Jean-Louis Dufier

Retinal Dehydrogenase 12 (RDH12) Mutations in Leber Congenital Amaurosis

Leber congenital amaurosis (LCA), the most early-onset and severe form of all inherited retinal dystrophies, is responsible for congenital blindness. Ten LCA genes have been mapped, and seven of these have been identified. Because some of these genes are involved in the visual cycle, we regarded the retinal pigment epithelium and photoreceptor-specific retinal dehydrogenase (RDH) genes as candidate genes in LCA. Studying a series of 110 unrelated patients with LCA, we found mutations in the photoreceptor-specific RDH12 gene in a significant subset of patients (4.1%). Interestingly, all patients harboring RDH12 mutations had a severe yet progressive rod-cone dystrophy with severe macular atrophy but no or mild hyperopia.

Spectrum of ABCR gene mutations in autosomal recessive macular dystrophies.

Stargardt disease (STGD) and late-onset fundus flavimaculatus (FFM) are autosomal recessive conditions leading to macular degenerations in childhood and adulthood, respectively. Recently, mutations of the photoreceptor cell-specific ATP binding transporter gene (ABCR) have been reported in Stargardt disease. Here, we report on the screening of the whole coding sequence of the ABCR gene in 40 unrelated STGD and 15 FFM families and we show that mutations truncating the ABCR protein consistently led to STGD. Conversely, all mutations identified in FFM were missense mutations affecting uncharged amino acids. These results provide the first genotype-phenotype correlations in ABCR gene mutations.

Complete exon-intron structure of the RPGR-interacting protein (RPGRIP1) gene allows the identification of mutations underlying Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is a genetically heterogeneous autosomal recessive condition responsible for congenital blindness or greatly impaired vision since birth. So far, six LCA loci have been mapped but only 4 out of 6 genes have been identified. A genome-wide screen for homozygosity was conducted in seven consanguineous families unlinked to any of the six LCA loci. Evidence for homozygosity was found in two of these seven families at the 14q11 chromosomal region. Two retinal specific candidate genes were known to map to this region, namely the neural retina leucine zipper (NRL) and the retinitis pigmentosa GTPase regulator interacting protein (RPGRIP1). No mutation of the NRL gene was found in any of the two families. Thus, we determined the complete exon-intron structure of the RPGRIP1 gene. RPGRIP1 encompasses 24 coding exons, nine of which are first described here with their corresponding exon-intron boundaries. The screening of the gene in the two families consistent with linkage to chromosome 14q11 allowed the identification of a homozygous null mutation and a homozygous missense mutation, respectively. Further screening of LCA patients unlinked to any of the four already identified LCA genes (n=86) identified seven additional mutations in six of them. In total, eight distinct mutations (5 out of 8 truncating) in 8/93 patients were found. So far this gene accounts for eight out of 142 LCA cases in our series (5.6%).

A gene for Usher syndrome type I (USH1A) maps to chromosome 14q

Usher syndrome (US) is an autosomal recessive disease characterized by congenital hearing impairment and retinitis pigmentosa. It is the most frequent cause of deaf-blindness in adults and accounts for 3 to 6% of deaf children. Here, we report the genetic mapping of a gene for US type I (USH1A), the most severe form of the disease, to the long arm of chromosome 14, by linkage to probe MLJ14 at the D14S13 locus in 10 families of Western France ancestry (Z = 4.13 at theta = 0). Among them, 8 families originated from a small area of the Poitou-Charentes region (Z = 3.78 at theta = 0), suggesting that a founder effect could be involved. However, since not all US type I families were found to be linked to this locus, the present study provides evidence for genetic heterogeneity of this condition (heterogeneity versus homogeneity test HOMOG, P < 0.05; heterogeneity versus no linkage, P < 0.01).

