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1.  Exome sequencing identifies novel and recurrent mutations in GJA8 and CRYGD associated with inherited cataract 
Human Genomics  2014;8(1):19.
Inherited cataract is a clinically important and genetically heterogeneous cause of visual impairment. Typically, it presents at an early age with or without other ocular/systemic signs and lacks clear phenotype-genotype correlation rendering both clinical classification and molecular diagnosis challenging. Here we have utilized trio-based whole exome sequencing to discover mutations in candidate genes underlying autosomal dominant cataract segregating in three nuclear families.
In family A, we identified a recurrent heterozygous mutation in exon-2 of the gene encoding γD-crystallin (CRYGD; c.70C > A, p.Pro24Thr) that co-segregated with ‘coralliform’ lens opacities. Families B and C were found to harbor different novel variants in exon-2 of the gene coding for gap-junction protein α8 (GJA8; c.20T > C, p.Leu7Pro and c.293A > C, p.His98Pro). Each novel variant co-segregated with disease and was predicted in silico to have damaging effects on protein function.
Exome sequencing facilitates concurrent mutation-profiling of the burgeoning list of candidate genes for inherited cataract, and the results can provide enhanced clinical diagnosis and genetic counseling for affected families.
Electronic supplementary material
The online version of this article (doi:10.1186/s40246-014-0019-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4240822  PMID: 25403472
Cataract; Exome sequencing; CRYGD; GJA8
2.  Aquaporin-0 Targets Interlocking Domains to Control the Integrity and Transparency of the Eye Lens 
Lens fiber cell membranes contain aquaporin-0 (AQP0), which constitutes approximately 50% of the total fiber cell membrane proteins and has a dual function as a water channel protein and an adhesion molecule. Fiber cell membranes also develop an elaborate interlocking system that is required for maintaining structural order, stability, and lens transparency. Herein, we used an AQP0-deficient mouse model to investigate an unconventional adhesion role of AQP0 in maintaining a normal structure of lens interlocking protrusions.
The loss of AQP0 in AQP0−/− lens fibers was verified by Western blot and immunofluorescence analyses. Changes in membrane surface structures of wild-type and AQP0−/− lenses at age 3 to 12 weeks were examined with scanning electron microscopy. Preferential distribution of AQP0 in wild-type fiber cell membranes was analyzed with immunofluorescence and immunogold labeling using freeze-fracturing transmission electron microscopy.
Interlocking protrusions in young differentiating fiber cells developed normally but showed minor abnormalities at approximately 50 μm deep in the absence of AQP0 in all ages studied. Strikingly, protrusions in maturing fiber cells specifically underwent uncontrolled elongation, deformation, and fragmentation, while cells still retained their overall shape. Later in the process, these changes eventually resulted in fiber cell separation, breakdown, and cataract formation in the lens core. Immunolabeling at the light microscopy and transmission electron microscopy levels demonstrated that AQP0 was particularly enriched in interlocking protrusions in wild-type lenses.
This study suggests that AQP0 exerts its primary adhesion or suppression role specifically to maintain the normal structure of interlocking protrusions that is critical to the integrity and transparency of the lens.
This study provides a novel finding that the water channel protein aquaporin-0 (AQP0) has a primary role in maintaining a normal structure of an interlocking system that is essential for maintaining structural order, stability, and transparency of the eye lens.
PMCID: PMC3941616  PMID: 24458158
aquaporin-0; lens; interlocking domain; AQP0-deficient mice; adhesion
3.  Mutation of the Melastatin-Related Cation Channel, TRPM3, Underlies Inherited Cataract and Glaucoma 
PLoS ONE  2014;9(8):e104000.
Inherited forms of cataract are a clinically important and genetically heterogeneous cause of visual impairment that usually present at an early age with or without systemic and/or other ocular abnormalities. Here we have identified a new locus for inherited cataract and high-tension glaucoma with variable anterior segment defects, and characterized an underlying mutation in the gene coding for transient receptor potential cation channel, subfamily M, member-3 (TRPM3, melastatin-2). Genome-wide linkage analysis mapped the ocular disease locus to the pericentric region of human chromosome 9. Whole exome and custom-target next-generation sequencing detected a heterozygous A-to-G transition in exon-3 of TRPM3 that co-segregated with disease. As a consequence of alternative splicing this missense mutation was predicted to result in the substitution of isoleucine-to-methionine at codon 65 (c.195A>G; p.I65 M) of TRPM3 transcript variant 9, and at codon 8 (c.24A>G; p.I8 M) of a novel TRPM3 transcript variant expressed in human lens. In both transcript variants the I-to-M substitution was predicted in silico to exert damaging effects on protein function. Furthermore, transient expression studies of a recombinant TRPM3-GFP reporter product predicted that the I-to-M substitution introduced an alternative translation start-site located 89 codons upstream from the native initiator methionine found in eight other TRPM3 transcript variants (1–8). Collectively, these studies have provided the first evidence that TRPM3 is associated with inherited ocular disease in humans, and further provide support for the important role of this cation channel in normal eye development.
