Insertional translocation of 15q25-q26 into 11p13 and duplication at 8p23.1 characterized by high resolution arrays in a boy with congenital malformations and aniridia

We report on a boy presenting submucous cleft palate, hydronephrosis, ventriculoseptal defect, aniridia, and developmental delay. Additional material on 11p13 was cytogenetically visible and array analyses identified a duplicated segment on 15q25-26 chromosome region; further, array analyses revealed a small deletion (49?kb) at 11p13 region involving the ELP4 gene and a duplication at 8p23.1. Results were confirmed with both molecular and molecular cytogenetics techniques. Possibilities for etiological basis of clinical phenotype are discussed. (c) 2012 Wiley Periodicals, Inc.

Long range physical map of the Wilms' tumor - aniridia region on human chromosome 11

Nephroblastoma or Wilms' tumor is a pediatric renal malignancy that is the most frequently occurring childhood solid tumor. Approximately 1-2% of children with Wilms' tumor also present with aniridia, a congenital absence of all or part of the iris of the eye. These children also have high rates of genitourinary anomalies and mental retardation resulting in what is called the WAGR (Wilms' tumor, aniridia, genitourinary anomaly, mental retardation) syndrome. Cytogenetic analysis of metaphase chromosomes from these patients revealed a consistent deletion of band P13 on chromosome 11. These observations suggest close physical linkage between the disease-related loci, and further imply that development of each phenotype results from the loss of normal gene function.^ The objective of this work is to understand the molecular events at chromosome band 11p13 that are essential to the development of sporadic Wilms' tumor and sporadic aniridia. Two human/hamster somatic cell hybrids have been used to identify sixteen independent DNA probes that map to this segment of the human genome. These newly identified DNA probes and four previously reported probes (CAT, FSHB, D11S16, and HBVIS) have been used to subdivide 11p13 into five intervals defined by overlapping constitutional deletions from several WAGR patients. A long-range physical map of 11p13 has been constructed using each of these probes in Southern blot analysis of genomic DNA after digestion with infrequently cutting restriction enzymes and pulse-field gel electrophoresis. This map, established primarily with MluI and NotI, spans approximately 13 $\times$ 10$\sp{6}$ bp and encompasses deletion and translocation breakpoints associated with genitourinary anomalies, aniridia, and sporadic Wilms' tumor. This complete physical map of human chromosome band 11p13 enables us to localize the genes for sporadic Wilms' tumor and sporadic aniridia to a small number of specific NotI fragments. ^

Positional cloning and characterization of the human aniridia and murine small eye genes

Aniridia (AN) is a congenital, panocular disorder of the eye characterized by the complete or partial absence of the iris. The disease can occur in both the sporadic and familial forms which, in the latter case, is inherited as an autosomal dominant trait with high penetrance. The objective of this study was to isolate and characterize the genes involved in AN and Sey, and thereby to gain a better understanding of the molecular basis of the two disorders.^ Using a positional cloning strategy, I have approached and cloned from the AN locus in human chromosomal band 11p13 a cDNA that is deleted in two patients with AN. The deletions in these patients overlap by about 70 kb and encompass the 3$\sp\prime$ end of the cDNA. This cDNA detects a 2.7 kb mRNA encoded by a transcription unit estimated to span approximately 50 kb of genomic DNA. The message is specifically expressed in all tissues affected in all forms of AN, namely within the presumptive iris, lens, neuroretina, the superficial layers of the cornea, the olfactory bulbs, and the cerebellum. Sequence analysis of the AN cDNA revealed a number of motifs characteristic of certain transcription factors. Chief among these are the presence of the paired domain, the homeodomain, and a carboxy-terminal domain rich in serine, threonine and proline residues. The overall structure shows high homology to the Drosophila segmentation gene paired and members of the murine Pax family of developmental control genes.^ Utilizing a conserved human genomic DNA sequence as probe, I was able to isolate an embryonic murine cDNA which is over 92% homologous in nucleotide sequence and virtually identical at the amino acid level to the human AN cDNA. The expression pattern of the murine gene is the same as that in man, supporting the conclusion that it probably corresponds to the Sey gene. Its specific expression in the neuroectodermal component of the eye, in glioblastomas, but not in the neural crest-derived PC12 pheochromocytoma cell line, suggests that a defect in neuroectodermal rather mesodermal development might be the common etiological factor underlying AN and Sey. ^

