Targeted resequencing identifies PTCH1 as a major contributor to ocular developmental anomalies and extends the SOX2 regulatory network.

Ocular developmental anomalies (ODA) such as anophthalmia/microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10,000 births. Mutations in SOX2 are the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted high-throughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes (four missense and one frameshift); targeted resequencing of PTCH1 in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Using multiple transient models and a CRISPR/Cas9-generated mutant, we show physiologically relevant phenotypes altering SHH signaling and eye development upon abrogation of ptch1 in zebrafish for which in vivo complementation assays using these models showed that all six patient missense mutations affect SHH signaling. Finally, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of the PTCH1 locus to regulate PTCH1 expression, findings that were validated both in vitro and in vivo. Together, these results demonstrate that PTCH1 mutations contribute to as much as 10% of ODA, identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, and indicate that ODA is likely the result of overactive SHH signaling in humans harboring mutations in either PTCH1 or SOX2.

Genetic characterization of congenital tufting enteropathy: epcam associated phenotype and involvement of SPINT2 in the syndromic form.

Congenital tufting enteropathy (CTE) is a rare and severe enteropathy recently ascribed to mutations in the epcam gene. Here we establish SPINT2, previously ascribed to congenital sodium diarrhea, as a second gene associated with CTE and report molecular and immunohistochemistry data in 57 CTE patients. Inclusion criteria were early onset diarrhea and intestinal insufficiency with the typical histological CTE abnormalities. The clinical phenotype was registered, the entire coding regions of epcam and SPINT2 sequenced, and immunostaining of EpCAM and SPINT2 performed on intestinal biopsies. An epcam mutation was involved in 41 patients (73 %) who mainly displayed isolated digestive symptoms. Mutations severely affected gene expression since the EpCAM signal on intestinal tissues was either undetectable or low and irregular. Twelve other patients (21 %) carried mutations in SPINT2, and were phenotypically characterized by systematic association with keratitis (p < 10(-4)) and, for half of them, with choanal atresia (p < 10(-4)). Dependency on parenteral nutrition (PN) was comparable in patients with epcam or SPINT2 mutations, but the frequent epcam mutation c.556-14A>G (abnormal splicing) was significantly associated with a better outcome (p = 0.032) with milder PN dependency to weaning in some cases. Finally, four patients (7 %) with isolated digestive symptoms had no detectable epcam or SPINT2 mutation. Two candidate genes, Elf3 and Claudin7, were excluded from this population. Our study allows us to separate CTE patients into at least three genetic classes, each with specific phenotypes. The genetics approach raises the question of the distinction between two congenital enteropathies. Our findings should help improve the diagnosis of CTE, guide toward strategies of long-term PN management, and limit indications for intestinal transplantation to life-threatening PN complications.

Acro-osteolysis, keloid like-lesions, distinctive facial features, and overgrowth: two newly recognized patients with premature aging syndrome, Penttinen type.

We report on two unrelated patients with a rare progeroid syndrome first described by Penttinen. Patients presented with prematurely aged appearance, delayed dental development, acro-osteolysis, diffuse keloid-like lesions, and ocular pterygia. Facial features are progressive but recognizable at birth. Premaxillary and maxillary retraction with pseudo-prognathism and palpebral malocclusion are characteristic. Thumbs and halluces are broad and spatulated. Linear growth is increased and intellectual functions are preserved. Skin retractions and joint contractures progressively developed during adolescence. Death occurred in the second decade in one of the patient due to restrictive respiratory insufficiency and cachexia. LMNA and ZMPSTE24 sequencing were normal. The molecular basis of the disorder remains unknown.

[From congenital glaucoma to chronic open angle glaucoma in adulthood: a clinical and genetic continuum]. / Du glaucome congénital au glaucome chronique de l'adulte: un continuum clinique et génétique.