Expression of myosin VIIA during mouse embryogenesis

Abstract The gene encoding myosin VIIA is responsible for the mouse shaker-1 phenotype, which consists of deafness and balance deficiency related to cochlear and vestibular neuroepithelial defects. In humans, a defective myosin VIIA gene is responsible for Usher syndrome type IB, which associates congenital deafness, vestibular dysfunction and retinitis pigmentosa. In an attempt to progress in the understanding of the function(s) of myosin VIIA, we studied the expression of the myosin VIIA gene during mouse embryonic development. Embryos from day 9 (E9) to E18 were analyzed by in situ hybridization and immunohistofluorescence. The myosin VIIA mRNA and protein were consistently detected in the same embryonic tissues throughout development. Myosin VIIA was first observed in the otic vesicle at E9, and later in a variety of tissues. The olfactory epithelium and the liver express it as early as E10. In the retinal pigment epithelium, choroid plexus, adrenal gland and tongue, expression begins at E12 and in the testis and the adenohypophysis at E13. In the small intestine, kidney and hair follicles of the vibrissae, expression of myosin VIIA starts only at E15. Myosin VIIA expression was observed only in epithelial cell types, most of which possess microvilli or cilia. Interestingly, myosin VIIA expression seems to be concomitant with the appearance of these structures in the epithelial cells, suggesting a role for this myosin in their morphogenesis. The cellular location of myosin VIIA within sensory hair cells and olfactory receptor neurons also argues for a role of this protein in the synaptic vesicle trafficking.

Mutations of the retinal specific ATP binding transporter gene (ABCR) in a single family segregating both autosomal recessive retinitis pigmentosa RP19 and Stargardt disease: evidence of clinical heterogeneity at this locus

Stargardt disease (STGD) is an autosomal recessive macular dystrophy of childhood characterised by bilateral loss of central vision over a period of several months. STGD has been mapped to chromosome 1p22.1 and recently ascribed to mutations in the retinal specific ATP binding transporter gene (ABCR). The fundus flavimaculatus with macular dystrophy (FFM), an autosomal recessive condition responsible for gradual loss of visual acuity in adulthood (second to third decade) has also been mapped to the same locus. However, a gene for autosomal recessive retinitis pigmentosa with distinctive features of choriocapillaris atrophy at an advanced stage (RP19) has been mapped to the genetic interval encompassing the STGD gene on chromosome 1p (D1S435-D1S236), raising the question of whether, despite striking differences in clinical course and presentation, RP19 and STGD might be allelic disorders at the ABCR locus. In a family segregating RP and STGD in two first cousins, we found that heterozygosity for a splicing mutation in the ABCR gene (1938-1 G→A) resulted in STGD while hemizygosity for this splice mutation resulted in RP, and when studying the RP patient’s parents, we found a maternal non-contribution with apparent segregation of a null allele ascribed to a partial deletion of the ABCR gene. The present study shows that, despite striking clinical differences, RP19 and STGD are allelic disorders at the ABCR locus.

Ophthalmologic findings in suspected child abuse victims with subdural hematomas

PURPOSE Shaken baby syndrome consists of intracranial and intraocular hemorrhages in young children in the absence of signs of direct head trauma. Because it has major medicolegal implications, it must be distinguished from accidental trauma. This study aimed to determine the ophthalmologic manifestations and their natural course in child abuse victims and whether ophthalmologic examination can help to distinguish shaken babies from children with accidental impact head trauma. DESIGN Prospective comparative observational case series. METHODS A prospective study was conducted from January 1996 to September 2001 on 241 consecutive infants hospitalized for a subdural hematoma to determine the frequency and the type of ocular abnormalities encountered. At admission, 186 children were highly presumed to have been shaken (group 1), 38 children had signs of direct head trauma without any relevant history of trauma (group 2), some of them having been possibly shaken, whereas 7 children had proven severe accidental head trauma (group 3). RESULTS Intraocular hemorrhages were the main finding. Their shape, laterality, and size were not significantly different in groups 1 and 2. However they were significantly more frequent in nonaccidental head trauma than in infants with head impact (77.5% versus 20%). None of the group 3 children had intraocular hemorrhage. Eighty-two percent of intraocular hemorrhages resolved within 4 weeks. CONCLUSIONS Intraocular hemorrhages are frequent in shaken babies but not specific of this syndrome. When associated with a subdural hematoma, they are strongly suggestive of shaken neglect. They are rare in pediatric accidental head trauma.