Structure-function analyses of {\it PAX6\/} and the molecular bases of aniridia

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. I dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems, the results show consistently that all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act cooperatively to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6 even though the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. I conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree, and that its full transactivation activity relies on the cooperative action of the four exon fragments.^ Most PAX6 mutations detected in patients with aniridia result in truncations of the protein. Some of the truncation mutations occur in the PST region of PAX6, resulting in mutant proteins that retain their DNA-binding ability but have no significant transactivation activity. It is not clear whether such mutants are true loss-of-function or dominant-negative mutants. I show that these mutants are dominant-negative if they are coexpressed with wild-type PAX6 in cultured cells and that the dominant-negative effects result from enhanced DNA-binding ability of these mutants due to removal of the PST domain. These mutants are able to repress the wild-type PAX6 activity not only at target genes with paired domain binding sites but also at target genes with homeodomain binding sites.^ Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis, and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, I performed a functional analysis of two missense mutations in the paired domain: the R26G mutation reported in a case of Peters' anomaly, and the I87R mutation identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. I found that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. My data support the haploinsufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele. ^

Aniridia congénita familiar : caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Diadenosine polyphosphates in the tears of aniridia patients

Purpose To quantify diadenosine polyphosphate levels in tears of congenital aniridia patients to estimate the ocular surface changes associated with congenital aniridia compared to normal individuals. Methods Fifteen patients diagnosed with congenital aniridia and a control group of forty volunteers were studied. Tears were collected to quantify the levels of diadenosine polyphosphates Ap4A and Ap5A by high-performance liquid chromatography (H.P.L.C). Break-up time (BUT), corneal staining, McMonnies questionnaire and the Schirmer I test were applied to both groups. Results Dinucleotides in congenital aniridia patients were higher than in control subjects. For the congenital aniridia group, under 15 years old, the values were 0.77 ± 0.01 μm and 0.17 ± 0.02 μm for Ap4A and Ap5A, respectively. The group aged from 15 to 40 years old provided concentrations of 4.37 ± 0.97 μm and 0.46 ± 0.05 μm for Ap4A and Ap5A, the group over 40 gave concentrations of 11.17 ± 5.53 μm and 0.68 ± 0.17 μm for Ap4A and Ap5A. Dinucleotide concentrations increased with age, being statistically significant different among the three age groups (p < 0.05). Congenital aniridia patients showed a normal tear secretion and no dry eye McMonnies scores, except for the group over 40 years old. BUT values decreased and corneal staining increased with age and correlated with the levels of diadenosine polyphosphates (p < 0.05). Conclusions The levels of dinucleotides in tears increase in aniridia patients compared with healthy subjects, and they seem to be related with the progression of corneal disorders in aniridia patients, both of which increase with ageing.

Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome

Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.

Unique Utilization of a Phosphoprotein Phosphatase Fold by a Mammalian Phosphodiesterase Associated with WAGR Syndrome

Metallophosphoesterase-domain-containing protein 2 (MPPED2) is a highly evolutionarily conserved protein with orthologs found from worms to humans. The human MPPED2 gene is found in a region of chromosome 11 that is deleted in patients with WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome, and MPPED2 may function as a tumor suppressor. However, the precise cellular roles of MPPED2 are unknown, and its low phosphodiesterase activity suggests that substrate hydrolysis may not be its prime function. We present here the structures of MPPED2 and two mutants, which show that the poor activity of MPPED2 is not only a consequence of the substitution of an active-site histidine residue by glycine but also due to binding of AMP or GMP to the active site. This feature, enhanced by structural elements of the protein, allows MPPED2 to utilize the conserved phosphoprotein-phosphatase-like fold in a unique manner, ensuring that its enzymatic activity can be combined with a possible role as a scaffolding or adaptor protein. (C) 2011 Elsevier Ltd. All rights reserved.

Limbal stem cell deficiency:Concept, aetiology, clinical presentation, diagnosis and management

Defects in renewal and repair of ocular surface as a result of limbal stem cell deficiency are now known to cause varying ocular, surface morbidity including persistent photophobia, repeated and persistent surface breakdown and overt conjunctivalisation of the cornea. Ocular conditions with abnormalities of ocular surface repair include pterygium, limbal tumours, aniridia, severe scarring following burns, cicatricial pemphigoid and Stevens-Johnson Syndrome, sequelae of mustard gas exposure and Herpes simplex epithelial disease, radiation keratopathy, contact lens induced keratopathy, neuroparalytic keratitis and drug toxicity. Restoring ocular health in these eyes has traditionally been frustrating. An understanding of these intricate cell renewal and maintenance processes has spurred the evolution in recent years of new treatment methods for several blinding diseases of the anterior segment; many more exciting modalities are in the offing. However, there is inadequate awareness among ophthalmologists about the current principles of management of ocular surface disorders. The purpose of this article is to help elucidate the important principles and current treatment methods relevant to ocular surface disorders.