Congenital glaucoma, a true hydrocephalus of the eye, is defined by ocular hypertension resulting in buphthalmos in children up to three years old, the elasticity of the eye wall allowing its expansion. Juvenile glaucoma in teenagers and chronic glaucoma in adults do not alter the external aspect of the eye, as the eyeball has lost its elasticity. However, chronic ocular hypertension always causes ischemic excavation of the optic nerve head, leading to insidious amputation of the visual field and, potentially, blindness. Like most ophthalmological disorders, the different types of glaucoma have been shown to be genetically determined, and alterations in several genes have been identified. These altered genes can be expressed more or less early in life, suggesting a role of modifier genes. The role of CYP1B1 alterations in classic primary congenital glaucoma is well known, as is the role of PITX2, FOXC1, PAX6 and LOXC1 alterations in secondary congenital glaucoma due to iridogoniodysgenesis, and of MYOC alterations in the genesis of chronic glaucoma in adulthood. An outbred family carrying CYP1B1 mutations in the compound heterozygous state includes two sibs with primary congenital glaucoma and two others who developed chronic glaucoma in adulthood.

TMEM126A, encoding a mitochondrial protein, is mutated in autosomal-recessive nonsyndromic optic atrophy.

Nonsyndromic autosomal-recessive optic neuropathies are rare conditions of unknown genetic and molecular origin. Using an approach of whole-genome homozygosity mapping and positional cloning, we have identified the first gene, to our knowledge, responsible for this condition, TMEM126A, in a large multiplex inbred Algerian family and subsequently in three other families originating from the Maghreb. TMEM126A is conserved in higher eukaryotes and encodes a transmembrane mitochondrial protein of unknown function, supporting the view that mitochondrial dysfunction may be a hallmark of inherited optic neuropathies including isolated autosomal-recessive forms.

[Light pollution]. / Pollution lumineuse.

LIGHT POLLUTION Artificial light can be a polluting agent deleterious for the retina, in relation to the toxicity of the blue band (380-500 nm) of the visible spectrum (380-700nm) specifically used in light-emitting diodes (LEDs). Photo-toxicity results from photochemical damage to the pigmented epithelium and retinal photoreceptors responsible for the visual function of the retina. Their photosensitive pigments, opsins for the cones and rhodopsin for the sticks, are consumed during the day and regenerated at night. Exposure to light at night seriously disrupts their metabolism. Photo-toxicity, along with heredity, is a major factor in degenerative diseases of the retina with, in addition to, the impact of age and tobacco for the most common of them, age-related macular degeneration: ARMD.Exposure to artificial light at night (LAN) has a deleterious effect on the internal clock. Intrinsically photosensitive ganglion cells (ipRGSs) are responsible for the non-visual functions of the retina, and perceive the light signal that is transmitted to the internal clock to reach the pineal gland. Light inhibits the secretion of melatonin and is able to advance or delay the clock depending on the time of exposure, causing desynchronization. Shift and night workers, like teenagers, are exposed to LAN. The incidence of breast cancer, higher in nurses exposed to LAN, is related to melatonin inhibition, sleep deprivation and desynchronization. The exposure of adolescents to screens is also questionable because the LEDs of the devices emit a blue light, the impact of which on the clock is considerable. The chronic desynchronizations of both shiftworkers and adolescents should be considered a major public health concern.

