Family 1, a five-generation Chinese Han family, and Family 2, a four-generation Chinese Han family, with autosomal dominant congenital cataracts were recruited for the present study from Peking University Third Hospital (Beijing, China; Fig. 1). A total of 100 healthy control individuals were also recruited from Peking University Third Hospital. The present study was approved by the ethics committee of Peking University Health Science Center. Informed consent was obtained from all participants. The present study followed the principles of the Declaration of Helsinki (7). The congenital cataract-affected status was determined by a history of cataract extraction or ophthalmologic examination, and the participants underwent ophthalmic examination, including visual acuity assessment, slit-lamp examination and intraocular pressure measurement. The phenotypes were documented using slit lamp photography (Topcon SL-1E; Topcon Medical Systems, Inc., Oakland, NJ, USA). Subsequently, 5 ml venous blood was obtained from each family member and control, and was collected in a BD Vacutainer (BD Biosciences, San Jose, CA, USA), containing EDTA. The genomic DNA was extracted using a QIAamp DNA Blood Mini kit (Qiagen, Germantown, MD, USA).

All coding exons and flanking splicing junctions of the candidate genes associated with congenital cataracts, including CRYAA, CRYAB, CRYBA1, CRYBB1, CRYBB2, CRYGC, CRYGD, CRYGS, GJA3, GJA8 and CRYBA4 were amplified using polymerase chain reaction (PCR), using the primers listed in Table I. Each reaction mixture (25 µl) contained 20 ng genomic DNA, 1X PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 0.5 µM forward primer, 0.5 µM reverse primer and 2.5 Units Taq DNA polymerase (Qiagen, Mississauga, ON, Canada). The following PCR program was used for DNA amplification: 95°C for 5 min; followed by 35 cycles at 95°C for 30 sec, 57–63°C for 30 sec (annealing temperature difference according to primer), 72°C for 30 sec, and a final extension at 72°C for 10 min. The PCR products of the probands from each family and one unaffected member were sequenced using an ABI3730 Automated Sequencer (PE Biosystems, Foster City, CA, USA). The sequencing results were analyzed using Chromas 2.33 (Technelysium Pty Ltd., South Brisbane, Australia) and were compared with the reference sequence in the NCBI database (http://www.ncbi.nlm.nih.gov/). Finally, mutations was screened for in the CRYAA and GJA8 genes from the family members and 100 ethnically matched controls to confirm the mutation.

The amino acid sequences of CRYAA and GJA8 from several different species were obtained from the NCBI GenBank (http://www.ncbi.nlm.nih.gov/genbank), and conservation analysis was performed using CLC Main Workbench 4.5.1 Software (Aarhus, Denmark). The function impact of the mutation was predicted using Polymorphism phenotyping (PolyPhen; http://genetics.bwh.harvard.edu/pph2/).

The human CRYAA and GJA8 open reading frame (ORF) cDNA was obtained from GeneCopoeia (Rockville, MD, USA). Site-directed mutagenesis was performed to generate CRYAA bearing the p.L139P mutation and GJA8 bearing the p.D47N mutation, using a QuickChange Lightning Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). DNA sequencing was used to confirm the introduced mutation (ABI 3730 Automated Sequencer; Applied Biosystems, Foster City, CA, USA). The ORFs of the wild-type (WT) and mutant (MT) sequences were amplified using PCR from the cDNAs, and were inserted into the HindIII- and XhoI-digested pEGFP-N1 vector (Invitrogen Life Technologies, Carlsbad, CA, USA) to produce the pEGFP-CRYAA-WT, pEGFP-CRYAA-MT, pEGFP-GJA8-WT and pEGFP-GJA8-MT expression plasmids. Each reaction mixture (25 µl) contained 200 ng plasmids, 2X GC buffer, 0.2 mM dNTPs, 0.5 µM forward primer, 0.5 µM reverse primer and 2.5 units of La-Taq DNA polymerase (Takara Bio., Inc., Beijing, China). The following PCR program was used for DNA amplification: 95°C for 3 min; followed by 35 cycles at 95°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec and a final extension at 72°C for 10 min.

Hela cells were provided by Professor Fan Yong at the Third Affiliated Hospital of Guangzhou Medical University (Guangzhou, China). In each well of a six-well plate, ~10⁻⁶ cells were added once the cells grew to 100% confluence. The Hela cells were maintained in Iscove's modified Dulbecco's medium, supplemented with 10% fetal bovine serum, 100 mg/ml penicillin and 100 mg/ml streptomycin, in a humidified atmosphere containing 5% CO₂ at 37°C. Transfection was performed using Lipofectamine 2000 (Invitrogen Life Technologies). The Hela cells were seeded into six-well tissue culture plates 24 h prior to transfection at ~60% confluence. The cells were transfected with eithr the pEGFP-CRYAA-WT, pEGFP-CRYAA-MT, pEGFP-GJA8-WT, pEGFP-GJA8-MT or GFP-control plasmid using Lipofectamine 2000, according to the manufacturer's instructions. At 48 h post-transfection, the cells were analyzed using fluorescence microscopy (Nikon Eclipse TS-10; Nikon Instruments, Amsterdam, Netherlands).

The slit-lamp examination revealed polymorphic cataracts in Family 1 (Fig. 2A–C). The proband in this family exhibited opacities involving the nucleus and peripheral cortex, and the slit lamp image of individual III:2 revealed a punctuate cataract in the central lens and opacities involving the peripheral cortex. The images of individual IV:4 revealed a nuclear cataract. The phenotypes of the three individuals all differed. The slit lamp image of the proband in Family 2 revealed nuclear cataracts (Fig. 2D and E). All affected individuals in this family exhibited bilateral cataracts, and the slit-lamp examination of the proband in this family revealed nuclear cataracts in the left and right eyes.

Through direct gene sequencing of the coding regions of the candidate genes, a novel missense mutation, c.416 T>C (p.L139P), was identified in the CRYAA gene in the affected individuals from Family 1. In the affected members from Family 2, the known mutation, c.139G>A (p.D47N), was detected in the GJA8 gene (Fig. 3A and B). These two mutations were not observed in any of the unaffected family members or in the 100 unrelated control individuals.

The CLC Main Workbench software revealed that leucine at amino acid position 139 of CRYAA and aspartic acid at amino acid position 47 of GJA8 were highly conserved among several species (Fig. 4A and B). The PolyPhen analysis demonstrated that either L139P of CRYAA or D47N of GJA8 produced a score of 1.000, which was predicted to be 'probably damaging'.

The subcellular localization of the wild-type and mutant proteins were assessed. The subcellular localization was determined using C-terminal green fluorescent protein (GFP) fusion constructs of CRYAA-WT, CRYAA-MT, GJA8-WT and GJA8-MT, followed by fluorescence microscopy. GFP, as a control, was located in the nucleus and cytoplasm. The cells transfected with CRYAA-WT demonstrated a homogenous distribution of expression in the cytoplasm alone, compared with CRYAA-MT. The expression of CRYAA-MT in these cells revealed significant protein aggregation (Fig. 5A). It was likely that the protein aggregation in the cytoplasm was due to protein conformational changes, which resulted from the L139P mutation. In addition, GJA8-WT was predominantly detected in the plasma membrane, whereas GJA8-MT was aberrantly expressed in the cytoplasm (Fig. 5B), indicating that the D47N mutation in GJA8 prevented its localization to the plasma membrane.