Molecular Vision 2007; 13:2096-2104 <http://www.molvis.org/molvis/v13/a237/>
Received 9 August 2007 | Accepted 5 November 2007 | Published 7 November 2007
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A novel deletion variant of γD-crystallin responsible for congenital nuclear cataract

Li-Yun Zhang, Gary Hin-Fai Yam, Dorothy Shu-Ping Fan, Pancy Oi-Sin Tam, Dennis Shun-Chiu Lam, Chi-Pui Pang
 
 

Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China

Correspondence to: Chi-Pui Pang, Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong Eye Hospital, 147K Argyle Street, Kowloon, Hong Kong, China; Phone: +852 2762 3169; FAX: +852 2715 9490; email: cppang@cuhk.edu.hk


Abstract

Purpose: To investigate a novel deletion variant of γD-crystallin (CRYGD) identified in a Chinese family with nuclear congenital cataract.

Methods: A Chinese family with five affected members diagnosed with nuclear cataract and four unaffected members were recruited for the mutational screening of 15 known candidate genes for autosomal dominant congenital cataract. Two-point linkage analysis with single nucleotide polymorphism markers and microsatellite markers flanking these genes together with direct sequencing was applied to identify the disease-causing mutation. Recombinant NH2-terminal FLAG-tagged wildtype or mutant γD-crystallin was expressed in COS-7 cells. The expression pattern, protein solubility and intracellular distribution were analyzed by western blotting and confocal double immunofluorescence.

Results: Linkage analysis located the candidate region in the γC-crystallin and γD-crystallin gene cluster. Direct sequencing identified a c.494delG in CRYGD, which cosegregated with the disease in all affected members. Neither the unaffected family members nor the 103 unrelated controls carried this deletion mutation, which causes a frameshift and an early termination of polypeptide to become G165fs. A significantly reduced solubility was observed for this mutant. Unlike wildtype γD-crystallin, which existed in both the nucleus and cytoplasm, G165fs was colocalized with lamin A/C on the nuclear envelope.

Conclusions: We have identified a novel mutation, c.494delG, in CRYGD, which was associated with nuclear cataract. This is the first deletion mutation of CRYGD found to cause autosomal dominant congenital cataract. The mutant protein with loss of solubility and localization to the nuclear envelope is hypothesized to impair nuclear transfiguration and degradation in lens fiber cell differentiation, leading to opacity formation during lens development.


Introduction

Congenital cataract refers to a lens opacity presented at birth or developing shortly thereafter. It is responsible for about 10% of childhood blindness worldwide [1,2]. If left untreated, permanent visual loss usually occurs. Congenital cataract tends to be inherited in a Mendelian mode. It is reported that around 18% of affected children have a family history of cataract. Among them, 63% of pedigrees are simple autosomal dominant, 6% are autosomal recessive, 10% are X-linked, 16% are associated with other diseases, and the rest are undetermined causes [3].

Seventeen functionally diversified genes have been identified to cause isolated autosomal dominant congenital cataract (ADCC). They include nine genes encoding different subtypes of crystallin (namely CRYAA, CRYAB, CRYBB1, CRYBB2, CRYBA1, CRYBA4, CRYGC, CRYGD, and CRYGS), three genes (MIP, GJA8, GJA3) encoding membrane transport proteins, one gene (BFSP2) encoding a cytoskeletal protein, three genes (PITX3, MAF, and HSF4) encoding transcription factors, and one gene (CHMP4B) encoding chromatin modifying protein. FOXE3 and EYA1 have also been reported to cause congenital cataract but also associated with other anterior segment anomalies [4,5]. Functional analysis of mutant gene products helps to understand the roles of these proteins during lens development and disclose the underlying mechanism of cataractogenesis.

In this study, we have identified a novel single base deletion, namely c.494delG, in exon 3 of the γD-crystallin gene (CRYGD) in a Chinese family diagnosed with autosomal dominant nuclear cataract. This change led to a truncated protein G165fs. Cell expression studies showed that the G165fs protein was detergent insoluble and localized to the nuclear envelope. To our knowledge, this is the first deletion mutation of CRYGD found to be disease-causing for ADCC.


Methods

Clinical examination and DNA specimens

A three-generation ADCC family was recruited in the University Eye Center, the Chinese University of Hong Kong. Nine family members, five affected and four unaffected, participated in this study (Figure 1A). Informed consent was obtained from all participants. Unrelated controls of 103 subjects were also recruited. Medical and family histories were documented. Complete ophthalmic examination was given. Peripheral blood samples and buccal swab samples were collected for DNA analysis. The research followed the tenets of the Declaration of Helsinki and approved by the Ethics Committee of the Chinese University of Hong Kong.