POLLUTION LUMINEUSE La lumière artificielle peut être un agent polluant délétère pour la rétine, en rapport avec la toxicité de la bande bleue (380-500 nm) du spectre visible (380-700 nm), notamment utilisée dans les diodes électroluminescentes (LED). La phototoxicité résulte de lésions photochimiques au niveau de l'épithélium pigmenté et des photorécepteurs rétiniens responsables de la fonction visuelle de la rétine. Leurs pigments photosensibles, opsines pour les cônes et rhodopsines pour les bâtonnets, sont consommés le jour et régénérés la nuit. L'exposition à la lumière la nuit perturbe gravement leur métabolisme. La phototoxicité constitue, avec l'hérédité, un facteur majeur pour les maladies dégénératives de la rétine avec, en plus, l'impact de l'âge et du tabac pour la plus fréquente d'entre elles, la dégénérescence maculaire liée à l'âge (DMLA). L'exposition à la lumière artificielle la nuit (LAN) dérègle l'horloge interne. Les cellules ganglionnaires intrinsèquement photosensibles (ipRPC), responsables des fonctions non visuelles de la rétine, perçoivent le signal lumineux qui est transmis à cette l'horloge interne pour aboutir à la glande pinéale. La lumière inhibe la sécrétion de mélatonine et est capable d'avancer ou de retarder l'horloge selon l'heure d'exposition, dans le cadre d'une désynchronisation. Les travailleurs postés et de nuit, comme les adolescents, sont exposés à la LAN. L'incidence de cancer du sein, plus élevée chez les infirmières exposées à la LAN, est attribuée à l'inhibition de la mélatonine, la privation de sommeil et la désynchronisation. L'exposition des adolescents aux écrans pose aussi question, car les diodes électroluminescentes (LED) des appareils émettent une lumière bleue dont l'impact sur l'horloge interne est considérable. Les désynchronisations chroniques des travailleurs postés, comme celles des adolescents, doivent être considérées comme des préoccupations importantes de santé publique.

Carotid artery dissection revealed by an oculosympathetic spasm.

Internal carotid dissection can be responsible for stroke and lead to severe neurological and functional complications. Thus, it must be diagnosed and treated with heparin as soon as possible. Horner syndrome is one of the most usual manifestations of internal carotid dissection. We report the case of a patient who presented with a unilateral non-reactive enlargement of the right pupil that did not last longer than 30 s. As a carotid dissection was not recognized from this atypical symptomatology, magnetic resonance angiography was performed only a few days later when Horner syndrome occurred. It disclosed a dissection of the internal carotid artery ipsilateral from its origin. The evolution and the duration of the pupil involvement suggest that the initial episode of mydriasis was caused by an oculosympathetic spasm, a rare form of sympathetic dysfunction that can be observed when the sympathetic nerve or the pericarotid plexus is irritated. It is important to recognize this oculosympathetic spasm because it has equal value as Horner syndrome for the diagnosis of internal carotid dissection.

[Under cover penetrating keratoplasty: a safer technique, especially for children]. / La kératoplastie transfixiante à ciel fermé, technique de sécurité dans certaines greffes de cornée, en particulier chez l'enfant.

Penetrating keratoplasty is usually performed when the cornea has lost all transparency Irrespective of the instruments used, the most awkward step occurs when the cornea is removed. In aphakic and infant eyes there is a high risk of lens expulsion, vitreous loss, or choroidal effusion. Under-cover penetrating keratoplasty never leaves the anterior segment exposed, and is therefore safer for infant and aphakic eyes and when large-diameter trepanation is necessary.

Population history and infrequent mutations: how old is a rare mutation? GUCY2D as a worked example.

The mosaic pattern of haplotypes observed around a single mutation results from one or several founder events. The difficulties involved in calculating the age of the variant are greatly reduced by assuming a single event, but this simplification may bias analysis of the genealogy of the mutation. However, if it is assumed that more than one founder event occurred, the number of genealogies is very large and the likelihood of every possible tree could not be realistically calculated. A multipoint approach is required, given the number of independent variables needed to describe a complex bifurcating genealogy. Starting from the observation that a limited number of parameters is needed for calculation of the simplest models of bifurcating genealogies, we show that the probability density of a two-ancestor model genealogy can be simply described as an algebraic function in a closed form, two coalescence times being calculated simultaneously without compromising accuracy. Implementation in a Bayesian framework is facilitated by the simplicity of the function, which describes the reciprocal relationship between the region of complete linkage disequilibrium and the branch length of the tree. We illustrate the use of haplotype information about allele-sharing decay around a mutation as a genetic clock, using data for two GUCY2D mutations in Mediterranean populations.

Spectrum of NPHP6/CEP290 mutations in Leber congenital amaurosis and delineation of the associated phenotype.

Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration responsible for congenital blindness. Hitherto, 13 LCA genes have been mapped, nine of which have been identified. Recently, mutations in the NPHP6/CEP290 gene were shown to account for Joubert and Senior-Loken syndromes and to represent a frequent cause of isolated LCA. All LCA patients shared an intronic mutation resulting in an aberrantly spliced transcript and low levels of wild-type transcript that was believed to explain the absence of cerebellar and renal involvement in these patients. Here, we confirm the high frequency of NPHP6/CEP290 mutations in our series of LCA families hailing worldwide (22%). However, we show that conversely to other LCA genes, NPHP6 is involved in families of European descent only (38/38). A total of 24 different mutations were found, 23 of which are novel (one founder mutation in the North region of France). All mutations but two were either nonsense, frameshift, or splice-site changes. The common NPHP6/CEP290 intronic mutation accounted for 43% (33/76) of all disease alleles. Twelve families did not carry this common intronic mutation. At least 10 out of them harboured two mutations expected to truncate the protein questioning the relevance of the assumption according to which the retinal-restricted phenotype in LCA patient could be due to a residual NPHP6/CEP290 activity. Finally, we show that all patients were affected with the cone-rod subtype of the disease whatever their NPHP6/CEP290 genotype.

Mutations in LCA5 are an uncommon cause of Leber congenital amaurosis (LCA) type II.

Leber congenital amaurosis (LCA) is the earliest and most severe form of inherited retinal dystrophy responsible for blindness or severe visual impairment at birth or within the first months of life. Up to date, ten LCA genes have been identified. Three of them account for ca. 43% of families and are responsible for a congenital severe stationary cone-rod dystrophy (Type I, 60% of LCA) while the seven remaining genes account for 32% of patients and are responsible for a progressive yet severe rod-cone dystrophy (Type II, 40% of LCA ). Recently, mutations in LCA5, encoding the ciliary protein lebercilin, were reported to be a rare cause of leber congenital amaurosis. The purpose of this study was to evaluate the involvement of this novel gene and to look for genotype-phenotype correlations. Here we report the identification of three novel LCA5 mutations (3/3 homozygous) in three families confirming the modest implication of this gene in our series (3/179; 1.7%). Besides, we suggest that the phenotype of these patients affected with a particularly severe form of LCA type II may represent a continuum with LCA type I.

Comprehensive survey of mutations in RP2 and RPGR in patients affected with distinct retinal dystrophies: genotype-phenotype correlations and impact on genetic counseling.

X-linked forms of retinitis pigmentosa (RP) (XLRP) account for 10 to 20% of families with RP and are mainly accounted for by mutations in the RP2 or RP GTPase regulator (RPGR) genes. We report the screening of these genes in a cohort of 127 French family comprising: 1) 93 familial cases of RP suggesting X-linked inheritance, including 48 out of 93 families with expression in females but no male to male transmission; 2) seven male sibships of RP; 3) 25 sporadic male cases of RP; and 4) two cone dystrophies (COD). A total of 5 out of the 93 RP families excluded linkage to the RP2 and RP3 loci and were removed form the cohort. A total of 14 RP2 mutations, 12 of which are novel, were identified in 14 out of 88 familial cases of RP and 1 out of 25 sporadic male case (4%). In 13 out of 14 of the familial cases, no expression of the disease was noted in females, while in 1 out of 14 families one woman developed RP in the third decade. A total of 42 RPGR mutations, 26 of which were novel, were identified in 80 families, including: 69 out of 88 familial cases (78.4%); 2 out of 7 male sibship (28.6%); 8 out of 25 sporadic male cases (32.0%); and 1 out of 2 COD. No expression of the disease was noted in females in 41 out of 69 familial cases (59.4%), while at least one severely affected woman was recognized in 28 out of 69 families (40.6%). The frequency of RP2 and RPGR mutations in familial cases of RP suggestive of X-linked transmission are in accordance to that reported elsewhere (RP2: 15.9% vs. 6-20%; RPGR: 78.4% vs. 55-90%). Interestingly, about 30% of male sporadic cases and 30% of male sibships of RP carried RP2 or RPGR mutations, confirming the pertinence of the genetic screening of XLRP genes in male patients affected with RP commencing in the first decade and leading to profound visual impairment before the age of 30 years.

