A mutated CRYGD associated with congenital coralliform cataracts in two Chinese pedigrees.

AIM: To investigate the causal gene mutation and clinical characteristics for two Chinese families with autosomal dominant congenital coralliform cataract. METHODS: Two Chinese pedigrees with congenital cataract were investigated. Routine ophthalmic examinations were performed on all patients and non-affected family members. Peripheral blood samples were collected, and the genomic DNAs were extracted. The coding regions of proband's DNAs were analyzed with cataract gene panel. The identified mutation was amplified by polymerase chain reaction, and automated sequencing was performed in other members of two families to verify whether the mutated gene was co-segregated with the disease. RESULTS: Congenital coralliform cataract was inherited in an autosomal dominant mode in both pedigrees. For each family, more than half of the family members were affected. All patients presented with severe visual impairment after birth as a result of bilateral symmetric coralliform lens opacification. An exact the same defect in the same gene, a heterozygous mutation of c.70C>A (p. P24T) in exon 2 of γD-crystallin gene, was detected in both probands from each family. Sanger sequencing analysis demonstrated that the mutated CRYGD was co-segregated in these two families. CONCLUSION: A c.70C>A (p. P24T) variant in CRYGD gene was reconfirmed to be the causal gene in two Chinese pedigrees. It is known that mutated CRYGD caused most of the congenital coralliform cataracts, suggesting that the CRYGD gene is associated with coralliform congenital cataract.

[The effects of chloride channel inhibitor on bFGF-induced proliferation of lens epithelial cells].

OBJECTIVE: To study the effect of chloride channel on proliferation induced by basic-fibroblast growth factor (bFGF) in human lens epithelial cells HLE B-3 (LEC). METHODS: HLE B-3 cells at Logarithmic growth phase were incubated in the 6 or 96 well plate overnight and the proliferation was induced by 10 µg/L bFGF. The cells were divided into bFGF group treated with bFGF and the blank control group with the regular cells culture. LEC were treated with different concentrations of chloride channel inhibitors: 5-nitro-2-[3-phenylpropylamino] benzoic acid (NPPB) at 50, 100, 150 µmol/L and 4 , 4'-2 diisothiocyanostilbene-2, 2' -disulfonic acid (DIDS) at 10, 50, 100 µmol/L with bFGF or without bFGF in 10% serum for 24, 48, 72 hours. The cell viability and the drug toxicity were detected by CCK-8 colorimetric assay. The expression of proliferating cell nuclear antigen (Ki-67) of LEC treated with NPPB or DIDS were observed by immunocytochemistry staining assay. The phase change of cell cycle was examined by flow cytometry. Each experiment group and control group were compared using two-way ANOVA, further pairwise comparisons using LSD-t test or by the Independent-Samples t test. RESULTS: 10 µg/L bFGF induced LEC proliferation and the values of A stage were gradually declined with the increase of chloride channel inhibitors' concentrations and time at 24 hours, 48 hours and 72 hours (bFGF+NPPB group: Ftime = 305.28, Fconcentrations = 18.76, P = 0.000;bFGF+ DIDS group:Ftime = 94.44, Fconcentrations = 24.42, P = 0.000). Different concentrations of chloride channel inhibitor reduced the values of a stage with 10 µg/L bFGF in a dose- and time-dependent manner. The expression of Ki-67 was significantly lowered compared with the bFGF group (bFGF+NPPB group: 18.32% ± 1.23%, F = 580.3, P = 0.000;bFGF+DIDS group: 11.21% ± 1.02%, F = 507.4, P = 0.000), when 150 µmol/L NPPB and 100 µmol/L DIDS with 10 µg/L bFGF were added at 48 hours. After 150 µmol/L NPPB and 100 µmol/L DIDS treatment at the 48th hour, the rate of G1 stage was significantly increased (F = 390.754, P = 0.000), where as that of S stage decreased significantly (F = 166.240, P = 0.000). CONCLUSIONS: These results indicate that chloride channel inhibitors play an important role in modulating the proliferation cycle of LEC treated with bFGF by limiting the cell at cycle S/G1 stage from dividing into S stage.

[Inhibition of lens epithelial cell cycle and PCNA mRNA expression by an antisense oligonucleotides complementary to the messenger RNA of proliferating cell nuclear antigen].

OBJECTIVE: To determine whether antisense oligonucleotides complementary to the messenger RNA of proliferating cell nuclear antigen (PCNA ASODN) inhibit the proliferation of bovine lens epithelial cell (BLEC) by changing the cell cycle and down-regulating the expression of PCNA mRNA and PCNA protein. METHODS: BLECs were cultured in vitro, and the second passage cells were used in this experiment. PCNA ASODN (30 micro mol/L), PCNA SODN (sense oligonucleotides, 30 micro mol/L), basic fibroblast growth factor (bFGF, 10 micro g/L), bFGF (10 ng/ml) + ASODN (30 micro mol/L), bFGF (10 micro g/L) + SODN (30 micro mol/L) were introduced respectively into the medium, and the same amount of PBS was added into the medium as a control. After 24 hours, the cell cycle and the PCNA expression were counted by flow cytometry, and the expression of PCNA mRNA was indicated by Northern ELISA hybridization. RESULTS: PCNA ASODN could decrease the rate of the cells of S phase and down-regulate the expression of PCNA mRNA and PCNA protein. Comparing with the control group, after 24 hours, the rate of cells of S phase was decreased from 15.67% to 7.96%, the expression of PCNA mRNA from 0.266 to 0.176 and the expression of PCNA protein from 55.27% to 12.32%. PCNA ASODN could also inhibit the proliferation of BLEC induced by bFGF, comparing with the bFGF group, the rate of cells of S phase was decreased from 23.4% to 19.9%, the expression of PCNA mRNA from 0.576 to 0.357 and the expression of PCNA protein from 76.4% to 35.48%. CONCLUSIONS: Our results demonstrate that the PCNA ASODN decreases expression of PCNA mRNA and PCNA protein, and stops the cell to enter and progress through S phase. These results provide an important impetus to initiate in vivo studies to determine the feasibility of antisense strategies in the prevention of posterior capsular opacification.