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Lumican and keratocan are members of the small leucine-rich proteoglycan (SLRP) family, and are the major keratan sulfate (KS) proteoglycans in corneal stroma. Both lumican and keratocan are essential for normal cornea morphogenesis during embryonic development and maintenance of corneal topography in adults. This is attributed to their bi-functional characteristic (protein moiety binding collagen fibrils to regulate collagen fibril diameters, and highly charged glycosaminoglycan (GAG) chains extending out to regulate interfibrillar spacings) that contributes to their regulatory role in extracellular matrix assembly. The absence of lumican leads to formation of cloudy corneas in homozygous knockout mice due to altered collagenous matrix characterized by larger fibril diameters and disorganized fibril spacing. In contrast, keratocan knockout mice exhibit thin but clear cornea with insignificant alteration of stromal collaegenous matrix. Mutations of keratocan cause cornea plana in human, which is often associated with glaucoma. These observations suggest that lumican and keratocan have different roles in regulating formation of stromal extracellular matrix. Experimental evidence indicates that lumican may have additional biological functions, such as modulation of cell migration and epithelium-mesenchyme transition in wound healing and tumorgenesis, besides regulating collagen fibrillogenesis.

Purpose: To evaluate the importance in anterior segment dysgenesis of genetic variation in Foxe3, a gene encoding a forkhead transcription factor specifically expressed in the lens., Methods: The phenotype of mice heterozygous for a mutation in the DNA-binding domain of Foxe3 was examined from histologic sections, and DNA binding by the encoded protein was investigated by gel-shift assay. FOXE3 from human patients with Peters' anomaly was PCR amplified and sequenced., Results: The dysgenetic lens (dyl) allele of Foxe3 was found to encode a protein unable to bind DNA. Approximately 40% of mice heterozygous for Foxe3(dyl) have corneal and lenticular defects. The phenotype is variable but typically consists of the equivalent of Peters' anomaly in humans, with central corneal opacity, keratolenticular adhesion, and, in some cases, anterior polar cataract. In a small cohort (n = 13) of patients with Peters' anomaly, shown to be normal in the PAX6 locus, one individual was found to be heterozygous for a nonconservative missense mutation in FOXE3. The mutation, which does not occur in 116 chromosomes from a control population, substitutes leucine for arginine 90 at a highly conserved position in the forkhead domain., Conclusions: Haploinsufficiency of Foxe3 in a mouse model causes anterior segment dysgenesis similar to Peters' anomaly. Although causality could not be shown in the human case, the presence of a rare, nonconservative substitution in FOXE3 of a patient with Peters' anomaly is interesting, in light of the phenotypic similarities with the mutant mice.

Purpose: To report a case of Avellino corneal dystrophy that increased in severity 1 year after uncomplicated laser in situ keratomileusis (LASIK) for myopia., Methods: Avellino dystrophy was confirmed by polymerase chain reaction sequencing of DNA from the patient and her parents., Results: Best spectacle-corrected visual acuity decreased from 20/20 to 20/30 12 to 20 months after LASIK owing to opacities that appeared centrally in the corneal stroma and the LASIK flap and remaining posterior stroma interface., Conclusions: LASIK is contraindicated in patients with Avellino corneal dystrophy because vision may be reduced by corneal opacities that appear in the interface of the flap and remaining posterior stroma postoperatively.

Purpose: To map the disease locus in a six-generation, consanguineous Pakistani family affected by nonsyndromic autosomal recessive persistent hyperplastic primary vitreous (arPHPV). All affected individuals had peripheral anterior synechiae and corneal opacities with variable degrees of cataract and a retrolenticular white mass behind the lens., Methods: Genomic DNA from family members was typed for alleles at more than 400 known polymorphic genetic markers, by polymerase chain reaction. Alleles were assigned to individuals, which allowed calculation of lod scores., Results: A maximum two-point lod score of 4.07 was obtained with marker D10S1225 with no recombination. Two recombinations with marker D10S208 and D10S537 localized the disease within a region of approximately 30 centimorgans (cM). However, homozygosity across the region refined the arPHPV locus to 13 cM., Conclusions: Linkage analysis shows localization of nonsyndromic arPHPV to chromosome10q11-q21.

Purpose: To investigate the opacity pattern in corneas with an Arg124His (R124H) homozygous mutation of the BIG-H3 gene., Methods: Slit-lamp examination was performed on eight patients with corneal dystrophy resulting from a genetically confirmed BIG-H3 R124H homozygous mutation. The birthplace of each patient also was determined., Results: Slit-lamp examination disclosed two types of opacity patterns in corneas with the BIG-H3 R124H homozygous mutation. Type I (n = 4) is a spot-like opacity present in the anterior stroma in which the lesions are confluent. Type I is the same pattern that previous reports have shown to be caused by the BIG-H3 R124H homozygous mutation. The type II corneal opacity pattern (n = 4) is a reticular opacity in the anterior stroma with round translucent spaces. Type II opacity has not been reported previously in association with any corneal dystrophy. The patients with the type I opacity do not share a common birthplace; however, interestingly, the patients with the type II opacity traced their origin to Tottori prefecture in western Japan., Conclusion: The BIG-H3 homozygous R124H mutation induces the development of two distinct patterns of corneal opacity, the recognition of which can establish an accurate diagnosis of corneal dystrophy caused by the homozygous BIG-H3 R124H mutation independent of genetic analysis. In addition, genetic factors or circumstantial influences other than the gene responsible for the corneal dystrophy may influence the pattern of corneal opacity.

