Mutational screening of Indian families with hereditary congenital cataract.

PURPOSE: To screen for pathogenic mutations in ten candidate genes in Indian families diagnosed with autosomal recessive and autosomal dominant cataracts. METHODS: Families with two or more affected individuals with bilateral familial congenital/developmental cataract were ophthalmically evaluated, and blood samples were obtained. Genomic DNA extracted from the blood leukocytes was screened with PCR amplification of the exons and the flanking intronic regions of various genes selected for analysis. The amplified products were subjected to single strand conformation polymorphism (SSCP) analysis. The variants in SSCP analysis were subjected to bidirectional sequencing by automated methods. RESULTS: We identified four novel sequence changes that cosegregated with the disease phenotype in each family and were absent in at least 50 ethnically matched unrelated normal controls. These changes include a homozygous missense change of c.649G>A (Val196Met) in GJA8/connexin 50 (Cx50) in a family with autosomal recessive cataract, two heterozygous missense changes, c.658C>T (Pro199Ser) in GJA8/Cx50 and c.589C>T (Pro197Ser) in GJA3/connexin 46 (Cx46) in two separate families with autosomal dominant cataract, and a silent change ( c.84G>A/p.Val28Val, predicted to result in the creation of a new potential branch point) in GJA8 one family with an autosomal dominant inheritance of cataract. Of the four novel mutations identified, three mutations, Val196Met (GJA8), Pro199Ser (GJA8), and Pro197Ser (GJA3), are predicted to be in the second extracellular domain of the respective connexin proteins. CONCLUSIONS: Our report extends the mutation spectrum of connexin genes GJA8 and GJA3 and confirms that connexin genes are among the most frequently mutated genes in hereditary cataracts. Our results suggest that connexin gene (GJA8 and GJA3) mutations occur in approximately 10% (4/40 families) of families with congenital hereditary cataracts in a population from southern India.

Deep lamellar keratoplasty for recurrent advanced pterygium.

The authors report a case of recurrent advanced pterygium managed by deep lamellar keratoplasty and conjunctival autograft. A woman presented with bilateral recurrent advanced pterygium (more prevalent in the left eye) associated with corneal thinning in the left eye. Two-thirds of the cornea was covered by pterygia. Her visual acuity was counting fingers at 1 meter in the right eye and light perception with projection of rays being accurate in all quadrants in the left eye. She underwent deep anterior lamellar keratoplasty and conjunctival autograft in her left eye. Clarity, state of corneal graft, and postoperative visual acuity were noted. The graft was clear and the patient's best-corrected visual acuity improved to 20/60 after the surgery. Deep lamellar keratoplasty with conjunctival autograft is a good option as a single-stage surgery for recurrent advanced pterygium.

Fungal keratitis following amniotic membrane transplantation.

PURPOSE: To report a case of fungal keratitis following amniotic membrane (AM) transplantation for pseudophakic bullous keratopathy. METHOD: A 55-year-old female underwent amniotic membrane transplantation (AMT) with epithelial debridement and anterior stromal puncture in the left eye for symptomatic bullous keratopathy. She developed fungal keratitis after 4 weeks. RESULT: Aspergillus sp. was isolated from the corneal scraping. The patient was treated with systemic and topical antifungal medication. CONCLUSION: Although AMT is safe, there is a risk of microbial infection after the procedure especially in poor ocular surfaces.

Mutation of the gap junction protein alpha 8 (GJA8) gene causes autosomal recessive cataract.

GJA8 encodes connexin-50, a gap junction protein in the eye lens. Mutations in GJA8 have been reported in families with autosomal dominant cataract. The objective of this report was to identify the disease gene in a family with congenital cataract of autosomal recessive inheritance. Eight candidate genes were screened for pathogenic alterations in affected and unaffected family members and in normal unrelated controls. A single base insertion leading to frameshift at codon 203 of connexin 50 was found to co-segregate with disease in the family. These results confirm involvement of GJA8 in autosomal recessive cataract.

Ultrastructural analysis of damage to nuclear fiber cell membranes in advanced age-related cataracts from India.

