Patients
The two-generation family includes eleven family members, nine of whom (Fig. 1a, I-1-4, II-1-5) participated in the study. Four family members were diagnosed with CCs with esotropia and nystagmus and underwent thorough ophthalmological examinations, including slit-lamp examination, fundus photochromy, optical coherence tomography (OCT) and visual evoked potential (VEP). Genomic DNA was extracted from blood samples from all nine participants. In Fig. 1a, genotype information with an asterisk indicates the individual whose sample was used for WES analysis.
Eighteen age-related cataract (ARC) patients (8 male and 10 female, aged 64.1 ± 4.5 years) agreed to donate the anterior capsule pieces from their cataract surgery to the study. The normal control capsular samples from 18 donors (12 male and 6 female, aged 60.2 ± 5.2 years) were provided by the eye bank of the Eye and ENT Hospital of Fudan University. The capsular piece of the normal donor lens was collected from the anterior part, the same site at which the capsule samples collected in the cataract surgery, as previously described [11].
Sequencing analysis
For WES, libraries for sequencing on a HiSeq2000 instrument (Illumina Inc., USA) were prepared from genomic DNA and exome sequences enriched with SureSelect Human All Exon 50 M (Agilent Technologies, USA) according to the manufacturers’ instructions. The bioinformatic analysis of the raw data (paired-end reads) has been described previously [12]. In addition, the variants with the allele frequency > 1% in gnomAD database were excluded (https://gnomad.broadinstitute.org/). After a series of filtering steps, 40 sites in 38 genes (Additional file 1: Table S1) were selected for further analysis. Both strands of the selected regions were sequenced by Sanger sequencing following polymerase chain reaction (PCR) amplification on an ABI 3730 Genetic Analyzer (Applied Biosystems Inc., USA). The results of Sanger sequencing were analyzed and presented using Chromas software (https://technelysium.com.au/wp/chromas/).
Cell culture and transfection
The human lens epithelial cell (LEC) line SRA01/04 (abbreviated as SRA in this study) was authenticated using short tandem repeat (STR) profiling, and the data of the STR analysis are provided in Additional file 2 (Shanghai Biowing Applied Biotechnology, China). SRA cells and embryonic kidney cell line 293 T cells were cultured in DMEM (#11995065, Gibco, USA) supplemented with 10% fetal bovine serum (#10099141, Gibco) under humidified air containing 5% CO2 at 37 °C. To overexpress the CRYBA1 constructs in these two cell lines, cDNA sequences of the human CRYBA1 (RefSeq NM_005208) wild-type and G91del mutant with the FLAG sequence “GATTACAAGGACGACGATGACAAG” at the N-terminus were cloned into the eukaryotic cell expression vector pcDNA3.1 (Invitrogen, USA). Cells were transfected using the transfection reagent (#C10511, RiboBio, China) according to the manufacturer’s instructions. In each well in a 6-well culture plate, 2.5 μg of plasmids were included in the transfection mixture.
Quantitative PCR (qPCR)
Capsular pieces from three subjects were pooled into one sample. Total RNA from the patients’ lens epithelium or cultured cells was extracted using an RNA extraction kit (#CW0581, CoWin Biosciences, China) and reverse transcribed into cDNA using a cDNA synthesis kit (#CW2582, CoWin Biosciences) according to the manufacturer’s protocols. mRNA levels of the selected genes were quantified by SYBR Green-based quantitative PCR (qPCR) kit (#CW2601, CoWin Biosciences) on an ABI 7500 analyzer (Thermo Fisher Scientific). The relative mRNA expression between the target genes and the internal β-actin control was calculated using the comparative cycle threshold (CT) method (2-△CT). The relative mRNA level between samples was calculated using 2-△△CT method.
Western blot
Capsular pieces from three subjects were pooled into one sample. Western blot analysis was performed according to standard methods as previously described [13]. The primary antibodies included CRYBA1 (1:1000, #NBP1–33010, Novus Biologicals, USA), FLAG (1:1000, #8146, CST, USA) and CRYBA4 (1:1000, NBP1–32741, Novus). β-Actin (#A3854, Sigma-Aldrich, USA) or GAPDH (#5174, CST) served as the internal control. Secondary antibodies were goat anti-mouse or goat anti-rabbit IgG (H + L) poly-horseradish peroxidase secondary antibody (Jackson Immunoresearch, USA). The dilution of the secondary antibodies was 1:10000.
Immunofluorescence staining
Cells used for immunofluorescence staining were seeded after a glass cover (#48380–080, VWR Scientific, USA) was placed into the bottom of the culture dish. Forty-eight hours after cell transfection, the glass covers were washed with phosphate buffer solution (PBS) and fixed with 4% paraformaldehyde for 15 min. Cells attached to the glass covers were then permeabilized with PBS containing 0.3% Triton X-100 for 15 min, followed by incubation in PBS containing 5% goat serum and 0.1% bovine serum albumin for 1 h to block nonspecific protein binding. Then, the cells were incubated with the FLAG primary antibody (1:100, #8146, CST), CRYBA1 (1:100, #NBP1–33010, Novus Biologicals) at 4 °C overnight. The next day, after washing with PBS, the cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:1000, #R37116, Thermo Fisher, USA) for 1 h at room temperature. Hoechst 33258 (1:2000, #H3569, Invitrogen, USA) was used to visualize the nucleus. Finally, the glass cover was removed from the bottom of the culture dish and placed onto a glass slide with the cells facing downward. The cells were then observed using the Cell Observer microscope (Zeiss, Germany), and the images were captured using ZEN 2012 software (blue edition).