Purpose: To report the recurrence pattern of corneal deposits after phototherapeutic keratectomy in patients with corneal dystrophies resulting from homozygous and heterozygous Arg124His (R124H) mutation of the BIG-H3 gene., Methods: Slit-lamp examination was performed on patients with corneal dystrophy resulting from a genetically confirmed BIG-H3 R124H mutation., Results: The recurrence of corneal deposits after phototherapeutic keratectomy in patient with heterozygous R124H mutation was mild; the granular opacities occurred as spot lesions in the central cornea. The patient with a homozygous mutation had a more severe pattern, and the recurrent lesions were diffuse and occurred in the periphery between the corneal epithelium and laser-ablated stroma. The recurrence-free interval in homozygous patients was shorter., Conclusion: The mutation genotype of BIG-H3 gene determines the recurrence pattern after phototherapeutic keratectomy.

Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na(+)-HCO(3)(-) cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na(+)-HCO(3)-cotransport activity has not been detected in mammalian lens epithelia, cell pH (pH(i)) measurements revealed the presence of Cl(-)-independent, electrogenic Na(+)-HCO(3)-cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pH(i) recovery from acid load in the presence of HCO(3)(-)/CO(2) was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO(3)-transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.

Infiltration of neutrophils and eosinophils into the mammalian cornea can result in loss of corneal clarity and severe visual impairment. To identify mediators of granulocyte recruitment to the corneal stroma, we determined the relative contribution of chemokine receptors CXC chemokine receptor (CXCR)-2 (IL-8R homologue) and CCR1 using a murine model of ocular onchocerciasis (river blindness) in which neutrophils and eosinophils migrate from peripheral vessels to the central cornea. CXCR2(-/-) and CCR1(-/-) mice were immunized s.c. and injected into the corneal stroma with Ags from the parasitic helminth Onchocerca volvulus. We found that production of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1 alpha was localized to the corneal stroma, rather than to the epithelium, which was consistent with the location of neutrophils in the cornea. CCR1 deficiency did not inhibit neutrophil or eosinophil infiltration to the cornea or development of corneal opacification. In marked contrast, neutrophil recruitment to the corneas of CXCR2(-/-) mice was significantly impaired (p < 0.0001 compared with control, BALB/c mice) with only occasional neutrophils detected in the central cornea. Furthermore, CXCR2(-/-) mice developed only mild corneal opacification compared with BALB/c mice. These differences were not due to impaired KC and MIP-2 production in the corneal stroma of CXCR2(-/-) mice, which was similar to BALB/c mice. Furthermore, although MIP-1 alpha production was lower in CXCR2(-/-) mice than BALB/c mice, eosinophil recruitment to the cornea was not impaired. These observations demonstrate the critical role for CXCR2 expression in neutrophil infiltration to the cornea and may indicate a target for immune intervention in neutrophil-mediated corneal inflammation.

We report a new variant type of Gaucher's disease characterized by hydrocephalus, corneal opacities, deformed toes, gastroesophageal reflux, and fibrous thickening of splenic and hepatic capsules. This patient had 1 D409H allele. He differed from other reported cases with a 1342G to C (D409H) homozygous mutation (onset at 4 months, no cardiac involvement until the age of 12 years, and massive hepatosplenomegaly with fibrous thickening of spleen and liver capsules). Enzyme replacement therapy was given for 4 years, resulting in an improvement of visceral and hematologic abnormalities but no neurologic improvement.

Purpose: The work forms part of a major project to study the genetics of mouse cataract mutants found during the course of mutagenesis experiments. The long-term aim is to find the underlying gene mutation in each cataract mutant. Here we report further studies of the mutant cataract and curly whiskers (Ccw), previously mapped to Chromosome 4, and also investigations of the corneal opacity (Coop) mutant, which is shown to involve a mutation in the Pax6 gene., Methods: For Ccw, the methods included mapping relative to microsatellite markers and histological studies. For the Coop mutant, breeding methods were used to show that Coop was allelic with Pax6. The Pax6 coding region in the mutant was then sequenced., Results: The Ccw locus was mapped to approximately position 45cM on the consensus map of Chr 4. Histologically, progressive degeneration of the lens was seen. In the Coop mutant, a base-pair change C->T was found at position 1033 in the Pax6 gene, which created a stop codon leading to premature termination of translation, and to a truncated Pax6 protein., Conclusions: The phenotype in Ccw/+ heterozygotes involves a new type of lens degeneration in the mouse. On the basis of the phenotype and the locus position, no candidate gene has yet been identified. The Pax6coop mutant differs in phenotype from known null alleles of Pax6, implying that it is a hypomorph.

Purpose: There is an ongoing discussion whether Lisch corneal dystrophy (band-shaped and whorled microcystic dystrophy of the corneal epithelium) represents a disorder that is different from Meesmann corneal dystrophy. The purpose of this study was to evaluate at the molecular level if Lisch and Meesmann corneal dystrophies are genetically distinct., Methods: We examined at the slit lamp a total of 48 members of a family with an aggregation of Lisch corneal dystrophy. Genomic DNA was extracted from leukocytes of the peripheral blood of seven affected and six unaffected members of this family. Mutational hotspots in the cornea-specific keratin genes K3 and K12 were scanned for mutations by single-strand conformation analysis. To test for linkage to the keratin K3 or K12 loci or for X-chromosomal inheritance, six (K3) and four (K12) microsatellite markers each flanking the keratin loci as well as 22 microsatellite markers covering the X-chromosome were typed. Linkage was analyzed using the MLINK and FASTMAP procedures., Results: A total of 19 trait carriers were identified in six generations of the family. No hereditary transmission from father to son was observed. Linkage was excluded for the keratin K3 and K12 genes. Furthermore, single-strand conformation analysis detected no mutations in these genes. Multipoint linkage analysis revealed linkage with a maximum likelihood of the odds (LOD) score of 2.93 at Xp22.3. Linkage was excluded for Xp22.2 to Xqter., Conclusions: Lisch corneal dystrophy is genetically different from Meesmann corneal dystrophy. Evidence was found for linkage of the gene for Lisch corneal dystrophy to Xp22.3.

