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Congenital or infantile cataract is a sight-threatening lens defect that affects about 1-6 cases per 10 000 live births and causes 10%-30% of all blindness in children.［1］ Approximately 25% of all cases are probably due to genetic defects. The majority of inherited non-syndromic cataracts show autosomal dominant (AD) inheritance. Autosomal recessive (AR) and X linked (XR) forms are seldom observed.
Developments in advanced molecular biological techni-ques have facilitated the identification of more than 30 independent cataract loci located on ten different human chromosomes, for which 20 mutations have been identified. Mutations in more than 10 genes have been identified as genetic causes for autosomal dominant cataracts (ADCs), including the crystalline genes (CRYAA, CRYAB, CRYBA, CRYBB, CRYGC, CRYGD), homeobox gene (PITX3), major intrinsic protein gene (MIP), beaded filament protein gene (BFSP2), connexin genes (CX50, CX46), and heat-shock factor 4 (HSF4) gene.［2-13］
The genetic defect associated with this condition remains unknown. We identified a four-generation family with coralliform cataracts transmitted in an autosomal dominant mode. To identify the disease locus in the family, we carried out whole-genome linkage analysis, eventually mapping the coralliform cataract locus to the γ-crystallin (CRYG) locus on chromosome 2q33-35. Mutation analysis of the four γ-crystallin genes (CRYGA, CRYGB, CRYGC, and CRYGD) revealed a missense mutation (P23T) in γD-crystallin (CRYGD).
METHODS
Clinical data and sample collection
Twenty-three selected family members received a careful examination, including a slit-lamp examination and photography of the lens, to record cataract type. After obtaining the written informed consent, 7 ml of blood was collected from affected and unaffected individuals. Genomic DNA was prepared using a QIAamp DNA blood mini kit (Qiagen, Germany).
Tissue processing
Tissue samples from the lens were extracted before intraocular lens implantation. The extracted lens obtained from two patients were immersed in 5% glutaraldehyde, then examined by transmission electron microscopy (Philips Tecnai 10, The Netherlands) and scanning electron microscopy (Cambridge Stereoscan 260, UK).
Gene scan and linkage analysis
We used 377 fluorescent dye-labeled dinucleotide repeat markers (PE Applied Biosystems, USA) for genotyping. DNA samples were amplified using GoldTaq DNA polymerase (AmpliTaq Gold, Applied Biosystems, USA) under “touchdown” PCR conditions: 94℃ for 12 minutes; followed by 10 cycles at 94℃ for 30 seconds, 62℃ for 1 minute (with a temperature decrease from 62℃ to 57℃ by 0.5℃ per cycle), and 72℃ for 50 seconds; followed by 30 cycles at 94℃ for 30 seconds, 57℃ for 40 seconds, and 72℃ for 50 seconds; with a final extension at 72℃ for 10 minutes. The PCR products were separated in 5% denaturing polyacrylamide gels using an Applied Biosystems 377 Sequencer. The Genescan 3.0 and Genotyper 2.1 software packages (Perkin-Elmer Corporation, USA) were used to generate genotypes, and Linkage 5.10 software was used for linkage analysis and to obtain two-point logarithm of odds (LOD) scores.
DNA sequencing and mutation screening
When evidence of linkage was obtained on chromosome 2q33-35, site of the γ-crystallin gene cluster or gene-specific PCR primers were designed to amplify the three exon sequences of CRYGA, CRYGB, CRYGC, and CRYGD ( Table ). Using Hotstar DNA Taq polymerase (Hotstar TaqTM, Qiagen, Germany), DNA templates were amplified on a GeneAmp PCR system 9700 (Perkin-Elmer Applied Biosystems, USA) with the following cycling parameters: 94℃ for 5 minutes; 35 cycles of 94℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 40 seconds; and extension at 72℃ for 10 minutes. Each 25 μl PCR reaction contained 25 ng DNA, 200 μmol/L of each dNTP, 10 pmol of each primer ( Table ), 2.5 μl Hotstar PCR 10×buffer, 1.5-3 mmol/L MgCl2, and 2 U Hotstar DNA polymerase. PCR products were subsequently purified with resin (Promega, Inc.), then analyzed on an Applied Biosystems 377 Sequencer. Sequences were assembled using Autoassemble software (PE version 1.4.0), and sequence variants were confirmed by sequencing in both directions.
