Mutation causing self-aggregation in human γC-crystallin leading to congenital cataract

Purpose: Many forms of congenital hereditary cataract are associated with mutations in the crystallin genes. The authors focus attention on congenital lamellar cataract, which is associated with the R168W mutation in γC-crystallin, and congenital zonular pulverulent cataract, which is associated with a 5-bp insertion in the γC-crystallin gene. Methods: To understand the molecular phenotypes-i.e., the functional defects that have occurred in the mutant γC-crystallin molecule in two cases described-the authors cloned, expressed, isolated, and compared the solution state structural features of these mutants with those of normal (wild-type) γC-crystallin. Structural models of the wild-type and mutant have been generated using comparative modeling. Circular dichroism and fluorescence spectroscopic methods were used to determine the conformation of the proteins, and temperature dependent self-aggregation was used to observe the quaternary structural features. The structural stability of the proteins was monitored with the use of chemical and thermal denaturation. Results: The authors found that the 5-bp insertion led to a loss of secondary and tertiary structures of the molecule and to an enhanced tendency of self-aggregation into light-scattering particles, offering a possible factor in lens opacification. The R168W mutant, on the other hand, was remarkably similar to the wild-type molecule in its conformation and structural stability, but it differed in its ability to aggregate and scatter light. Conclusions: These results support the idea that unfolding or structural destabilization is not always necessary for crystallin-associated cataractogenesis

Mutation Causing Self-Aggregation in Human ␥C-Crystallin Leading to Congenital Cataract

PURPOSE. Many forms of congenital hereditary cataract are associated with mutations in the crystallin genes. The authors focus attention on congenital lamellar cataract, which is associated with the R168W mutation in ␥C-crystallin, and congenital zonular pulverulent cataract, which is associated with a 5-bp insertion in the ␥C-crystallin gene. METHODS. To understand the molecular phenotypes-i.e., the functional defects that have occurred in the mutant ␥C-crystallin molecule in two cases described-the authors cloned, expressed, isolated, and compared the solution state structural features of these mutants with those of normal (wild-type) ␥C-crystallin. Structural models of the wild-type and mutant have been generated using comparative modeling. Circular dichroism and fluorescence spectroscopic methods were used to determine the conformation of the proteins, and temperature dependent self-aggregation was used to observe the quaternary structural features. The structural stability of the proteins was monitored with the use of chemical and thermal denaturation. RESULTS. The authors found that the 5-bp insertion led to a loss of secondary and tertiary structures of the molecule and to an enhanced tendency of self-aggregation into light-scattering particles, offering a possible factor in lens opacification. The R168W mutant, on the other hand, was remarkably similar to the wild-type molecule in its conformation and structural stability, but it differed in its ability to aggregate and scatter light. CONCLUSIONS. These results support the idea that unfolding or structural destabilization is not always necessary for crystallinassociated cataractogenesis. (Invest Ophthalmol Vis Sci. 2006; 47:5212-5217

Modified helix-loop-helix motifs of calmodulin: the influence of the exchange of helical regions on calcium-binding affinity

The four calcium-binding sites, called the helix-loop-helix, or the EF-hand motifs, of calmodulin differ in their ion-binding affinities; this has been thought to arise due to the variations in the sequences of the loop regions where the ion binds. We focus attention here on the role of the flanking helical regions on the calcium-binding affinities. Peptides were synthesized in a manner that simulates the E and F helical flanks of site 4 (the strongest calcium-binding site of the calmodulin) to sandwich the loop sequences of sites 1, 2, 3 and 4 so as to produce peptides named 414, 424, 434 and 444, as well as using the helical flanks of site 1 (the weakest site) to produce peptides 111, 121, 131 and 141. Calcium binding was monitored using the calcium-mimic dye Stains-all (4,4,4',5'-dibenzo-3,3'-diethyl-9-methyl-thiacarbocya-nine bromide). Binding abilities were seen to increase several-fold when the E and F helices of site 1 were replaced by those of site 4 (i.e., 111-414). In contrast, the intensity of circular dichroism induced in the absorption bands of the bound achiral dye decreased significantly when the helical flanks of site 4 were replaced with those of site 1 (i.e., 444-141). The helical flanks of site 4 impart greater binding ability to a given loop region, while the helical flanks of site 1 tend to weaken it

Novel mutations of the carbohydrate sulfotransferase-6 (CHST6) gene causing macular corneal dystrophy in India

Purpose: Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by progressive central haze, confluent punctate opacities and abnormal deposits in the cornea. It is caused by mutations in the carbohydrate sulfotransferase-6 (CHST6) gene, encoding corneal N-acetyl glucosamine-6-O-sulfotransferase (C-GlcNAc-6-ST). We screened the CHST6 gene for mutations in Indian families with MCD, in order to determine the range of pathogenic mutations. Methods: Genomic DNA was isolated from peripheral blood leukocytes of patients with MCD and normal controls. The coding regions of the CHST6 gene were amplified using three pairs of primers and amplified products were directly sequenced. Results: We identified 22 (5 nonsense, 5 frameshift, 2 insertion, and 10 missense) mutations in 36 patients from 31 families with MCD, supporting the conclusion that loss of function of this gene is responsible for this corneal disease. Seventeen of these mutations are novel. Conclusions: These data highlight the allelic heterogeneity of macular corneal dystrophy in Indian patients

Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant

Background Optineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process. Results We show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N) in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking. Conclusions Our results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis

Identification of R368H as a predominant CYP1B1 allele causing primary congenital glaucoma in Indian patients

Purpose: To investigate the predominant mutation in the CYP1B1 gene in patients in India with primary congenital glaucoma (PCG), using PCR-restriction fragment length polymorphism (RFLP) methods and to characterize the molecular defect in two generations of an affected family. Methods: DNA samples from 146 patients with PCG from 138 pedigrees were analyzed for several distinct mutations in CYP1B1 by PCR-RFLP. Results: PCR-RFLP screening revealed that 30.8% of patients were positive for any one of the six mutations (376insA, 528G→A, 923C→T, 959G→A, 1449G→A, and 1514C→A), and 17.8% of the patients were found to have the rarely reported mutation R368H (1449G→A). All mutations were confirmed by DNA sequencing. Conclusions: The results suggest extensive allelic heterogeneity in the Indian patients with PCG, with the predominant allele being R368H among the 146 Indian patients tested. It appears possible to use this approach for carrier detection in pedigrees with a positive family history and in population screening. The approach also offers a method for rapid screening of potential carriers and affected individuals

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 µm 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 µm 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 µm3) 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 µm 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 mm3 –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

Microsporidial keratitis in India: 16S rRNA gene-based PCR assay for diagnosis and species identification of microsporidia in clinical samples

Purpose: To evaluate 16S rRNA-based polymerase chain reactions for the detection and species identification of the microsporidia that cause keratitis. Methods: Of the 5892 cases of microbial keratitis seen between September 2002 and December 2005, 31 (0.5%) microscopically diagnosed cases of microsporidial keratitis were included in the test group; 103 patients with nonmicrosporidial keratitis constituted the control group. A 16S rRNA-based pan-microsporidian PCR was chosen for the detection of microsporidian DNA. Species level identification was made using species-specific primer sets of Encephalitozoon spp (E. cuniculi, E. hellem, and E. intestinalis). Sequencing and BLAST analysis of amplicons obtained with pan-microsporidian primers were performed for validation. Results: The corneal scrapings from 26 of 31 cases in the test group and 2 of 103 cases in the control group showed a 250- to 280-bp amplicon in PCR by pan-microsporidian primers (sensitivity of 83% and specificity of 98%). The amplicons of 13 of 26 test group samples were identified by species-specific PCR: E. cuniculi, n = 7 (549 bp); E. hellem; n = 3 (549 bp); E. intestinalis; n = 1 (520 bp). The two cases in the control group were identified to be E. cuniculi. The remaining 15 cases (test group) were confirmed to be Vittaforma corneae by sequencing and BLAST analysis. All species were confirmed by sequencing and database homology comparison. Conclusions: This study is the first to validate PCR-based assays for detection of microsporidial DNA in corneal scrapings. Pan microsporidian PCR can be a useful adjunct with smear examination in the diagnosis of microsporidial keratitis

Mutation spectrum of the CYP1B1 gene in Indian primary congenital glaucoma patients

Purpose: The human Cytochrome P450 gene CYP1B1 has been implicated in primary congenital glaucoma worldwide. The aim of this study was to understand the role of CYP1B1 mutations in causing primary congenital glaucoma in Indian populations. Methods: The study included 64 new and unrelated cases of primary congenital glaucoma from different ethnic groups of India. Direct sequencing screened the coding and the promoter regions of CYP1B1. Results: Sixteen pathogenic mutations were observed in 24 cases, of which 7 were novel. These included two frameshift mutations leading to deletions of 23 bp (g.3905del23bp) and 2 bp (g.7900-7901delCG) in exons II and III, respectively. Four novel missense mutations viz. A115P, M132R, Q144P, S239R were noted in exon II, and one in exon III (G466D), whose residue is a part of the "signature sequence" (NH2-FXXGXXXCXG-COOH) and is present in all heme binding cytochromes. Overall, CYP1B1 was involved in 37.50% (24/64) cases and homozygosity of the mutant allele was seen in 29.68% (19/64) and compound heterozygosity in 3.12% (2/64) of the cases, respectively. The frequency of CYP1B1 mutations was comparatively lower than Saudi Arabian, Slovakian Gypsys, and Turkish populations, largely due to genetic heterogeneity and ethnic diversities in Indian populations. Genotype-phenotype correlation indicated variable prognosis that could be due to the type of mutation, leading to alteration of CYP1B1 protein. Conclusions: This study provides a mutation spectrum of CYP1B1 causing primary congenital glaucoma in Indian populations that has implications in devising molecular diagnostics for rapid screening