Genotyping and linkage analysis

Genomic DNA was extracted using the QIAamp DNA kit (Qiagen, Valencia, CA). Linkage analysis with both single nucleotide polymorphism (SNP) markers and microsatellite markers was performed to screen 15 known ADCC causative genes. First, SNP markers within these genes were selected with ABI SNPbrowser version 3.5 (Applied Biosystems, Foster City, CA) and genotyped by the TaqMan SNP genotyping assay (Applied Biosystems). The results were analyzed on an ABIPRISM 7000 sequence detection system (Applied Biosystems) and used to screen for the possible causative genes through cosegregation between markers and disease and two-point linkage analysis. For genes without informative SNP markers or that cannot be excluded by SNP analysis, flanking microsatellite markers were chosen from the Marshfield genetic map. All SNPs and microsatellite markers are listed in Table 1. Genescan was conducted on an ABIPRISM® 377 genetic analyzer (Applied Biosystems). ABI GenoPedigree 1.0 and GeneBase 2.0.1 software were used to draw pedigree and to export genotyping data. Two-point LOD scores were calculated by MLINK of FASTLINK v4.1P software. A gene frequency of 0.0001 and a penetrance of 100% were assumed for ADCC.

Gene sequencing

By linkage analysis, we excluded all known candidate genes except CRYGC and CRYGD for which all exons and splicing regions were sequenced with specific primers (Table 2) and the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems) on the ABIPRISMTM 377 DNA sequencer. Sequence results were compared to reference sequences from the NCBI (GeneBank NM_006891).

γD-crystallin expression constructs, epitope tagging, and site-directed mutagenesis

To create a eukaryotic expression vector encoding human wildtype CRYGD cDNA, a 545 base pair EcoR1/Xho1 fragment encompassing the full-length 525 base pair open reading frame was subcloned directionally into the EcoR1/Xho1 sites of the plasmid p3XFLAG-myc-CMVTM-25 (Sigma, St Louis, MO). The wildtype γD-crystallin expression construct was named as pFLAG/myc-CRYGDWT. Correct sequence was verified by direct sequencing with primers: sense 5'-ATG AGC AGC CCA ACT ACT CG-3'; antisense 5'-CCT GAA GAC AGG AGC AGT CC-3'. The mutation, c.494delG, was created by a site-directed mutagenesis kit (Stratagene, Lo Jolla, CA) using oligonucleotides: sense 5'-GGG CCA CGA ATG CCA GAG TGG *CT CTC TGA GGA GAG TCA TAG; antisense 5'-CTA TGA CTC TCC TCA GAG AG* CCA CTC TGG CAT TCG TGG CCC (the asterisk indicates the position of the base deletion). Due to the creation of a STOP codon before the myc sequence, the mutant construct was named as pFLAG-CRYGDG165fs. Specific base changes were confirmed by direct sequencing.

Cell culture and transfection

COS-7 cells (ATCC, Manassas, VA) were maintained in Eagle's Minimum Essential Medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% antibiotics and incubated at 37 °C in a humidified chamber with 5% CO2 balanced with air. Before transfection, the absence of endogenous CRYGD mRNA and protein in COS-7 cells was verified by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting (data not shown). To prepare for DNA transfection, COS-7 cells were plated at a density of 5x105 cells per 60 mm (diameter) culture dish (Nalge Nunc, Rochester, NY). Transient transfection was done with pFLAG/myc-CRYGDWT and pFLAG-CRYGDG165fs DNA with transfection reagent FuGene HD (Roche, Basel, Switzerland; using conditions suggested by the manufacturer). A ratio of 1 μg DNA mixed with 3 μl FuGene HD reagent was used to incubate in Opti-MEM® I supplemented with GlutaMAXTM-I (Invitrogen) for 30 min followed by transfection of COS-7 cells for 24-48 h.

Total protein analysis

Cells were lysed at a concentration of 2.5x106 cells/ml in a radioimmunoprecipitation (RIPA) buffer containing 50 mM Tris-HCl (Sigma, St Louis, MO), 150 mM sodium chloride, 1% Nonidet P-40 (Sigma), 0.25% sodium deoxycholate (Sigma), protease inhibitor cocktail (Roche) and 1 mM phenylmethyl sulfonylfluoride (PMSF; Sigma) for 30 min and centrifuged. The supernatant was collected and denatured in sample buffer with a final concentration of 2% sodium dodecylsulfate (SDS, BioRad, Hercules, CA) and 50 mM DL-dithiothreitol (DTT; Sigma). The cell pellet was washed once with ice-cold PBS and denatured and sonicated in SDS sample buffer containing 9 M urea (BioRad). Both protein samples (equivalent to 7.5x104 cells) were mixed together and analyzed by 15% SDS-polyarcylamide gel electrophoresis (PAGE) and western blotting using antibodies against FLAG (Sigma), GAPDH (Ambion, Austin, TX), and β-actin (Chemicon, Temecula, CA), and appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Bioscience, Piscataway, NJ). The signals were detected by enhanced chemiluminescence (Amersham Bioscience, Buckinghamshire, UK).

