Marc Abitbol

Mutation of the bone morphogenetic protein GDF3 causes ocular and skeletal anomalies

Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel-Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.

Incomplete penetrance and phenotypic variability characterize Gdf6-attributable oculo-skeletal phenotypes

Proteins of the bone morphogenetic protein (BMP) family are known to have a role in ocular and skeletal development; however, because of their widespread expression and functional redundancy, less progress has been made identifying the roles of individual BMPs in human disease. We identified seven heterozygous mutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocular and vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, and demonstrated comparable phenotypes in model organisms with reduced Gdf6 function. We observed a spectrum of ocular and skeletal anomalies in morphant zebrafish, the latter encompassing defective tail formation and altered expression of somite markers noggin1 and noggin2. Gdf6(+/-) mice exhibited variable ocular phenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evident between patients and animal models included pleiotropic effects, variable expressivity and incomplete penetrance. These data establish the important role of this determinant in ocular and vertebral development, demonstrate the complex genetic inheritance of these phenotypes, and further understanding of BMP function and its contributions to human disease.

A new dimension for the human genome project: towards comprehensive expression maps

The current Human Genome Project is largely devoted to structural characterisation of our genome. We now need international co-ordination of a second phase of genome analysis, the systematic construction of expression maps using both basic and high-resolution expression assays. Databases recording different types of expression pattern for a variety of human cell types need to be established and co-ordinated. There is a compelling need for a database of gene expression in early human development, but the scarcity of human material for study requires optimisation of research strategies and co-ordination of expression studies in early human and mouse development.

Sirt1 Involvement in rd10 Mouse Retinal Degeneration

PURPOSE Sirtuin1 (Sirt1) is an NAD(+)-dependent deacetylase involved in development, cell survival, stress resistance, energy metabolism, and aging. It is expressed in the mammalian central nervous system (CNS) and is activated during processes associated with neuroprotection. The retinal degeneration 10 (rd10) mouse model of retinitis pigmentosa (RP) was used to investigate the possible role of Sirt1 in this type of retinal degeneration. METHODS Eyes from control and rd10 mice were used. Sirt1 mRNA was detected by in situ hybridization, and its abundance was estimated by semiquantitative RT-PCR. The presence of Sirt1 protein was investigated by immunohistofluorescence and Western blot analysis. The apoptosis of photoreceptor cells was analyzed by terminal dUTP transferase nick-end labeling (TUNEL). Immunolabeling for Sirt1, apoptosis-inducing factor (Aif), and caspase-12 (Casp-12) was performed on retinal tissue sections. RESULTS Sirt1 mRNA and immunoreactivity were observed in normal adult mouse eyes. In the control retina, Sirt1 was immunolocalized mostly to the nucleus. In rd10 mice with retinal degeneration, changes in Sirt1 immunolabeling were observed only in the retinal outer nuclear layer (ONL). The pathologic pattern of Sirt1 immunoreactivity correlated with the start of retinal degeneration in rd10 mice. CONCLUSIONS The results suggest a link between Sirt1 production and retinal degeneration in rd10 mice. The anti-apoptotic, neuroprotective role of Sirt1 in the mouse retina is based on the involvement of Sirt1 in double DNA strand-break repair mechanisms and in maintaining energy homeostasis in photoreceptor cells. The results suggest that the neuroprotective properties of Sirt1 may gradually weaken in rd10 mouse photoreceptor cells.

Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet.

PURPOSE To evaluate functional and ultrastructural changes in the retina of scavenger receptor B1 (SR-BI) knockout (KO) mice consuming a high fat cholate (HFC) diet. METHODS Three-month-old male KO and wild-type (WT) mice were fed an HFC diet for 30 weeks. After diet supplementation, plasma cholesterol levels and electroretinograms were analyzed. Neutral lipids were detected with oil red O, and immunohistochemistry was performed on cryostat ocular tissue sections. The retina, Bruchs membrane (BM), retinal pigment epithelium (RPE), and choriocapillaris (CC) were analyzed by transmission electron microscopy. RESULTS Using the WT for reference, ultrastructural changes were recorded in HFC-fed SR-BI KO mice, including lipid inclusions, a patchy disorganization of the photoreceptor outer segment (POS) and the outer nuclear layer (ONL), and BM thickening with sparse sub-RPE deposits. Within the CC, there was abnormal disorganization of collagen fibers localized in ectopic sites with sparse and large vacuolization associated with infiltration of macrophages in the subretinal space, reflecting local inflammation. These lesions were associated with electroretinographic abnormalities, particularly increasing implicit time in a- and b-wave scotopic responses. Abnormal vascular endothelial growth factor (VEGF) staining was detected in the outer nuclear layer. CONCLUSIONS HFC-fed SR-BI KO mice thus presented sub-RPE lipid-rich deposits and functional and morphologic alterations similar to some features observed in dry AMD. The findings lend further support to the hypothesis that atherosclerosis causes retinal and subretinal damage that increases susceptibility to some forms of AMD.