In order to test the hypothesis that fish-eye disease (FED) is due to a deficient activation of lecithin:cholesterol acyltransferase (LCAT) by its co-factor apolipoprotein (apo) A-I, we overexpressed the natural mutants T123I, N131D, N391S, and other engineered mutants in Cos-1 cells. Esterase activity was measured on a monomeric phospholipid enelogue, phospholipase A(2) activity was measured on reconstituted high density lipoprotein (HDL), and acyltransferase activity was measured both on rHDL and on low density lipoprotein (LDL). The natural FED mutants have decreased phospholipase A(2) activity on rHDL, which accounts for the decreased acyltransferase activity previously reported. All mutants engineered at positions 131 and 391 had decreased esterase activity on a monomeric substrate and decreased acyltransferase activity on LDL. In contrast, mutations at position 123 preserved these activities and specifically decreased phospholipase A(2) and acyltransferase activites on rHDL. Mutations of hydrophilic residues in amphipathic helices alpha 3;-4 and alpha His to an alanine did not affect the mutants' activity on rHDL. Based upon the 3D model built for human LCAT, we designed a new mutant F382A, which had a biochemical phenotype similar to the natural T123I FED mutant. These data suggest that residues T123 and F382, located N-terminal of helices alpha 3-4 and alpha His, contribute specifically to the interaction of LCAT with HDL and possibly with its co-factor apoA-I. Residues N131 and N391 seem critical for the optimal orientation of the two amphipathic helices necessary for the recognition of a lipoprotein substrate by the enzyme.

Purpose: To report the chronic clinical course of two patients with homozygous R124H mutations in the betaig-h3 gene., Methods: Case reports., Results: Two patients with homozygous R124H mutations in the betaig-h3 gene developed severe juvenile corneal opacities that required keratoplasty. After surgery, corneal opacities recurred and limited the recovery of visual acuity in the chronic follow-up., Conclusion: In patients with homozygous R124H mutations in the betaig-h3 gene, recurrence of corneal opacities after keratoplasty limits the recovery of visual acuity in the chronic follow-up.

Intracameral fibrin formation, a complication of ocular inflammation and intraocular operations, sometimes results in glaucoma and/or corneal damage leading to permanent visual loss. We transferred a therapeutic gene to the corneal endothelium in order to use it as a therapeutic organ. A plasmid encoding tissue plasminogen activator (tPA) was injected into the anterior chamber of rats and electric pulses (EPs) were given subsequently, which transferred a plasmid gene to a highly selected area of corneal endothelium with no inflammation. The biologically active tPA was clearly present for 4 days after treatment. Fibrin formation induced by YAG laser-generated bleeding in the anterior chamber decreased significantly more in treated eyes than in control eyes. Corneal opacity was significantly lower in treated eyes than in control eyes and histological damage was not apparent in the treated eyes. This genetic modification allows us to use the corneal endothelium to treat various ocular diseases and could be a new and effective type of pharmacologic gene therapy.

Lecithin: cholesterolacyltransferase (LCAT) transacylates the fatty acid at the sn-2 position of lecithin to the 3beta-OH group of cholesterol forming lysolecithin and the majority of cholesteryl ester found in plasma. LCAT participates in the reverse cholesterol transport pathway in man where it esterifies tissue-derived cholesterol following efflux from peripheral cells into HDL. Only 38 unique mutations in the human LCAT gene have been reported worldwide. Our French female proband presented with corneal opacity and no detectable plasma LCAT activity using either endogenous or exogenous assays. Her total plasma cholesterol and HDL cholesterol were low (2.34 mmol/l and 0.184 mmol/l, respectively) with a very high cholesterol/cholesteryl ester molar ratio (10.9:1). Plasma triglycerides were 0.470 mmol/l with low apo B (40.5 mg/dl), apo A-I (14.7 mg/dl), apo A-II (6.8 mg/dl) and apo E (2.1 mg/dl) levels. Plasma lipoprotein analysis by ultracentrifugation showed very low HDL concentrations and a characteristic shift of the lipoprotein profile towards larger, less dense particles. No proteinuria, renal dysfunction or signs of atherosclerosis were noted at age 45. Sequence analysis of her LCAT gene showed a novel homozygous TG-deletion at residues 138-139 that resulted in a frameshift causing the generation of a stop codon and premature termination of the LCAT protein at amino acid residue 144. Western blotting of the patient's plasma using a polyclonal IgY primary antibody against human LCAT failed to demonstrate the presence of a truncated LCAT protein. A 53 bp mismatched PCR primer was designed to generate an Fsp 1 restriction site in the wild type sequence of exon 4 where the mutation occurred. The 155 bp PCR product from the wild type allele produced a 103 bp and 52 bp fragment with Fsp 1 and no cleavage products with the mutant allele thus permitting rapid screening for this novel mutation.

Background: Congenital microphthalmia (OMIM: 309700) may occur in isolation or in association with a variety of systemic malformations. Isolated microphthalmia may be inherited as an autosomal dominant, an autosomal recessive, or an X linked trait., Methods: Based on a whole genome linkage analysis, in a six generation consanguineous family with autosomal recessive inheritance, the first locus for isolated microphthalmia was mapped to chromosome 14q32. Eight members of this family underwent clinical examination to determine the nature of the microphthalmia phenotype associated with this locus., Results: All affected individuals in this family suffered from bilateral microphthalmia in association with anterior segment abnormalities, and the best visual acuity achieved was "perception of light". Corneal changes included partial or complete congenital sclerocornea, and the later development of corneal vascularisation and anterior staphyloma. Intraocular pressure, as measured by Schiotz tonometry, was greatly elevated in many cases., Conclusions: This combination of ocular defects suggests an embryological disorder involving tissues derived from both the neuroectoderm and neural crest. Other families with defects in the microphthalmia gene located on 14q32 may have a similar ocular phenotype aiding their identification.