PMCID: PMC4121231  PMID: 25090642
4.  Focus on Molecules: Major Intrinsic Protein 
Experimental eye research  2010;101:107-108.
PMCID: PMC3261317  PMID: 21134370
lens; aquaporin; thin-junction; cataract
5.  Pharmacogenetics of Complement Factor H (Y402H) and treatment of exudative age-related macular degeneration with ranibizumab 
To determine whether complement factor H (CFH) genotypes have a pharmacogenetic effect on the treatment of exudative age-related macular degeneration (AMD) with ranibizumab.
A retrospective study of 156 patients with exudative AMD treated with intravitreal ranibizumab monotherapy was conducted. AMD phenotypes were characterized by clinical examination, visual acuity, fundus photography, fluorescein angiography, and injection timing. Patients received intravitreal ranibizumab injections as part of routine ophthalmologic care and were followed for a minimum of nine months. Each patient was genotyped for the single nucleotide polymorphism rs1061170 (Y402H) in the CFH gene.
Baseline lesion size and angiographic type, as well as mean visual acuities at baseline, 6 months, and 9 months were similar among the three CFH genotypes. Over 9 months, patients with both risk alleles received approximately one more injection (p = 0.09). In a recurrent event analysis, patients homozygous for the CFH Y402H risk allele had a 37% significantly higher risk of requiring additional ranibizumab injections (p = 0.04)
In our cohort, response to treatment of AMD with ranibizumab differed according to CFH genotype, suggesting that determining patients' CFH genotype may be helpful in the future in tailoring treatment for exudative AMD with intravitreal ranibizumab.
PMCID: PMC3490485  PMID: 19091853
Complement Factor H; Ranibizumab; Age-Related Macular Degeneration; Pharmacogenetics
6.  A Role for Epha2 in Cell Migration and Refractive Organization of the Ocular Lens 
Epha2, an abundant component of the lens fiber cell membrane, is required for tissue patterning and refractive organization of the lens.
The Epha2 receptor is a surprisingly abundant component of the membrane proteome of vertebrate lenses. In humans, genetic studies have linked mutations in EPHA2 to inherited and age-related forms of cataract, but the function of Epha2 in the lens is obscure. To gain insights into the role of Epha2, a comparative analysis of lenses from wild-type and Epha2−/− mice was performed.
Epha2 distribution was examined using immunocytochemistry and Western blot analysis. Lens optical quality was assessed by laser refractometry. Confocal microscopy was used to analyze cellular phenotypes.
In wild-type lenses, Epha2 was expressed by lens epithelial cells and elongating fibers but was degraded during the later stages of fiber differentiation. Epha2-null lenses retained their transparency, but two key optical parameters, lens shape and internal composition, were compromised in Epha2−/− animals. Epha2-null lenses were smaller and more spherical than age-matched wild-type lenses, and laser refractometry revealed a significant decrease in refractive power of the outer cell layers of mutant lenses. In the absence of Epha2, fiber cells deviated from their normal course and terminated at sutures that were no longer centered on the optical axis. Patterning defects were also noted at the level of individual cells. Wild-type fiber cells had hexagonal cross-sectional profiles with membrane protrusions extending from the cell vertices. In contrast, Epha2−/− cells had irregular profiles, and protrusions extended from all membrane surfaces.
These studies indicate that Epha2 is not required for transparency but does play an indispensable role in the cytoarchitecture and refractive quality of the lens.
PMCID: PMC3317406  PMID: 22167091
7.  A recurrent missense mutation in GJA3 associated with autosomal dominant cataract linked to chromosome 13q 
Molecular Vision  2011;17:2255-2262.
To map and identify the genetic defect underlying autosomal dominant cataract segregating in a 5-generation Caucasian American family.
Genomic DNA was prepared from blood leukocytes, genotyping was performed using microsatellite markers, and logarithm of the odds (LOD) scores were calculated using the LINKAGE programs. Mutation profiling was performed using direct exon cycle-sequencing and restriction fragment analysis. Protein function effects were evaluated using in silico prediction algorithms.