Allo-limbal transplantation in patients with limbal stem cell deficiency

Aim - To report the outcome of a series of patients with stem cell deficiency who underwent allo-limbal transplantation and to describe a technique for this procedure. Methods - Six consecutive patients underwent allo-limbal stem cell transplantation. The primary diagnosis included alkali burn (n = 2), trachoma (n = 1), chronic rosacea blepharitis and keratoconjunctivitis (n = 1), aniridia (n = 1), and Stevens-Johnson syndrome (n = 1). The limbal rim consisted of peripheral cornea and perilimbal sclera, FK-506 was used postoperatively for immunosuppression. Results - The length of follow up ranged from 3 to 24 months (mean follow up 11.8 (SD 9.3) months). The outcome was considered satisfactory in five of six cases. The corneal surface was completely epithelialised within 2 weeks, and there was a substantial improvement in vision and symptoms. One patient had recurrent epithelial defects related to eyelid abnormalities. No side effects associated with systemic immunosuppression were noted. Conclusion - Allo-limbal transplantation, with systemic immunosuppression with FK-506 is useful in reconstruction of the ocular surface with improvement in vision in patients with severe stem cell deficiency.

Transcriptional regulation of the human {\it PAX6\/} gene

PAX6, a member of the paired-type homeobox gene family, is expressed in a partially and temporally restricted pattern in the developing central nervous system, and its mutation is responsible for human aniridia (AN) and mouse small eye (Sey). The objective of this study was to characterize the PAX6 gene regulation at the transcriptional level, and thereby gain a better understanding of the molecular basis of the dynamic expression pattern and the diversified function of the human PAX6 gene.^ Initially, we examined the transcriptional regulation of the PAX6 gene by transient transfection assays and identified multiple cis-regulatory elements that function differently in different cell lines. The transcriptional initiation site was identified by RNase protection and primer extension assays. Examination of the genomic DNA sequence indicated that the PAX6 promoter has a TATA like-box (ATATTTT) at $-$26 bp, and two CCAAT-boxes are located at positions $-$70 and $-$100 bp. A 38 bp ply (CA) sequence was located 992 bp upstream from the initiation site. Transient transfection assays in glioblastoma cells and leukemia cells indicate that a 92 bp region was required for basal level PAX6 promoter activity. Gel retardation assays showed that this 92 bp sequence can form four DNA-protein complexes which can be specifically competed by a 31-mer oligonucleotide containing a PAX6 TATA-like sequence or an adenovirus TATA box. The activation of the promoter is positively correlated with the expression of PAX6 transcripts in cells tested.^ Based on the results obtained from the in vitro transfection assays, we did further dissection assay and functional analysis in both cell-culture and transgenic mice. We found that a 5 kb upstream promoter sequence is required for the tissue specific expression in the forebrain region which is consistent with that of the endogenous PAX6 gene. A 267 bp cell-type specific repressor located within the 5 kb fragment was identified and shown to direct forebrain specific expression. The cell-type specific repressor element has been narrowed to a 30 bp region which contains a consensus E-box by in vitro transfection assays. The third regulatory element identified was contained in a 162 bp sequence (+167 to +328) which functions as a midbrain repressor, and it appeared to be required for establishing the normal expression pattern of the PAX6 gene. Finally, a highly conserved 216 bp sequence identified in intron 4 exhibited as a spinal cord specific enhancer. And this 216 bp cis-regulatory element can be used as a marker to trace the differentiation and migration of progenitor cells in the developing spinal cord. These studies show that the concerted action of multiple cis-acting regulatory elements located upstream and downstream of the transcription initiation site determines the tissue specific expression of PAX6 gene. ^

Developmental anomalies of the eye: the genetic link

As in other areas of the body, developmental anomalies of the eye arise as a result of the disturbance of events during embryology and in a proportion of cases these anomalies are genetically inherited. Developmental anomalies that occur early in embryonic life may be so severe that the embryo may not survive but others result in the birth of healthy babies but with developmental eye defects of varying severity. The most dramatic developmental defects of the eye include anophthalmos (complete absence of an eye), microphthalmos (a general failure of the eye to develop resulting in a small, undeveloped eye), coloboma (caused by failure of the optic vesicle to invaginate), and aniridia (complete or partial loss of the iris). The present article does not provide an exhaustive review of the topic but considers the major types of developmental anomaly to affect the eye and will discuss how recent progress in genetics has increased our understanding of these disorders. The major genes linked to the developmental anomalies are discussed as well as how defects in these genes might lead to specific problems.