Peters anomaly is a congenital corneal opacity with underlying defects in the posterior stroma, Descemets membrane and corneal endothelium. It is a disorder resulting from abnormal migration or function of neural crest cells and may include abnormalities of other anterior segment structures, such as the lens and iris. We report a family in which anterior segment abnormalities, including Peters anomaly and cataracts, were inherited in an autosomal dominant fashion. Although the PAX6 gene on chromosome 11 has been shown to be involved in some cases of anterior segment developmental defects, we found no evidence that the condition in this family is linked to the PAX6 gene. Identification of this gene will indicate another gene with major involvement in the development of the anterior segment of the eye.

Fish-eye disease is a familial syndrome with corneal opacification, major high density lipoprotein (HDL) deficiency in plasma, significant cholesterol esterification in plasma on non-HDL lipoproteins, generally without premature coronary disease. This first British male case from unrelated British parents had infarcts when aged 49 and 73 years but was asymptomatic at age 81 years, with plasma cholesterol 4.3-7.1 mmol/litre, triglycerides 1.8-2.2 mmol/litre, HDL cholesterol < 0.1 mmol/litre, apolipoprotein A-I < 0.16 g/litre, lipoprotein(a) 0.61 g/litre. Cholesterol esterification was impaired using HDL-3 and A-I proteoliposomes but not using VLDL/IDL/LDL. The findings are those of LCAT deficiency with the classic fish-eye disease defect. Most of the 22 reported cases were homozygous or heterozygous for a Thr-Ile mutation at codon 123 of the lecithin:cholesterol acyltransferase (LCAT) gene. This patient was a double heterozygote for this mutation and a second new incompletely defined mutation affecting LCAT expression as defined by reduced mass and activity in plasma.

We encountered 2 siblings with corneal opacities, anemia, decreased concentration of plasma esterified cholesterol, chronic renal failure and markedly reduced levels of plasma lecithin:cholesterol acyltransferase. Renal biopsy in 1 patient showed clear lacunae containing characteristic dense bodies in the glomerular and tubular basement membranes and the interstitium. One of the 2 siblings received a kidney transplant and is doing well after 9 years. This is the first report of this rare metabolic disease from India.

Loss-of-function mutations in the gene (CSTB) encoding human cystatin B, a widely expressed cysteine protease inhibitor, are responsible for a severe neurological disorder known as Unverricht-Lundborg disease (EPM1). The primary cellular events and mechanisms underlying the disease are unknown. We found that mice lacking cystatin B develop myoclonic seizures and ataxia, similar to symptoms seen in the human disease. The principal cytopathology appears to be a loss of cerebellar granule cells, which frequently display condensed nuclei, fragmented DNA and other cellular changes characteristic of apoptosis. This mouse model of EPM1 provides evidence that cystatin B, a non-caspase cysteine protease inhibitor, has a role in preventing cerebellar apoptosis.

Purpose: Two patients were diagnosed with Reis-Bücklers corneal dystrophy (RBCD), although the pattern and severity of corneal opacification differed. To see whether there was a genetic basis for these phenotypic variations, we analyzed beta ig-h3, the gene that codes for kerato-epithelin and that contains a mutation (Arg555Gln) that causes RBCD., Methods: A 30-year-old man with honeycomb-shaped subepithelial opacities in his central cornea and a 25-year-old man with progressive subepithelial geographic opacities were both considered to have RBCD. We isolated genomic DNA from leukocytes of the two patients and their family members and screened for an Arg555Gln kerato-epithelin mutation. Then we analyzed all exons of the gene using the single-strand conformation polymorphism (SSCP) technique to search for any other kerato-epithelin mutations., Results: The patient with honeycomb-shaped opacities had an Arg555Gln kerato-epithelin mutation that caused his RBCD, whereas the patient with geographic opacities did not; instead, he had a new kerato-epithelin mutation (Arg124Leu), which cosegregated with his family members., Conclusions: The variant of RBCD characterized by honeycomb-shaped opacities is caused by an Arg555Gln kerato-epithelin mutation. On the other hand, a new kerato-epithelin mutation, Arg124Leu, was found to cause the RBCD variant characterized by recurrent epithelial erosions and progressive geographic subepithelial opacification. Codon 124 is a hot spot for kerato-epithelin mutations, where the mutations responsible for three autosomal dominant corneal dystrophies--lattice type I (Arg124Cys), Avellino (Arg124His), and the variant of RBCD with geographic rather than honeycomb opacities (Arg124Leu)--are located.

Purpose: Bsk (bare skin) is an autosomal dominant mutation linked to the Krt 1 (type 1 keratin) locus of mouse chromosome 11. The adult Bsk mouse manifests hair loss and corneal opacity. To identify and characterize the keratin genes involved in this mutation, we examined the hypothesis proposing that the Bsk mutation might involve a recombination event between cornea-specific (K12) and hair-specific (mHa 1, 2, 3 and 4) type I keratin genes., Methods: The Bsk phenotype was examined by histochemical analysis, using light and electron microscopy. RFLP was used for their genotyping, and possible keratin gene expression was examined by immunohistochemical staining, Western analysis, RT-PCR and Northern hybridization., Results: Northern hybridization, RT-PCR and Western blot analysis revealed that mHa 1, 2, 3 and 4 keratins are expressed in the skin, but not in cornea, whereas the expression of K12 is limited to the corneas of the Bsk mice. These data ruled out the hypothesis that Bsk phenotype results from a recombination event between K12 and mHa 1, 2, 3 and 4. Ultrastructural and biochemical analyses also indicated that Bsk does not involve negative dominant mutations of keratin 12, mHa 1, 2, 3 and 4, epidermal-specific keratin 10, or basal cell-specific keratin 14. Expression of an acidic 50 kD keratin, recognized by monoclonal antibody AK 2, was up-regulated in the injured corneas of normal mice as well as Bsk corneas., Conclusion: The gene linked to the Bsk mutation remains unknown. The pathological changes in the skin and corneas may be secondary to the loss of protecting hairs and lashes by an unknown mechanism.