Renal coloboma syndrome

OBJECTIVE To characterize the ocular features of renal coloboma syndrome. DESIGN Prospective, observational case series. PARTICIPANTS Twelve patients referred by the pediatric nephrology clinic and the ophthalmic records of five additional patients. METHODS For each patient, age at the time of examination, gender, renal function, and presence of a mutation in the PAX2 gene were noted. All patients underwent measurement of visual acuity and anterior and posterior segment examination with fundus photography. Goldmann visual fields were tested in four cases. MAIN OUTCOME MEASURES Visual acuity, optic disc abnormalities, and mutation in the PAX2 gene. RESULTS Mean age was 21.5 years. Renal failure was mild in 6 patients and severe in 11 patients. A mutation in the PAX2 gene was identified in nine patients, without correlation to the ocular phenotype. Ocular features could be divided into five groups: optic disc dysplasia limited to an unusual pattern of retinal vessels without functional consequence; optic disc pit with normal visual acuity and blind spot enlargement; large optic disc coloboma; large coloboma of the optic disc and adjacent retina; morning glory anomaly (these last three conditions were accompanied by poor visual acuity). Fundus abnormalities were symmetrical in most cases and unrelated to renal status. CONCLUSIONS Ophthalmic and renal characteristics of the renal coloboma syndrome are highly variable. The need for dialysis or renal transplantation can occur early in life or several years later. A wide range of ocular abnormalities located in the posterior segment can be observed. Mild optic disc dysplasia or pit have no functional consequence and can be underdiagnosed. More severe colobomas or related abnormalities, such as morning glory anomaly, often lead to poor visual acuity. Molecular biology allows detection of the mutations in the PAX2 gene, but can be negative in approximately 50% of cases. The observation of an optic disc coloboma or related abnormality stimulates the ophthalmologist to propose simple nephrologic investigations to check for renal hypoplasia, a potentially life-threatening disease. Conversely, renal hypoplasia stimulates the nephrologist to ask for a fundus examination to confirm the diagnosis and check for complications such as retinal detachment.

Quantitative analysis of intravitreal injections in the rat

Intravitreal injections are currently used in the rat to introduce a therapeutic factor in the eye, especially for experimental treatments of retinal degenerations. The injected volume and its location can influence the quantification of results. We have investigated the quantitative effect of a single intravitreal injection in rats at different ages and for different volumes. Albinos rats aged three weeks or two months received intravitreal injections of 1, 3, 5 or 10 µl China ink. Animals were sacrificed immediately after injection, eyes were enucleated, fixated, embedded in paraffin and microtomy was performed in a sagittal plane. Regularly spaced sections were analyzed to reconstruct the vitreous and injected dye volumes. The measured vitreous volume was 6.76 ± 0.37 mm 3 in three weeks old rats and 13.36 ± 0.64 mm 3 in two months old rats. Mean intravitreal ink volumes immediately after injection were 0.8 mm 3 for 1 µl injections, 2 mm 3 for 3 µl, 2.3 to 2.6 mm 3 for 5 µl and 3.2 mm 3 for 10 µl. The percentage of vitreous volume involved by the injection ranged from 4.4% to 33.2%. The injected volume is limited by the large lens size of the rat. Extraocular loss of injected solution increases for higher injected volumes, with larger standard deviations. In this model, the dye tends to localize behind the lens. A 3 or 5 µl volume appears to have the best reproducibility with minimum loss of solution.

Dominant X linked retinitis pigmentosa is frequently accounted for by truncating mutations in exon ORF15 of the RPGR gene

Retinitis pigmentosa (RP) is a group of progressive hereditary disorders of the retina in which various modes of inheritance have been described. The X linked forms of retinitis pigmentosa (XLRP, MIM 268000) are among the most severe owing to their early onset, leading to significant vision loss before the fourth decade. Five XLRP loci have been localised by linkage: RP2 (MIM 312600), RP3 (MIM 312610), RP6 (MIM 312612), RP23,1 and RP24 (MIM 300155). The major loci, RP2 and RP3, map to Xp11.4 and Xp21.1, respectively. RP3 is accounted for by mutations in the retinitis pigmentosa GTPase regulator ( RPGR ) gene.2 RP3 accounts for 70% of XLRP,3, 4 but until recently only 20% of mutations were identified in RP3 families, suggesting genetic heterogeneity at this locus. This hypothesis has been excluded by the discovery of a mutational hot spot in a new RPGR exon, ORF15.5 In 1997, we reported on X linked RP in nine families with constant and severe expression in carrier females.6 In this series, onset was delayed and sometimes milder in females than in hemizygous males. …