating brain cell cultures. Using quantitative reverse transcriptase-polymerase chain reaction to analyze changes in gene expression, we found that in astrocytes, non cytotoxic concentrations of OTA down-regulated glial fibrillary acidic protein, while it up-regulated vimentin and the peroxisome proliferator-activated receptor-gamma expression. OTA also up-regulated the inducible nitric oxide synthase and the heme oxygenase-1. These OTA-induced alterations in gene expression were more pronounced in cultures at an advanced stage of maturation. The natural peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-delta(12,14) prostaglandin J2, and the cyclic AMP analog, bromo cyclic AMP, significantly attenuated the strong induction of peroxisome proliferator-activated receptor-gamma and inducible nitric oxide synthase, while they partially reversed the inhibitory effect of OTA on glial fibrillary acidic protein. The present results show that OTA affects the cytoskeletal integrity of astrocytes as well as the expression of genes pertaining to the brain inflammatory response system, and suggest that a relationship exists between the inflammatory events and the cytoskeletal changes induced by OTA. Furthermore, these results suggest that, by inducing an atypical glial reactivity, OTA may severely affect the neuroprotective capacity of glial cells.

Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina.

PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.

Trabecular bone score improves fracture risk prediction in non-osteoporotic women: the OFELY study.

The use of areal bone mineral density (aBMD) for fracture prediction may be enhanced by considering bone microarchitectural deterioration. Trabecular bone score (TBS) helped in redefining a significant subset of non-osteoporotic women as a higher risk group. INTRODUCTION: TBS is an index of bone microarchitecture. Our goal was to assess the ability of TBS to predict incident fracture. METHODS: TBS was assessed in 560 postmenopausal women from the Os des Femmes de Lyon cohort, who had a lumbar spine (LS) DXA scan (QDR 4500A, Hologic) between years 2000 and 2001. During a mean follow-up of 7.8 ± 1.3 years, 94 women sustained 112 fragility fractures. RESULTS: At the time of baseline DXA scan, women with incident fracture were significantly older (70 ± 9 vs. 65 ± 8 years) and had a lower LS_aBMD and LS_TBS (both -0.4SD, p < 0.001) than women without fracture. The magnitude of fracture prediction was similar for LS_aBMD and LS_TBS (odds ratio [95 % confidence interval] = 1.4 [1.2;1.7] and 1.6 [1.2;2.0]). After adjustment for age and prevalent fracture, LS_TBS remained predictive of an increased risk of fracture. Yet, its addition to age, prevalent fracture, and LS_aBMD did not reach the level of significance to improve the fracture prediction. When using the WHO classification, 39 % of fractures occurred in osteoporotic women, 46 % in osteopenic women, and 15 % in women with T-score > -1. Thirty-seven percent of fractures occurred in the lowest quartile of LS_TBS, regardless of BMD. Moreover, 35 % of fractures that occurred in osteopenic women were classified below this LS_TBS threshold. CONCLUSION: In conclusion, LS_aBMD and LS_TBS predicted fractures equally well. In our cohort, the addition of LS_TBS to age and LS_aBMD added only limited information on fracture risk prediction. However, using the lowest quartile of LS_TBS helped in redefining a significant subset of non-osteoporotic women as a higher risk group which is important for patient management.

Effect of endotoxin on the angiotensin II receptor in cultured vascular smooth muscle cells.

1. In some tissues, a decrease in the number of cell surface receptors and alterations of the receptor coupling have been proposed as possible mechanisms mediating the deleterious effects of bacterial endotoxin in septic shock. 2. The effects of bacterial lipopolysaccharide (Escherichia coli 0111-B4; LPS) on vascular angiotensin II and vasopressin receptors have been examined in cultured aortic smooth muscle cells (SMC) of the rat by use of radioligand binding techniques. 3. In vascular SMC exposed to 1 micrograms ml-1 endotoxin for 24 h, a significant increase in angiotensin II binding was found. The change in [125I]-angiotensin II binding corresponded to an increase in the number of receptors whereas the affinity of the receptors was not affected by LPS. In contrast, no change in [3H]-vasopressin binding was observed. 4. The pharmacological characterization of angiotensin II binding sites in control and LPS-exposed cells demonstrated that LPS induced an increase in the AT1 subtype of the angiotensin II receptors. Receptor coupling as evaluated by measuring total inositol phosphates was not impaired by LPS. 5. The effect of LPS on the angiotensin II receptor was dose-, time- and protein-synthesis dependent and was associated with an increased expression of the receptor gene. 6. The ability of LPS to increase angiotensin II binding in cultured vascular SMC was independent of the endotoxin induction of NO-synthase. 7. These results suggest that, besides inducing factors such as cytokines and NO-synthase, endotoxin may enhance the expression of cell surface receptors. The surprising increase in angiotensin II binding in LPS exposed VSM cells may represent an attempt by the cells to compensate for the decreased vascular responsiveness. It may also result from a non-specific LPS-related induction of genes.