We analyzed Spanish Collaborative Study of Congenital Malformations (ECEMC) data on a series of 1,124,654 consecutive births to study congenital eye malformations from an epidemiological standpoint. We studied their frequencies as well as some causal and clinical aspects. Four hundred fourteen infants had eye malformations, for an overall prevalence of 3.68/10,000 newborns. Most frequent were: anophthalmia/microphthalmia (21.34/100,000), congenital cataract (6.31), coloboma (4.89), corneal opacity (3.11), and congenital glaucoma (2.85). In our data, the tendency of eye malformations to be associated with other congenital abnormalities is evident (only 21.01% of cases were isolated). Eye defects are heterogeneous, since we have observed them in clinical patterns with all modes of inheritance or caused by different environmental agents. Chromosomal syndromes represent 60% of total syndromes, followed by syndromes of autosomal-recessive inheritance (15%), environmental syndromes (10%), autosomal-dominant syndromes (5.83%), and other types which have a lower frequency. Regarding defects associated with eye malformations, most frequent are limb anomalies (affecting 59.3% of multiply malformed cases), auricular/facial (47.1%), central nervous system (42.5%), osteomuscular excluding limbs (42.2%), genital defects (30.6%), oral clefts (29.4%), and the rest of the body systems, which are less frequent. Using the method outlined by Prieto and Martínez-Frías [1996: Am J Med Genet 62:61-67], it was demonstrated that the association of coloboma and anophthalmia/microphthalmia was specific, as was the combination of cataract and anophthalmia/microphthalmia, and that of anophthalmia/microphthalmia with holoprosencephaly. From these statistical associations some pathogenetic relationships in human embryos can be inferred, supporting several previously proposed mechanisms.

Mutations in the PAX 6 gene are known to cause many cases of inherited and sporadic aniridia. Although embryologically similar to aniridia, the cause of Peters' anomaly has received far less attention. Two reports have been published demonstrating mutations in the PAX 6 gene in Peters' anomaly. We have analysed the PAX 6 gene in 15 individuals with Peters' anomaly (7 familial, 8 sporadic). This is the largest cohort of Peters' anomaly described. The PAX 6 gene was screened using a combination of single-strand conformational polymorphism gel electrophoresis and direct sequencing. No mutations were found in the coding region of the PAX 6 gene. We feel that Peters' anomaly is a heterogeneous condition and that for the majority of cases PAX 6 is not the 'Peters' anomaly gene'.

We report on a patient with a de-novo deletion of chromosome 6p. This male infant presented with multiple systemic congenital defects together with an unusual ocular phenotype. Slit-lamp examination revealed thin, opaque, rectilinear bands within the anterior segment partially connecting iris to corneal endothelium. These were associated with bilateral hyperopia and optic nerve hypoplasia. Ocular abnormalities in such patients have been documented although the number of individuals is small and identical cytogenetic defects are rarely encountered. We compare the clinical findings in this case with previously described phenotypes. Characterisation of such cases is important as it is becoming apparent that deletion of genetic information encoded on chromosome 6p has implications for ocular embryogenesis.

The genetic and biochemical basis of fish-eye disease (FED) was investigated in a 63-year-old female proband with low plasma HDL cholesterol. Analyses of corneal and plasma lipids of the proband were consistent with impaired lecithin:cholesterol acyltransferase (LCAT) activity. Free cholesterol and phospholipid levels were elevated relative to control values, whereas cholesteryl ester levels were greatly reduced. Fatty acid compositions of corneal lipids from the proband and control subjects differ from the respective fatty acid compositions of their plasma lipids. This suggests that the metabolic pathways and acyl chain specificities for phospholipid, cholesteryl ester, and triglyceride metabolism within the cornea are distinct from those of plasma. Sequencing of the LCAT gene from the proband revealed a novel mutation at nucleotide 399, corresponding to an Arg99-->Cys substitution. Secretion of LCAT (Arg99-->Cys) by transfected COS-6 cells was approximately 50% of that of the wild type, but its specific activity against reassembled HDL was 93% lower than that of wild-type LCAT. The specific activities of wild-type and LCAT (Arg99-->Cys) against LDL were reduced similarly, suggesting that the appearance of the FED phenotype does not require enhanced activity against LDL. Our data support the hypothesis that FED is a partial LCAT deficiency in which poor esterification in specific types of HDL particles may contribute to the appearance of the corneal opacities.

Cat4 is the second largest allelism group in the collection of mouse dominant eye mutations recovered in Neuherberg and carriers express anterior polar cataract, central corneal opacity, and lens-corneal adhesions. We have mapped the Cat4 locus of the mouse to central Chromosome (Chr) 8 at position cM 31. Histological characterization of Cat4(a) heterozygotes and homozygotes indicates failure of separation of the lens vesicle from the surface ectoderm. Human anterior segment ocular dysgenesis (ASOD) is autosomal dominant, carriers express an eye phenotype similar to that of Cat4(a) carriers, and it has been mapped to a region of 4q homologous to mouse central Chr 8. Thus, on the basis of phenotype and map position, Cat4 may be a mouse model of human ASOD. The genes Junb, Jund1, Mel, and Zfp42 are discussed as possible candidates for Cat4.