The primary goal was to characterize the structural alterations that occur at the fiber cell interfaces in nuclei of fully opaque cataracts removed by extracapsular cataract surgery in India. The dark yellow to brunescent nuclei, ages 38-78 years, were probably representative of advanced age-related nuclear cataracts. Thick tissue slices were fixed, en bloc stained and embedded for transmission electron microscopy. Stained thin sections contained well-preserved membranes and junctions, although the complex cellular topology often made it necessary to tilt the grid extensively to visualize the membranes. Damage to the fiber cell membranes was noted in all regions of the nucleus. The most important damage occurred within undulating membrane junctions where the loss of membrane segments was common. These membrane breaks were not sites of fusion as membrane edges were detected and cytoplasm appeared to be in contact with extracellular space, which was enlarged in many regions. Dense deposits of protein-like material were frequently observed within the extracellular space and appeared to be similar to protein in the adjacent cytoplasm. The deposits were often 20-50 nm thick, variable in length and located on specific sites on plasma membranes and between clusters of cells or cell processes. In addition, low density regions were seen within the extracellular space, especially within highly undulating membranes where spaces about 100 nm in diameter were observed. The membrane damage was more extensive and extracellular spaces were larger than in aged transparent donor lenses. Because high and low density regions contribute equally to the fluctuations in refractive index, the changes in density due to the observed damage near membranes are likely to produce significant light scattering based on theoretical analysis. The dimensions of the fluctuations in the range 20-100 nm imply that the scattering is probably similar to that of small particles that would increase high-angle scattering visible in the slit lamp. Such damage to membranes would be expected to contribute to the total opacification of the nucleus as the cataract matures. The main sources of the fluctuations appear to be the degradation of membranes and adjacent cytoplasmic proteins, as well as the redistribution of proteins and fragments.

Mie light scattering calculations for an Indian age-related nuclear cataract with a high density of multilamellar bodies.

PURPOSE: Multilamellar bodies (MLBs) are lipid-coated spheres (1-4 microm in diameter) found with greater frequency in the nuclear region of human age-related cataracts compared with human transparent lenses. Mie light scattering calculations have demonstrated that MLBs are potential sources of forward light scattering in human age-related nuclear cataracts due to their shape, size, frequency, and cytoplasmic contents, which often differ in refractive index from their surroundings. Previous studies have used data from several non-serial tissue sections viewed by light microscopy to extrapolate a volume and have assumed that MLBs are random in distribution. Currently, confocal microscopy is being used to examine actual tissue volumes from age-related nuclear cataracts and transparent lenses collected in India to confirm MLB shape, size, frequency, and randomness. These data allow Mie scattering calculations to be done with directly observed MLBs in intact tissue. METHODS: Whole Indian donor lenses and Indian lens nuclei after extracapsular cataract extraction were immersion-fixed in 10% formalin for 24 h and in 4% paraformaldehyde for 24 h before sectioning with a Vibratome. The 160 microm thick sections were stained for 24 h in the lipid dye DiI (1,1'-dilinoleyl-3,3,3',3' tetramethylindocarbocyanine, 4-chlorobenzenesulfonate), washed, stabilized in Permount under coverslips and examined with a Zeiss LSM 510 confocal microscope. Individual volumes of tissue (each typically 500,000 microm(3)) were examined using a plan-apochromat 63X oil (NA=1.4) lens. Other lenses were prepared for electron microscopy and histological examination using previously described procedures. RESULTS: Analysis of tissue volumes within Indian age-related nuclear cataracts and transparent lenses has confirmed that most MLBs are 1-4 microm in diameter and typically spherical with some occurring as doublets or in clusters. Most Indian cataracts and transparent lenses are similar to samples obtained in the United States. One cataract contained as many as 400,000 MLBs per mm(3) -100 times more than in cataracts collected in the United States. Pairwise distribution analysis has revealed that MLBs even in this exceptional case are found with a distribution that appears to be random. Mie calculations indicate that more than 90% of the incident light could be scattered by the high density of MLBs. CONCLUSIONS: An important finding was that one advanced Indian cataract contained many more MLBs than cataracts examined from India and previously from the United States. This indicates that specific conditions or susceptibilities may exist that promote the formation of excessive MLBs. Based on the extremely high frequency, as well as their spherical shape, large size, and apparent random distribution, the MLBs are predicted according to Mie light scattering calculations to cause high amounts of forward scattering sufficient to produce nuclear opacity.

Microbial keratitis in a case of lamellar ichthyosis.