Three new PAX6 mutations including one causing an unusual ophthalmic phenotype associated with neurodevelopmental abnormalities.

PURPOSE: The PAX6 gene was first described as a candidate for human aniridia. However, PAX6 expression is not restricted to the eye and it appears to be crucial for brain development. We studied PAX6 mutations in a large spectrum of patients who presented with aniridia phenotypes, Peters' anomaly, and anterior segment malformations associated or not with neurological anomalies. METHODS: Patients and related families were ophthalmologically phenotyped, and in some cases neurologically and endocrinologically examined. We screened the PAX6 gene by direct sequencing in three groups of patients: those affected by aniridia; those with diverse ocular manifestations; and those with Peters' anomaly. Two mutations were investigated by generating crystallographic representations of the amino acid changes. RESULTS: Three novel heterozygous mutations affecting three unrelated families were identified: the g.572T>C nucleotide change, located in exon 5, and corresponding to the Leucine 46 Proline amino-acid mutation (L46P); the g.655A>G nucleotide change, located in exon 6, and corresponding to the Serine 74 Glycine amino-acid mutation (S74G); and the nucleotide deletion 579delG del, located in exon 6, which induces a frameshift mutation leading to a stop codon (V48fsX53). The L46P mutation was identified in affected patients presenting bilateral microphthalmia, cataracts, and nystagmus. The S74G mutation was found in a large family that had congenital ocular abnormalities, diverse neurological manifestations, and variable cognitive impairments. The 579delG deletion (V48fsX53) caused in the affected members of the same family bilateral aniridia associated with congenital cataract, foveal hypolasia, and nystagmus. We also detected a novel intronic nucleotide change, IVS2+9G>A (very likely a mutation) in an apparently isolated patient affected by a complex ocular phenotype, characterized primarily by a bilateral microphthalmia. Whether this nucleotide change is indeed pathogenic remains to be demonstrated. Two previously known heterozygous mutations of the PAX6 gene sequence were also detected in patients affected by aniridia: a de novo previously known nucleotide change, g.972C>T (Q179X), in exon 8, leading to a stop codon and a heterozygous g.555C>A (C40X) recurrent nonsense mutation in exon 5. No mutations were found in patients with Peters' anomaly. CONCLUSIONS: We identified three mutations associated with aniridia phenotypes (Q179X, C40X, and V48fsX53). The three other mutations reported here cause non-aniridia ocular phenotypes associated in some cases with neurological anomalies. The IVS2+9G>A nucleotide change was detected in a patient with a microphthalmia phenotype. The L46P mutation was detected in a family with microphthalmia, cataract, and nystagmus. This mutation is located in the DNA-binding paired-domain and the crystallographic representations of this mutation show that this mutation may affect the helix-turn-helix motif, and as a consequence the DNA-binding properties of the resulting mutated protein. Ser74 is located in the PAX6 PD linker region, essential for DNA recognition and DNA binding, and the side chain of the Ser74 contributes to DNA recognition by the linker domain through direct contacts. Crystallographic representations show that the S74G mutation results in no side chain and therefore perturbs the DNA-binding properties of PAX6. This study highlights the severity and diversity of the consequences of PAX6 mutations that appeared to result from the complexity of the PAX6 gene structure, and the numerous possibilities for DNA binding. This study emphasizes the fact that neurodevelopmental abnormalities may be caused by PAX6 mutations. The neuro-developmental abnormalities caused by PAX6 mutations are probably still overlooked in the current clinical examinations performed throughout the world in patients affected by PAX6 mutations.

Nonpenetrating external trabeculectomy for congenital glaucoma: a retrospective study.