Molecular model prediction
It has been observed that different γ-crystallins have highly conserved tertiary structures. Bovine CRYGD, for example, is 87% identical to human CRYGD. γD crystallin was modeled using the program Swiss-Model based on the experimentally derived coordinates of γD-crystallin (PDB: 1ELP), bovine γF-crystallin (PDB: 1A45), and rat γE-crystallin (PDB: 1A5D). The surface of wild-type γD-crystallin and its mutant were simulated using the program Swiss Model to observe alterations caused by the mutation.
RESULTS
Clinical findings
Examination of the pedigree suggested that the cataracts in this family were inherited as an autosomal dominant trait. The cataract phenotype was characterized by a large number of shiny, slice-like, punctiform opacities exten-ding radially from the lens nucleus, with an appearance resembling sea coral ( Fig. 1 ). Of the 23 examined family members, 11 were found to be affected by bilateral cataracts; the remaining 12 were unaffected ( Fig. 2 ).
Ultrastructure
Results of transmission electron microscopy
The ultrastructure of many fiber cells was observed as normal in extracted lens tissues. The cytoplasm was homogeneous with uniformly distributed fine granules, and the outline of the cell borders appeared either linear or undulating. Some distinct, giant, oval structures with irregular borders containing extensive globular cytoplasmic granules with different densities and with diameters ranging from 0.1 to 1.0 μm were present along with these normal cells ( Fig. 3A ). Some granules were membrane-coated, surrounded by single- or double-layered membrane systems, while others were not. In addition, there were single, giant granules and some irregular membrane-coated vacuoles.
Results of scanning electron micrography
Except in normal lens fiber cells, crystal deposits of different sizes (maximum side length, 160 μm) were observed in the lens tissue samples ( Fig. 3B ).
Linkage and mutation analysis
After a genome-wide examination, linkage analysis gave a significantly positive two-point LOD score at marker D2S325 (2q33-35) (maximum Z［Zmax］=3.5; maximum recombination frequency［θmax］=0.1), the site of the γ-crystallin gene cluster. Sequence analysis of the CRYGA, CRYGB, CRYGC, and CRYGD genes showed a C→A transversion at nucleotide position 70 in exon 2 of CRYGD. This mutation leads to an amino acid alteration from proline to threonine (P23T) ( Fig. 4 ). The sequence change co-segregated perfectly with the disease phenotype: it was observed in all affected family members, but was absent from unaffected family member and 200 control individuals. Residue 23 was conserved as either a proline or serine in all γ-crystallin isoforms.
Structure and stability
A molecular model of CRYGD was produced using the experimentally derived coordinates of γD-crystallin (PDB: 1ELP), bovine γF-crystallin (PDB: 1A45), and rat γE-crystallin (PDB: 1A5D) ( Figs. 5A-5D ). The sequences and structures of γ-crystallins are well conserved, allowing for simple modeling based on residue replacement. The total molecular energy of CRYGD and its mutant were computed to be -515.986 kJ/mol and -575.320 kJ/mol, respectively ( Figs. 5E and 5F ).
DISCUSSION
In this study, we suggest that a C→A transversion at nucleotide position 70 in the CRYGD gene is responsible for a rare autosomal dominant form of a congenital coralliform cataract affecting a Chinese family. This is the first identification of a molecular defect related to a coralliform cataract.