We report a case of microbial keratitis in a patient with lamellar ichthyosis. An 11-year-old boy, a known case of lamellar ichthyosis, presented with microbial keratitis. Microbiological evaluation of corneal scraping revealed a mixed infection caused by gram-negative bacilli and gram-positive cocci. He was treated with topical ciprofloxacin and fortified cefazolin eyedrops. Microbial keratitis in patients with lamellar ichthyosis has a poor prognosis.

Deep lamellar keratoplasty in keratoconus with healed hydrops.

PURPOSE: To report a case of successful deep lamellar keratoplasty (DLK) in a patient with keratonous after healed hydrops. METHODS: A 17-year-old boy underwent DLK for corneal scar secondary to healed hydrops in a case of keratoconus. RESULTS: The graft was clear, with a postoperative best spectacle-corrected visual acuity of 20/20. CONCLUSIONS: DLK may be a viable option in healed hydrops.

Mutational spectrum of the SLC4A11 gene in autosomal recessive congenital hereditary endothelial dystrophy.

PURPOSE: Autosomal recessive congenital hereditary endothelial dystrophy (AR-CHED or CHED2) is a bilateral corneal disorder manifesting at birth or in early childhood. CHED2 is caused by mutations in the sodium bicarbonate transporter-like solute carrier family 4 member 11 (SLC4A11) gene on chromosome 20p13. We screened 42 unrelated families with CHED2 in order to establish the spectrum of mutations in SLC4A11 and to look for genotype-phenotype correlations. METHODS: Forty-two families (49 affected and 73 unaffected members) with recessive CHED were recruited according to predefined diagnostic criteria. Clinical data including age at onset and presentation, pre- and post-operative visual acuities, and presence of nystagmus were taken from patient records. Histopathologic parameters such as corneal thickness, Descemet membrane thickness, and endothelial cell counts were assessed on corneal sections. DNA from patients was screened for sequence changes by polymerase chain reaction (PCR)-amplification of coding regions of SLC4A11 and single strand conformation polymorphism analysis followed by sequencing. Sequence changes found were tested in 50 unrelated normal controls. RESULTS: Twenty-seven different mutations were identified in 35 unrelated families, 19 of which were not previously reported. The mutations identified consisted of 13 missense, 5 nonsense, 7 deletions, 1 complex (deletion plus insertion) mutation, and 1 splice site mutation. Both mutant alleles were identified in 33 families and only one mutant allele in two families. No correlations were evident between clinical or histopathologic parameters and SLC4A11 mutations. CONCLUSIONS: These data add to the mutational repertoire of SLC4A11 and establish the high degree of mutational heterogeneity in autosomal recessive CHED.

Mutation of the gap junction protein alpha 8 (GJA8) gene causes autosomal recessive cataract.

BACKGROUND: GJA8 encodes connexin-50, a gap junction protein in the eye lens. Mutations in GJA8 have been reported in families with autosomal dominant cataract. OBJECTIVE: To identify the disease gene in a family with congenital cataract of autosomal recessive inheritance. METHODS: Eight candidate genes were screened for pathogenic alterations in affected and unaffected family members and in normal unrelated controls. RESULTS: A single base insertion leading to frameshift at codon 203 of connexin 50 was found to co-segregate with disease in the family. CONCLUSIONS: These results confirm involvement of GJA8 in autosomal recessive cataract.

Coexistent congenital hereditary endothelial dystrophy and congenital glaucoma.

PURPOSE: To retrospectively evaluate the coexistence of congenital glaucoma with congenital hereditary endothelial dystrophy. METHODS: Ten infants presented to our hospital with diffuse corneal edema and bilaterally elevated intraocular pressure (IOP). These patients were diagnosed with congenital glaucoma. All patients underwent trabeculotomy with trabeculectomy for control of IOP. Although IOP was normalized in all patients, corneal edema persisted. These patients underwent penetrating keratoplasty, and the buttons were subjected to histopathologic examination. RESULTS: The corneal grafts remained clear in all patients. The histopathologic examination of the excised corneal buttons showed diffuse stromal edema, loss of the endothelial cell layer, and thickening of the posterior non-banded portion of the Descemet membrane, suggestive of congenital hereditary endothelial dystrophy. CONCLUSIONS: Congenital hereditary endothelial dystrophy may coexist with congenital glaucoma. This combination should be suspected where persistent and total corneal opacification fails to resolve after normalization of IOP.