We describe a new case of a rare syndrome characterized by ocular abnormalities and pathognomonic linear skin defects. This syndrome is the result of an unbalanced translocation resulting in a deletion of the distal end of the short arm of the X chromosome. We report the thirteenth case and review the clinical and cytogenetic aspects of this disorder. In addition we discuss new findings pertaining to the histopathology of the skin lesions.

The trisomy 8 mosaic syndrome (Tr8MS), karyotype 46,XY/47,XY, +8, is a rare multisystem disorder that may be associated with corneal opacity. We report the case of a dysmorphic infant with multiple congenital abnormalities referred to our unit with a congenital corneal opacity. Subsequent chromosomal analysis of peripheral leucocytes demonstrated constitutional Tr8MS. At 4 years of age, lamellar keratoplasty was performed. Histological examination confirmed the lesion to be consistent with a corneal choristoma. Cytogenetic studies using in situ hybridisation techniques showed the presence of trisomic cells in cell culture derived from the tissue in higher proportion (92%) than in the blood (44%). Amplification of the c-myc oncogene on chromosome-8 could not be detected in cells cultured from the corneal lesion. Although not proof, these findings lend support to the concept of the corneal lesion representing a focus of viable trisomic cells rather than an inflammatory response to a nidus of effete cells.

An 8-month-old girl was examined because of corneal clouding and microphthalmos. The fundi of both eyes could not be visualized because of corneal clouding. Orbital and cranial computerized tomographic scanning and magnetic resonance imaging demonstrated bilateral microphthalmos and presumed retinal dysplasia, hypoplasia of the optic nerves and chiasm, agenesis of the septum pellucidum, thinning of corpus callosum, and a normal pituitary infundibulum. Cerebral cortex and white matter were unremarkable. Other ocular malformations were anterior segment dysgenesis in the right eye and congenital cataract or lens abnormality in the left eye. Endocrine studies revealed normal serum hormone levels. There were no colobomatous lesions and systemic anomalies suggestive of a coloboma syndrome. This case represents the rare association of septo-optic dysplasia with complex microphthalmos.

Morquio disease (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Patients commonly present in early infancy with growth failure, spondyloepiphyseal dysplasia, corneal opacification, and keratan sulfaturia, but milder forms have been described. We report on a patient who grew normally until age 5 years. Her keratan sulfaturia was not detected until adolescence, and she now has changes restricted largely to the axial skeleton. She has experienced only mildly impaired vision. At age 22, thin-layer chromatography of purified glycosaminoglycans showed some keratan sulfaturia. GALNS activity in fibroblast homogenate supernatants was 20 +/- 5% of controls (as compared to 5 +/- 3% of controls in severe MPS IVA, P < .003). Kinetic analysis of residual fibroblast GALNS activity in patient and parents revealed decreased K(m) and increased Vmax in the mother and daughter, but not in the father, compatible with compound heterozygosity. GALNS exons were amplified from patient genomic DNA and screened by SSCP. Two missense mutations, a C to T transition at position 335 (predicting R94C) and a T to G transversion at position 344 (predicting F97V), were found on sequencing an abnormally migrating exon 3 amplicon. Digestion of the amplicon with FokI and AccI restriction enzymes (specific for the R94C and F97V mutations, respectively) confirmed heterozygosity. In fibroblast transfection experiments, heterozygous R94C and F97V mutants independently expressed as severe and mild GALNS deficiency, respectively. We interpret these findings to indicate that our patient bears heteroallelic GALNS missense mutations, leading to GALNS deficiency and mild MPS IVA. Our findings expand the clinical and biochemical phenotype of MPS IVA, but full delineation of the genotype-phenotype relationship requires further study of native and transfected mutant cell lines.

The analysis of twenty-one observations with Peter Syndrome, shows that the disease was established by a disorder in the development of the mesoderm who gives birth at the posterior, stratum of cornea, iris stroma and angle of the posterior chamber. The disease was associated with cataract (nine cases), strabismus (seven cases), congenital glaucoma (five cases), microophthalmia (three cases), microcornea (four cases), remainders of the pupillary membrane (one case), vitreous opacities (one case), facial malformations (one case), congenital dacryocystitis (one case), nystagmus (four cases). The presence at the same patient of the posterior keratoconus or staphiloma of cornea at one eye and Peter syndromme at the other eye, is possible to say that these diseases are evolutionary stages by one and the same mesodermal disgenesia.

Purpose: To describe a new mouse model of corneal surface disease and neovascularization., Methods: Anatomic changes were demonstrated in corn1 and control A.By/SnJ mice from day 10 of gestation of 8 months of age by routine techniques of light microscopic and scanning electron microscopy. Corneal epithelial cell kinetics were evaluated by labeling cells in the "S" phase of the cell cycle by intraperitoneal injection of tritiated thymidine. Labeled cells were counted under 250X magnification, and the length of the corneal epithelial chord was measured by morphometric techniques. Results were expressed as labeled cells per linear millimeter of corneal epithelium. The corn1 locus was mapped using selected back-crosses., Results: Corn1 is characterized by early, irregular thickening of the corneal epithelium, development of stromal neovascularization by 20 days of age, and cataract by 48 days of age. Corneal epithelial cell kinetics demonstrated prominent labelling of corn1 mice at 30 days of age compared to the control mice. Corn1 behaves as an autosomal recessive gene and is located on mouse chromosome 2, approximately 5.2 cM from the agouti locus. Heterozygotes have no corneal disease., Conclusions: Corn1 mice, with genetically determined corneal epithelial hyperplasia and stromal neovascularization, may be particularly useful in studies of neovascularization and corneal surface proliferative disease.