Near‐complete adaptation of the PRiMA knockout to the lack of central acetylcholinesterase

J. Neurochem. (2012) 122, 1065–1080.

Pituitary function and glucose tolerance in a family with a PAX6 mutation

BACKGROUND PAX6 is a transcription factor involved in the regulation of eye and islet cell development in humans and has also been shown to be an early marker of the pituitary gland in mice. While some subjects with PAX6 mutations were found to have impaired glucose tolerance or diabetes in two previous studies, there has been no report of systematic pituitary function assessment in these patients. AIM The objective of this report was to assess pituitary function and glucose tolerance in five related patients with a heterozygous PAX6 mutation and an unusual ocular and neurological phenotype. SUBJECTS AND METHODS Pituitary function (static and dynamic exploration of the five ante-pituitary axes) and glucose tolerance (oral glucose tolerance test) were explored in all patients. RESULTS Glucose tolerance was normal in all patients. We found no obvious pituitary deficiency in four of the five patients. However, borderline cortisol levels were observed in three out of these patients, with subnormal values, at baseline and/or after stimulation test. Basal and stimulated cortisol levels were both more clearly diminished in one subject. CONCLUSIONS We report here the first complete pituitary function assessment, together with glucose tolerance evaluations, in five related patients with a PAX6 mutation. We cannot rule out subtle corticotrope deficiency induced by PAX6 mutation. The conflicting results with the literature about glucose tolerance could be explained by genotype/phenotype correlations.

Usefulness of cocaine drops in investigating infant anisocoria

INTRODUCTION Whereas apraclonidine has eclipsed cocaine test in the exploration of unilateral miosis in adults, its use in infants is avoided because of the risk of central nervous system depression. This chart review evaluates the usefulness of cocaine drops in infants. METHODS Infants under the age of one referred for unilateral miosis between November 1, 2009 and November 1, 2015, were reviewed. Patients underwent the following protocol: (1) in case of isolated miosis, cocaine test was performed. If the miotic pupil did not dilate, imaging was performed. Dilation in both eyes led to simple clinical follow-up. (2) In case of miosis associated with ptosis or iris heterochromia, imaging of the brain, neck and chest was directly performed. RESULTS Twenty-six children were included. Twenty-two presented an isolated miosis; three had ipsilateral ptosis, and one had no pupillary light reflex in the miotic eye. Cocaine tests performed in the 22 patients led to imaging in four, which was always normal. No side effect of the test was noticed. Imaging found one neuroblastoma and one intraorbital hemolymphangioma in two patients presenting miosis plus another sign. Imaging was avoided for 18 patients thanks to negative cocaine test. DISCUSSION Urgent imaging is mandatory in infants presenting with miosis associated with other localizing sign on the sympathetic nerve pathway (Horner syndrome). Since the uselessness of complementary investigations in isolated infantile miosis cannot be proven so far, cocaine test remains the gold standard, as it is safe, cheaper and less stressful than systematic imaging.

Homozygous Nonsense Mutations in RBP3 Gene Cause Early-Onset Retinal Dystrophies Associated With High Myopia

The very detailed report by Arno et al. 1 of early-onset autosomal recessive dystrophies associated with high myopia occurring in young children carrying homozygous nonsense mutations of the RBP3 gene highlights the power of the combination of meticulous phenotyping and whole-exome sequencing analysis in the field of inherited retinal dystrophies. A homozygous missense mutation of the RBP3 gene was previously associated with autosomal recessive retinitis pigmentosa (RP) and high myopia in adult patients. 2 The recent findings emphasize the importance of longitudinal phenotypic studies based on the addition of the most modern methods of investigation, such as fundus autofluorescence imaging and spectral-domain optical coherence tomography (OCT), to the classical methods used for the diagnosis of retinal dystrophies. The authors not only have discovered novel phenotypes of retinal dystrophies caused by different mutations in the same gene RBP3, but also show unequivocally that the loss of the inner segment ellipsoid band over peripheral macular areas is a key marker of the novel dystrophies found. Whether these novel phenotypes will evolve toward RP remains to be demonstrated by rigorous follow-up studies of each affected patient. This report illustrates the necessity of increasing our knowledge on the unknown and probably diverse functions of the interphotoreceptor retinoid binding protein (IRBP). 3 The roles of IRBP in the normal development of human eye and retina are far from being fully understood. The exploration of the role of IRBP in the pathophysiology of nonsyndromic myopia requires the urgent implementation of cleverly designed innovative experiments. Finally, this important study points toward the possibility of a window of opportunity for gene therapy or pharmacological therapies in some patients affected by RBP3 homozygous mutations.

Novel insights provided by spectral-domain coherent tomography in pediatric ophthalmology

Tilted disk syndrome (TDS) is observed in approximately 1 % to 2 % of the population. This percentage appears to be highly variable according to the clinical and imaging features retained for establishing the accurate diagnosis. It is of paramount importancetodistinguish isolated tiltedoptic diskfrom TDS. This syndrome has a number of alternative nomenclatures such as the nasal fundus ectasia syndrome, inversion of the optic disk, inverse myopia, and dysversion of the optic disk. TDS may be a variant of colobomatous defects. The definition of TDS has often been inaccurate in many studies which did not take account of the antero-posterior tilt and torsion of the optic disk. This so-called torsion corresponds to the rotation of the optic nerve around its sagittal axis. Additionally, until now there was neither an objective index of tilt nor any objective index of rotation. Surrogate indexes wereproposedbasedonthe ellipticformofthe optic disk. The ratio of the minimal diameter and the maximal diameter of the optic disk or the inverse ratio have been used for evaluating the tilt. Normal optic disks are minimally oval, with the vertical diameter slightly longer than the horizontal. Optic disk torsion can be viewed and measured in twodimensional space. The longest diameter usually falls within 15° of the vertical meridian; axes beyond 15° are, by definition, torted. Most tilted disks appear to rotate excessively about the sagittal axis of the optic nerve, such that the longest diameter of the disk falls outside this 30° boundary. This apparent rotation is often accentuated by the orientation of