OBJECTIVE: To evaluate the results of nonpenetrating external trabeculectomy (NPET) for primary congenital glaucoma (CG). DESIGN: Retrospective, noncomparative, interventional case series. PARTICIPANTS: Forty-three eyes of 27 consecutive patients with primary CG. METHODS: Initial intent of NPET with intraoperative conversion to trabeculectomy in cases where aqueous humor outflow was deemed insufficient or where Schlemm's canal appeared to be absent. MAIN OUTCOME MEASURES: Intraocular pressure (IOP). Success was defined as an IOP less than 12.5 mmHg at 1 year after the procedure or later, using adjunctive pressure-lowering topical medications whenever needed. RESULTS: Mean initial measures of IOP were 18.8 mmHg. Of the 43 eyes, a total of 13 required conversion to trabeculectomy: 9 because of insufficient filtration, 3 because of an apparent absence of Schlemm's canal, and 1 because of accidental perforation. Of these 13 eyes that ultimately underwent trabeculectomy, 11 achieved successful IOP control, 3 in association with topical therapy. In 1 eye, a retinal detachment developed, and in 7 eyes, other complications were observed. Among the 30 remaining eyes that underwent NPET, 2 underwent the procedure twice, and 1 eye underwent the procedure 3 times. Postoperative complications were not noted in this NPET-only group. Mean postoperative IOP was 10.8 mmHg, with final IOP controlled in 28 of the 30 eyes (93%; P<0.0001), occasionally after repeat surgeries and in combination with topical therapy. CONCLUSIONS: Nonpenetrating external trabeculectomy may be considered as an initial procedure for some cases of primary CG. It appears to be an alternative to trabeculectomy with fewer risks of postoperative complications.

[Organic causes of bad visual development in the child]. / Causes organiques de mauvais développement visuel chez l'enfant.

The eye trouble of organic origin must be diagnosed and treated before the installation of an irreversible amblyopia. This early diagnosis rests on the observation of the child by his parents but especially on the knowledge of the various clinical signs by the general practitioner or the pediatrist. Thus, the blindness and the behaviors which is referred to it (leucocorie, strabism, nystagmus, or sometimes exophtalmy) must alert the doctor who will then address the child towards the ophthalmologist. This one will be able to highlight a malformation of the anterior segment, a cataract or glaucoma, an ocular inflammation or its after-effects, retinal, orbital or optical ways anomaly. The treatment will have to then allow a fast visual rehabilitation in order to treat the residual amblyopia as fast as possible. In some cases, it will direct the child and his parents towards a structure specialized in order to develop to the maximum of the sometimes unexpected visual capacities.

Identification of the first intragenic deletion of the PITX2 gene causing an Axenfeld-Rieger Syndrome: case report.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is characterized by bilateral congenital abnormalities of the anterior segment of the eye associated with abnormalities of the teeth, midface, and umbilicus. Most cases of ARS are caused by mutations in the genes encoding PITX2 or FOXC1. Here we describe a family affected by a severe form of ARS. CASE PRESENTATION: Two members of this family (father and daughter) presented with typical ARS and developed severe glaucoma. The ocular phenotype was much more severe in the daughter than in the father. Magnetic resonance imaging (MRI) detected an aggressive form of meningioma in the father. There was no mutation in the PITX2 gene, determined by exon screening. We identified an intragenic deletion by quantitative genomic PCR analysis and characterized this deletion in detail. CONCLUSION: Our findings implicate the first intragenic deletion of the PITX2 gene in the pathogenesis of a severe form of ARS in an affected family. This study stresses the importance of a systematic search for intragenic deletions in families affected by ARS and in sporadic cases for which no mutations in the exons or introns of PITX2 have been found. The molecular genetics of some ARS pedigrees should be re-examined with enzymes that can amplify medium and large genomic fragments.

Eight previously unidentified mutations found in the OA1 ocular albinism gene.

BACKGROUND: Ocular albinism type 1 (OA1) is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene. METHODS: The ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments. RESULTS: We sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found. CONCLUSION: The identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand.

Identification of four new PITX2 gene mutations in patients with Axenfeld-Rieger syndrome.