A 53-year-old man with a severely reduced HDL cholesterol level, dense corneal opacities, normal renal function, and premature coronary artery disease was investigated together with 16 members of his family. The proband was diagnosed with fish eye disease. As in previously reported patients with fish eye disease, the endogenous plasma cholesterol esterification rate was near normal, yet lecithin:cholesterol acyltransferase (LCAT) activity was almost absent when measured with exogenous HDL analogues used as substrate. Direct sequencing of the LCAT gene revealed two novel missense mutations in exon 1 and exon 4, resulting in the substitution of Pro10 with Gln (P10Q) and Arg135 with Gln (R135Q), respectively. Both missense mutations were located on different alleles. Genetic analysis by polymerase chain reaction revealed 4 carriers of the P10Q and 3 carriers of the R135Q defect. Functional assessment of both missense mutations revealed that when exogenous HDL analogues were used as substrate, the specific activity of rLCAT p10Q was 18% of wild type (WT); however, when LDL was used as substrate, the activity was 146% of WT. By contrast, rLCATR135Q was inactive against both substrates. Thus, we conclude that the LCATR135D mutation is causative for complete LCAT deficiency and that the clinical phenotype of fish eye disease seen in this patient is due to the Pro10 mutation. The presence of premature coronary artery disease in the absence of other risk factors in this new case of fish eye disease raises questions regarding the risk of atherosclerosis, which has previously been reported to be nonexistent.

Purpose/methods: Focal dermal hypoplasia is an inherited dermatologic disorder commonly associated with skeletal and dental abnormalities. Ocular abnormalities frequently found in patients with focal dermal hypoplasia include microphthalmos, anophthalmos, and colobomas. Corneal abnormalities rarely have been described in patients with focal dermal hypoplasia. We examined a mother and daughter with focal dermal hypoplasia with distinctive corneal lesions., Results/conclusion: Several discrete vascularized peripheral subepithelial corneal opacifications were present bilaterally in both patients with focal dermal hypoplasia. No ocular abnormalities that would predispose to these abnormalities were found. These corneal lesions appear to represent an unusual manifestation of focal dermal hypoplasia.

Trabeculotomy is a well-documented procedure for reducing intraocular pressure in primary infantile glaucoma with cloudy corneas. However, the efficacy of the procedure is reduced in glaucoma associated with other ocular disorders. We report a child with lens-corneal adhesions in whom trabeculotomy produced a secondary cataract.

We want to present a 47-years-old patient suffering of a histologically and biochemically confirmed M. Fabry. Besides the ocular findings, we list the systemic findings and discuss the diagnostic and therapeutic possibilities. We especially want to point out the interdisciplinary collaboration in dealing with this condition.

Fish-eye disease (FED) and familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) are rare disorders of lipid metabolism linked to mutations in the LCAT gene. Eleven LCAT cDNA constructs associated with FED and FLD were prepared by site-directed mutagenesis and expressed in COS-6 cells. Analysis of total RNA from wild-type, FED, and FLD transfectants revealed that all contained LCAT-specific mRNA. Western blot analysis demonstrated that all LCAT transfectants synthesized LCAT. Mean LCAT secretion by FED transfectants was slightly lower than secretion by wild-type transfectants, whereas secretion by FLD transfectants was much lower. The specific activities of FED and FLD LCAT against model high density lipoproteins were 6% and 11%, respectively, of wild-type activity. The ratios of the LCAT activities against low density lipoproteins to those against model high density lipoproteins decreased in the order FED mutants > FLD mutants approximately wild type. FED and FLD LCAT mutants are different: the former are more active against low density lipoproteins, and the latter are less secretion-competent. The greater reactivity of FED LCAT against low density lipoproteins may explain the relative mildness of the clinical manifestations of FED compared to those of FLD.

Purpose: During the generation of transgenic mice (TGs) introduced with mouse T-cell receptor delta (TCR delta) gene, the authors found a TG line with corneal opacity that coincided with the presence of the transgene. The authors investigated the pathogenesis and molecular mechanisms of this corneal opacity in this line., Methods: The pathologic features and pathogenesis of the corneal opacity in TGs were examined histologically using transmission and scanning electron microscopy, as well as light microscopy. DNA and RNA blot analyses were performed to examine the copy number and the expression of the transgenes, respectively., Results: Histologically, edema of the corneal epithelium and adhesion of the iris to the cornea were observed in adult TGs. In the developmental analysis, the authors first observed relative hypoplasia of the ciliary body on day 18 of gestation and dysgenesis of the anterior chamber angle from postnatal day 2. Corneal opacity was observed from postnatal day 8, coinciding with the histologic vesicular change of the epithelium. No inflammation was observed through its life. In the sublines that have different copy numbers of the transgene, the occurrence of the opacity depended on the copy number of the transgene. Expression of the transgene in the thymus was consistent with the number of the introduced transgene., Conclusion: In a TCR delta TG line, the overexpression of transgenes coincided with abnormal development of the ocular anterior segment and the corneal opacity. Pathogenesis is described, and possible molecular mechanisms are discussed.

We report a 12-year-old girl with an unusual phenotype of Gaucher disease type 3. Liver glucocerebrosidase activity was 20% of the normal. In addition to common manifestations such as hepatomegaly, she showed primary communicating hydrocephalus, corneal opacities, deafness, left ventricle hypertrophy, and clinodactyly of hands and feet. We suspect cardiomyopathy to be due to myocardiac infiltration with Gaucher cells, and corneal opacities to result from an accumulation of lipid-like inclusions into the posterior stromal keratinocytes. We were only able to find one previously published sibship disclosing similar features, which could allow the delineation of a new clinical variant of Gaucher disease.