A cluster of Nocardia keratitis after LASIK.

PURPOSE: To report a cluster of Nocardia asteroides keratitis cases after LASIK. METHODS: Retrospective review of the history and examination of three patients (four eyes) operated on the same day at a single center who developed postoperative keratitis. All patients underwent lifting of the superficial flap for microbiologic evaluation of the corneal scrapings. The operating surgeon was contacted to identify the possible source of contamination. RESULTS: Two patients underwent simultaneous bilateral LASIK; however, only one developed postoperative keratitis in both eyes. One patient had unilateral surgery and developed keratitis in the operated eye. Microscopic examination of smears from all eyes revealed thin, branching, acid-fast, filamentous bacteria that were identified as Nocardia asteroides after culture. The infiltrates resolved with topical administration of amikacin sulphate (2.5%) and topical and oral trimethoprim-sulfamethoxazole. Final visual acuity ranged between 20/25 and 20/80. The operating surgeon had used the same blade and microkeratome in all patients. CONCLUSIONS: Nocardia, a relatively unusual organism, can cause an epidemic of infection after LASIK.

Autosomal recessive corneal endothelial dystrophy (CHED2) is associated with mutations in SLC4A11.

OBJECTIVE: To map and identify the gene for autosomal recessive congenital hereditary endothelial dystrophy (CHED2, OMIM 217700), a disorder characterised by diffuse bilateral corneal clouding that may lead to visual impairment and requiring corneal transplantation. METHODS: Members of 16 families with autosomal recessive CHED were genotyped for 13 microsatellite markers at the CHED2 locus on chromosome 20p13-12. Two-point linkage analysis was carried out using the FASTLINK version of the MLINK program. Mutation screening was carried out by amplification of exons and flanking regions by polymerase chain reaction, followed by direct automated sequencing. RESULTS: Linkage and haplotype analysis placed the disease locus within a 2.2 cM (1.3 Mb) interval flanked by D20S198 and D20S889, including SLC4A11. The maximum LOD score of 11.1 was obtained with D20S117 at theta = 0. Sequencing of SLC4A11 showed homozygotic mutations in affected members from 12 of 16 families. CONCLUSION: These results confirm that mutations in the SLC4A11 gene cause autosomal recessive CHED.

Spontaneneous hydrops in pellucid marginal degeneration: documentation by OCT-III.

Corneal pellucid marginal degeneration (PMCD) is an idiopathic condition characterized by non-inflammatory, non-ulcerative thinning of inferior peripheral cornea. PMCD has been reported occasionally complicated with hydrops owing to break in descemet membrane. We herein report a 38-year-old man, who presented with sudden dimness of vision in right eye. Clinical findings and Orbscan II were suggestive of PMCD in both eyes with hydrops in right eye. Slit-lamp and optical coherence tomography of right eye showed central descemet's detachment without any break. Patient underwent descemetopexy by isoexpansile C3F8 (14%) and is doing well with significant improvement in the hydrops.

Outcome of corneal transplant rejection: a 10-year study.

PURPOSE: To study the incidence of graft rejection and the predictive factors for its reversibility. METHODS: It is a retrospective study of 1927 consecutive penetrating keratoplasties performed between January 1990 and January 2000 with more than 6 months follow up. A total of 224 rejection episodes were noted in 183 patients. Of these, 184 first rejection episodes were included in this study. RESULTS: The incidence of first rejection episode was 9.55%. Of patients 87% were symptomatic during the episode with vision loss being the commonest. The average time of onset of rejection was 15.25 +/- 14.4 months (median 11.7 months). In total, 53.3% of rejections occurred within 1 year after surgery. Of the patients who completed minimum 3 months follow up after the rejection episode, the rate of reversibility was 63.3%. Major predictive factors for a poor outcome after rejection were repeated grafting and associated anterior vitrectomy during surgery. The reversibility of the episode did not differ significantly with the modality of treatment used, but treatment with intravenous steroids within 7 days of onset of rejection may have a role in reducing the recurrences of rejection episodes, thus increasing the graft survival. CONCLUSION: Regrafts and associated anterior vitrectomy were significant risk factors for a poor outcome following rejection episode.