PURPOSE: Axenfeld Rieger syndrome (ARS) is an autosomal dominant inherited disorder affecting development of the ocular anterior chamber, abdomen, teeth and facial structures. The PITX2 gene is a major gene encoding a major transcription factor associated with ARS. METHODS: ARS patients were collected from six unrelated families. Patients and their families were ophthalmologically phenotyped and their blood was collected for DNA extraction. We screened the coding region of human PITX2 gene by direct sequencing. The consequences of the mutations described were investigated by generating crystallographic representations of the amino acid changes. In order to better understand the occurrence of glaucoma in ARS patients, we studied the PITX2 gene expression in human embryonic and fetal ocular tissue sections. RESULTS: We identified four novel PITX2 genetic alterations in four unrelated families with ARS. These mutations included two nonsense mutations (E55X and Y121X), an eight nucleotides insertion (1251 ins CGACTCCT) and a substitution (F58L), in familial and sporadic cases of ARS. We also showed for the first time that PITX2 is expressed at early stages of the human embryonic and fetal periocular mesenchyme, as well as at later stages of human development in the fetal ciliary body, ciliary processes, irido corneal angle and corneal endothelium. The human fetal eye PITX2 gene expression pattern reported here for the first time provides a strong basis for explaining the frequent occurrence of glaucoma in patients affected by PITX2 gene mutations. CONCLUSIONS: Two mutations identified affect the homeodomain (E55X and F58L). The E55X nonsense mutation is likely to alter dramatically the DNA-binding capabilities of the PITX2 homeodomain. Furthermore, there is a complete loss of the carboxy-terminal part of the PITX2 protein beyond the site of the mutation. The phenylalanine F58 is known to contribute to the hydrophobic network of the homeodomain. The crystallographic representations of the mutation F58L show that this mutation may change the conformation of the helical core. The F58L mutation is very likely to modify the homeodomain conformation and probably alters the DNA binding properties of PITX2. The other mutations (Y121X and the eight-nucleotide insertion (1251 ins CGA CTC CT) CGA CTC CT, at position 224 in PITX2A) result in partial loss of the C-terminal domain of PITX2. Pitx2 synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. Pitx2 activity is regulated by its own C-terminal tail. This region contains a highly conserved 14-amino-acid element involved in protein-protein interactions. The C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2 interactions with other transcription factors, for Pitx2-Pit-1 interaction and Pit-1synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Thus, the partial or complete loss of the C terminus tail can lead to decreased or absent DNA binding activity and trigger severe ARS phenotypes. Our in situ hybridization results obtained on human embryonic and fetal ocular tissue sections constitute the first molecular histological data providing an explanation for the occurrence of precocious glaucoma in human patients affected by ARS caused by PITX2 mutations. Further structural and biochemical studies are needed for understanding the wide spectrum of clinical phenotypes caused by the increasing number of new PITX2 mutations found in ARS affected patients.

Recurrent lacrimal gland choristoma of the ciliary body in an infant mimicking a medulloepithelioma.

PURPOSE: Choristoma is a congenital tumor made up of ectopic normal tissue. Different histopathologic subtypes have been described. Among them, lacrimal gland choristoma is found mainly in infants and can affect the iris, the ciliary body, or the choroid and epibulbar region. Our aims were to report a case of lacrimal gland choristoma, review the published cases, and present the main differential diagnoses. METHODS: A local resection of a limited mass of the ciliary body was performed on a 12-month-old girl who had a 6-month history of visual loss, leukocoria, and pupillary deformation. RESULTS: Histopathologically, we observed a well-demarcated lesion involved under the epithelium of the ciliary body. It was composed of acini delineated by a well-differentiated epithelium without atypia and mitotic figures. Immunohistochemical analyses confirmed the lacrimal nature with the expression of epithelial markers (cytokeratin 7 positive and cytokeratin 20 negative) and neuron-specific enolase without immunoreactivity for other neuronal markers. Two years later, a local recurrence appeared and was resected. It showed nearly the same histopathologic features. CONCLUSIONS: Lacrimal gland choristoma is a very rare lesion of the infant. Diagnosis is based on a histopathologic analysis with immunohistochemical studies to exclude other differential diagnoses such as a more common malignant tumor in childhood, medulloepithelioma. This observation shows an atypical clinical presentation of this benign lesion characterized by local recurrences.