Significant evidence of linkage was obtained at marker D13S175 (maximum LOD score [Zmax]=3.67; maximum recombination fraction [θmax]=0.04) and D13S1316 (Zmax=2.80, θmax=0.0). Haplotyping indicated that the disease lay in the ~170 Kb physical interval between D13S1316 and D13S175, which contained the gene for gap-junction protein alpha-3 (GJA3) or connexin-46. Sequencing of GJA3 detected a heterozygous transition (c.130G>A) in exon-2 that resulted in gain of an Hsp92 II restriction site. Allele-specific PCR amplification and restriction analysis confirmed that the novel Hsp92 II site co-segregated with cataract in the family but was not detected in 192 normal unrelated individuals. The c.130G>A transition was predicted to result in a non-conservative substitution of valine-to-methionine at codon 44 (p.V44M) with damaging effects on protein function.
These data confirm GJA3 as one of the most frequently mutated genes that underlie autosomal dominant cataract in humans, and further emphasize the importance of connexin function in maintaining lens transparency.
PMCID: PMC3164684  PMID: 21897748
8.  Cat-Map: putting cataract on the map 
Molecular Vision  2010;16:2007-2015.
Lens opacities, or cataract(s), may be inherited as a classic Mendelian disorder usually with early-onset or, more commonly, acquired with age as a multi-factorial or complex trait. Many genetic forms of cataract have been described in mice and other animal models. Considerable progress has been made in mapping and identifying the genes and mutations responsible for inherited forms of cataract, and genetic determinants of age-related cataract are beginning to be discovered. To provide a convenient and accurate summary of current information focused on the increasing genetic complexity of Mendelian and age-related cataract we have created an online chromosome map and reference database for cataract in humans and mice (Cat-Map).
PMCID: PMC2965572  PMID: 21042563
9.  Clinical phenotypes associated with the Complement Factor H Y402H variant in age-related macular degeneration 
American journal of ophthalmology  2007;144(3):404-408.
To determine whether the complement factor H (CFH) Y402H variant is associated with specific age-related macular degeneration (AMD) clinical phenotypes.
Retrospective, case-control study.
188 Caucasian subjects with AMD and 189 control subjects were genotyped for the T-to-C polymorphism in exon-9 of the CFH gene by restriction-fragment length analysis and DNA sequencing using genomic DNA from mouthwash samples. AMD phenotypes were characterized by clinical examination, fundus photography, and fluorescein angiography.
Heterozygosity for the at-risk genotype (TC) increased the likelihood for AMD 2.1-fold (95% CI 1.3–3.3) while homozygosity for the genotype (CC) increased the likelihood for AMD 6.5-fold (95% CI 3.4–12.5) in our population. The C allele was significantly associated with predominantly classic choroidal neovascularization (OR 2.01, 95% CI 1.34–3.30). Neovascular lesion size was similar among the three genotypes (p=0.67).
The Y402H CFH variant carried a significantly increased risk for developing AMD in our population. Genotype/phenotype correlations regarding choroidal neovascular lesion type were observed
PMCID: PMC2140051  PMID: 17631852
10.  The EPHA2 gene is associated with cataracts linked to chromosome 1p 
Molecular Vision  2008;14:2042-2055.
Cataracts are a clinically and genetically heterogeneous disorder affecting the ocular lens, and the leading cause of treatable vision loss and blindness worldwide. Here we identify a novel gene linked with a rare autosomal dominant form of childhood cataracts segregating in a four generation pedigree, and further show that this gene is likely associated with much more common forms of age-related cataracts in a case-control cohort.
Genomic DNA was prepared from blood leukocytes, and genotyping was performed by means of single nucleotide polymorphism (SNP) markers, and short tandem repeat (STR) markers. Linkage analyses were performed with the GeneHunter and MLINK programs, and association analyses were performed with the Haploview and Exemplar programs. Mutation detection was achieved by PCR amplification of exons and di-deoxy cycle-sequencing.
Genome-wide linkage analysis with SNP markers, identified a likely disease-haplotype interval on chromosome 1p (rs707455-[~10 Mb]-rs477558). Linkage to chromosome 1p was confirmed using STR markers D1S2672 (LOD score [Z]=3.56, recombination distance [θ]=0), and D1S2697 (Z=2.92, θ=0). Mutation profiling of positional-candidate genes detected a heterozygous transversion (c.2842G>T) in exon 17 of the gene coding for Eph-receptor type-A2 (EPHA2) that cosegregated with the disease. This missense change was predicted to result in the non-conservative substitution of a tryptophan residue for a phylogenetically conserved glycine residue at codon 948 (p.G948W), within a conserved cytoplasmic domain of the receptor. Candidate gene association analysis further identified SNPs in the EPHA2 region of chromosome 1p that were suggestively associated with age-related cataracts (p=0.007 for cortical cataracts, and p=0.01 for cortical and/or nuclear cataracts).
These data provide the first evidence that EPHA2, which functions in the Eph-ephrin bidirectional signaling pathway of mammalian cells, plays a vital role in maintaining lens transparency.
PMCID: PMC2582197  PMID: 19005574

Results 1-10 (10)