An overview of eye-related photophobias

This chapter provides an overview of the various eye-related causes of photophobia and the likely mechanisms responsible. Photophobia is the experience of discomfort affecting the eyes as a result of exposure to light. It has a variety of causes, including the result of eye or brain disease, or it can be a side effect of various drugs or laser surgery. Photophobia can also be a symptom of a more serious disorder such as meningitis and therefore, requires appropriate investigation, diagnosis, and treatment. Trauma or disease affecting several structures of the eye are a common cause of photophobia and can be associated with: (1) the ocular adnexia, such as blepharitis and blepharospasm, (2) the cornea, including abrasion, ulcerative keratitis, and corneal dystrophy, (3) problems in eye development, such as aniridia, buphthalmos, coloboma, and aphakia, (4) various eye inflammations, including uveitis, and (5) retinal disorders, such as achromatopsia, retinal detachment, and retinal dystrophy. There may be two main explanations for photophobia associated with these conditions: (1) direct stimulation of the trigeminal nerve due to damage, disease, or excessive light entering the eye and (2) overstimulation of the retina including a specific population of light-sensitive ganglion cells.

Eye-related photophobia

This article provides an overview of the various eye-related causes of photophobia and the likely mechanisms responsible. Photophobia is an experience of discomfort affecting the eyes due to exposure to light. It has a variety of causes including the result of eye or brain disease, or it can be a side effect of various drugs or laser surgery. Photophobia can also be a symptom of a more serious disorder such as meningitis and therefore, requires appropriate investigation, diagnosis, and treatment. Trauma or disease affecting several structures of the eye are a common cause of photophobia and can be associated with: (1) the ocular adnexia, such as blepharitis and blepharospasm, (2) the cornea, including abrasion, ulcerative keratitis, and corneal dystrophy, (3) problems in eye development, such as aniridia, buphthalmos, coloboma, and aphakia, (4) various eye inflammations, including uveitis, and (5) retinal disorders, such as achromatopsia, retinal detachment, and retinal dystrophy. There may be two main explanations for eye-related photophobia: (1) direct stimulation of the trigeminal nerve due to damage, disease, or excessive light entering the eye and (2) overstimulation of the retina including a specific population of light-sensitive ganglion cells.

The role of dinucleoside polyphosphates on the ocular surface and other eye structures

Dinucleoside polyphosphates comprises a group of dinucleotides formed by two nucleosides linked by a variable number of phosphates, abbreviated NpnN (where n represents the number of phosphates). These compounds are naturally occurring substances present in tears, aqueous humour and in the retina. As the consequence of their presence, these dinucleotides contribute to many ocular physiological processes. On the ocular surface, dinucleoside polyphosphates can stimulate tear secretion, mucin release from goblet cells and they help epithelial wound healing by accelerating cell migration rate. These dinucleotides can also stimulate the presence of proteins known to protect the ocular surface against microorganisms, such as lysozyme and lactoferrin. One of the latest discoveries is the ability of some dinucleotides to facilitate the paracellular way on the cornea, therefore allowing the delivery of compounds, such as antiglaucomatous ones, more easily within the eye. The compound Ap4A has been described being abnormally elevated in patient's tears suffering of dry eye, Sjogren syndrome, congenital aniridia, or after refractive surgery, suggesting this molecule as biomarker for dry eye condition. At the intraocular level, some diadenosine polyphosphates are abnormally elevated in glaucoma patients, and this can be related to the stimulation of a P2Y2 receptor that increases the chloride efflux and water movement in the ciliary epithelium. In the retina, the dinucleotide dCp4U, has been proven to be useful to help in the recovery of retinal detachments. Altogether, dinucleoside polyphosphates are a group of compounds which present relevant physiological actions but which also can perform promising therapeutic benefits.