Electron microscopy analysis revealed many crystals of different sizes deposited in the extracted lens tissue samples from affected individuals and found extensive granules dispersed within oval structures. This pattern is different from the normal lens structure, consisting of homogeneous fiber-like or strand-like cells in a regular array, with no crystal formations. Lens transparency is dependent on a critical biophysical balance between proteins and water within a regular array of cells. The lens crystallins provide the main cellular cytoplasmic proteins, and any disruption in oligomerization or destabilization of the tertiary structure of the crystallins can result in cataract formation.［14］ Further study is needed to clarify if the mutant protein aggregates into the dense amorphous granules and subsequently forms crystals.
The lens crystallins constitute 80%-90% of soluble proteins in lens cells, and, in most species, α-, β-, and γ-crystallins constitute three main families. The human γ-crystallin gene family has seven highly related members: γA-, γB-, γC-, γD-, γE-, and γF- crystallins, and a gene fragment of γG resides as a gene cluster in the region 2q33-35. γ-S crystallin is localized on chromosome 3. Only 2 of the gene family members, CRYGC and CRYGD, express abundant proteins. Almost 90 % of the gamma-crystallins synthesized in the human lens are the products of these two genes.［15］ Several mutations in CRYGD have been described in association with human congenital cataracts. Stephan et al discovered a 1-base alteration that resulted in an Arg14Cys substitution in CRYGD, causing progressive juvenile-onset punctate cataracts in a three-generation family.［16］ Heon et al found that an Arg58His substitution in CRYGD co-segregated with the phenotype of aculeiform cataract.［6］ Kmoch et al revealed an Arg36Ser substitution in CRYGD in a 5-year-old boy with a unique congenital cataract caused by the deposition of macroscopically prismatic crystals.［7］ After studying seven families with autosomal dominant childhood cataracts in India, Santhiya et al identified two novel mutations in CRYGD.［17］ Interestingly, the Pro23Thr substitution observed in exon 2 of CRYGD in two patients affected with lamellar cataracts is identical with the one observed in this study.
Wild-type and mutant γD-crystallin model calculations show that replacing proline 23 with threonine may cause a decrease in the total molecular energy of the protein. It is possible that the mutant protein would be more prone to crystallization than the wild-type protein. Another effect of the P23T mutation is observable on the surface of γD-crystallin. The P23T replacement changes the orientation of a groove, which may affect the way the mutant γD interacts with other proteins, destroying the well-regulated fiber cell structure in the lens, ultimately resulting in a lens transparency deficiency.
Lens development begins with the formation of an embryonic lens nucleus during morphogenesis, around which lens fibers are deposited throughout life, initially forming the fetal nuclear region and, thereafter, the cortex. Animal models suggest that the genes associated with cataractogenesis are expressed in a time-ordered, sequential fashion.［18］ The location of opacification is time-specific. In terms of the location of lens opacities and their respective developmental histories, congenital nuclear cataracts and lamellar cataracts are distinct clinical entities. Opacities in the nucleus suggest abnormal gene expression during early lens development (usually in the embryonic period). The concentric deposition of secondary lens fibers that occurs during growth of the normal lens results in the formation of lamellae. Therefore, opacities confined to a specific lamella reflects a short period of developmental disturbance, usually during the fetal period.
The genetic heterogeneity of cataracts is evident, with more than one gene reported to cause identical phenotypes.［6,19］ On the other hand, Gill D et al found that an identical mutation can result in different phenotypes.［19］ It has been previously observed, and then supported by this study, that all cataract phenotypes caused by mutations in CRYGD are characterized by opacities in the lens nucleus. The only exception was reported by Santhiya et al, who investigated a lamellar cataract.［17］ It is interesting that an identical mutation appears to affect different phases of human lens development. It is not clear whether the P23T mutant can play different roles in lens development directly, or whether some modifier genes or factors modulate the cataract phenotype caused by the genetic defect. Insights gained from further studies of cataracts may help clarify the pathogenic mechanisms associated with γ-crystallin gene defects in humans.
Acknowledgment ： The authors thank Li Yue-bing of the Chinese National Human Genome Center at Shanghai, Cai Shang-rong of the Cancer Institute of Zhejiang University, and Shentu Xing-chao of the Eye Center of Zhejiang University.
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