Congenital hereditary endothelial dystrophy is characterized by a diffuse bilateral corneal opacity (edema). Its inheritance has been reported to be both autosomal recessive and dominant. All our cases seemed to be autosomal recessive. There seems to be a prevalence of the recessive gene in the Iranian population. The dystrophy might be misdiagnosed as congenital glaucoma, as in several of our cases. We have operated on 37 eyes of 21 patients during the past 10 years. Our visual and anatomical success rate has been very good, with 92% clear grafts and only an 8% rejection rate, in contrast to poor prognosis that has been previously reported. In children, suture removal should be started 3 months postoperatively. Partial amblyopia was preexistent in all cases, but deep amblyopia was not common.

We present findings in the ninth known Japanese family with lecithin:cholesterol acyltransferase (LCAT) deficiency. A 54-year-old man (proband) and his 58-year-old brother presented with corneal opacity. Both subjects showed a marked decrease in serum high density lipoprotein (HDL)-cholesterol and in the cholesteryl ester ratio. Although apo A-I and A-II were low, apo E tended to be high. Serum LCAT activity and mass were not detectable. Urinary examination showed microhematuria or proteinuria. Renal function was normal and no anemia was demonstrated, but blood smears showed poikilocytosis with target cells. The serum LCAT activity of the proband's three sons, obligate heterozygotes of LCAT deficiency, was about one-half the normal level, and HDL-cholesterol and apo A-I levels were low normal.

Fleck dystrophy of the cornea is characterized by numerous, tiny, small opacities scattered throughout the entire corneal stroma. The mode of inheritance is autosomal dominant, and this dystrophy is considered to be bilaterally symmetric. This report describes five members from three-generations of the same family with corneal fleck dystrophy. Their clinical features and genetic inheritance pattern are discussed. Visual acuity in all patients was normal and the density of opacities were similar except for two patients with less density. Except for one who presented with mild photophobia all patients were asymptomatic. The inheritance pattern appeared as autosomal dominant with variable expression.

Mutation or deletion of the PAX6 gene underlies many cases of aniridia. Three lines of evidence now converge to implicate PAX6 more widely in anterior segment malformations including Peters' anomaly. First, a child with Peters' anomaly is deleted for one copy of PAX6. Second, affected members of a family with dominantly inherited anterior segment malformations, including Peters' anomaly are heterozygous for an R26G mutation in the PAX6 paired box. Third, a proportion of Sey/+ Smalleye mice, heterozygous for a nonsense mutation in murine Pax-6, have an ocular phenotype resembling Peters' anomaly. We therefore propose that a variety of anterior segment anomalies may be associated with PAX6 mutations.

The eyes of 200 rats (Mol:SPRD, Moellegaard Ltd., Skensvet, Denmark), 8 weeks of age and of both sexes, were examined routinely with a photo-slitlamp microscope (Zeiss) and opthalmoscope (Heine) 3-4 times in the course of four different 12-week toxicity studies. The animals were kept under constant lighting conditions at a room temperature of 20 +/- 2 degrees C and 55 +/- 5% humidity on a standard diet. More than 70% of the animals were found to have more or less prominent corneal opacities already at the beginning of the study. These were morphologically characterized as meshwork-like alterations of the deeper corneal epithelium, mostly located in the central and nasal region. The temporal, upper and lower periphery remained always unaffected. Male animals were more frequently and more intensively affected than the females. The occurrence of the opacity was totally independent of the treatment scheme (controls and drug dosages, respectively), showing a slight increase in density in some of the animals of all groups and remaining stable in others. Regression was rarely observed. Light and electron microscopical investigations demonstrated focal degenerations of the basal epithelial cell layer as well as alterations of its basement membrane. The lesion was not associated with inflammation or irritation. Therefore, we considered that a genetically determined metabolic disorder of the basal epithelial cells might have led to the impairment of basement membrane synthesis. Further screenings performed in conjunction with the breeder evidenced that these opacities are probably caused by a spontaneous mutation with a complex, not X-linked genetic background.

Background: The results of penetrating keratoplasty in patients with systemic mucopolysaccharidosis (MPS) type VI-A are controversial. Clouding of the transplant was often observed and was thought to be related to storage of glycosaminoglycans also in the donor button., Patients and Methods: We performed penetrating keratoplasties successfully in three children with MPS VI-A (severe type) at the age of 7-11 years., Results: The transplants remained clear during the follow-up of 2.5-5 years and the longterm visual acuity was encouraging. In two patients we could observe a partial, circular clearing of the host's cornea adjacent to the transplant. The related pathomechanism and the clinical, histopathological and ultrastructural findings of the cornea will be discussed., Conclusion: As the intellectual development is normal, decrease of vision impairs the patient's life extremely. Thus, the indication of penetrating keratoplasty should be made early to improve the patient's quality of living, who have a reduced life-span.

Bilateral microphthalmia with blepharophimosis, linear lesions of dermal aplasia involving the face, and microcephaly were present in a newborn girl who died at age 9 months from cardiomyopathy resulting in ventricular fibrillation. Autopsy showed an atrial septum defect, persistent gross trabeculation of the left ventricle, and an arteria lusoria. This case represents a further example of a new entity for which we propose the term MIDAS syndrome. The acronym stands for microphthalmia, dermal aplasia, and sclerocornea. Our patient is the second with this syndrome to have a major congenital heart defect. Cytogenetic studies reported in previous cases indicate that the underlying gene defect can be assigned to Xp22.3. This new X-linked male-lethal trait should be distinguished from focal dermal hypoplasia that will be found to map elsewhere on the X-chromosome.