Monitoring detergent solubility of γD-crystallin

Cells expressing either wildtype or mutant γD-crystallin were washed twice with ice-cold PBS and lysed for 2 min on ice at 2.5x106 cells/ml lysis buffer, which contained 100 mM Tris-HCl (pH 7.4), 3 mM EGTA (Sigma), 5 mM MgCl2, 0.5% Triton X-100 (Tx; Sigma), protease inhibitor cocktail, and 1 mM PMSF [6]. After centrifugation, the supernatant containing Tx-soluble proteins was denatured in SDS buffer containing 50 mM DTT. The pellet containing Tx-insoluble proteins was washed twice with ice-cold PBS, sonicated, and denatured in SDS buffer containing 9 M urea. Tx-soluble and Tx-insoluble proteins from samples equivalent to 7.5x104 cells were analyzed by 15% SDS-PAGE and western blotting using antibodies against FLAG, γD-crystallin (Abnova, Heidelberg, Germany), GAPDH, or α-tubulin (Sigma).

Immunofluorescence

Cells grown on glass coverslips were fixed with freshly prepared 2% paraformaldehyde (Sigma), permeabilized with 0.05% Tx, and processed for immunofluorescence as described previously [6]. γD-crystallin expression in cells was detected by either mouse monoclonal antibody against FLAG or mouse monoclonal antibody against γD-crystallin. For double immunofluorescence, FLAG immunoreactivity was detected with a mouse monoclonal anti-FLAG antibody followed by Rhodamine Red-X goat anti-mouse IgG (Invitrogen). The open antigen binding sites on goat anti-mouse IgG were first blocked with normal mouse serum and then incubated with the Fab fragment of goat anti-mouse IgG (Jackson Immunoresearch Lab, West Grove, PA). Nuclear envelopes were labeled using a mouse monoclonal antibody against lamin A/C (Sigma) followed by an Alexa 488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen). After nuclear staining by DAPI, the samples were examined by fluorescence microscopy (DMRB, Leica, Germany) equipped with Spot RT color system (Diagnostic Instruments, Sterling Heights, MI) and confocal scanning microscopy (LSM 510 META, Carl Zeiss, Gottingen, Germany).


Results

Clinical features of the nuclear cataract

The cataract exhibited an autosomal dominant inheritance pattern in this family (Figure 1A,B). Among seven affected individuals, five attended our study. Four patients were recorded to have bilateral congenital cataract and had undergone lens surgery. The remaining patient without cataract removal showed an opacity in the central nucleus region of both lenses. The Y-sutures were also involved with prominent opaque (Figure 1B). The diagnosis of bilateral nuclear cataract was made by a clinical ophthalmologist. The patient's uncorrected visual acuities are 20/70 in the right eye and 20/50 in the left eye. During eight years of follow up, he did not experience any obvious vision loss. None of the five affected family members have other ocular or systemic abnormalities.

Identification of a novel deletion mutation in CRYGD

Linkage analysis with SNP and microsatellite markers excluded most of the candidate genes except for CRYGC and CRYGD. Direct sequencing identified a heterozygous single G deletion at the coding nucleotide position of 494 in exon 3 of CRYGD (c.494delG; Figure 2A) when compared to wildtype sequence (GeneBank NM_006891; Figure 2B). This deletion led to a frameshift starting at the 165th amino acid position with the substitution of Gly with Ala followed by a Lys residue before a termination codon at the 167th position (G165fs). A truncated protein with eight amino acids shorter as compared to wildtype was encoded. This sequence alteration was observed in all patients of this family but not detected in any unaffected family member and 103 unrelated control subjects.

Detergent insolubility of G165fs γD-crystallin

Recombinant FLAG/myc-tagged wildtype and FLAG-tagged G165fs γD-crystallin were transiently expressed in COS-7 cells and were detected by anti-FLAG antibody. Western blot analysis in samples with same number of cells showed similar expression levels of wildtype and mutant γD-crystallin protein (Figure 3). The molecular size of recombinant wildtype protein is about 28 kDa whereas the recombinant mutant was about 24 kDa. This verified the truncation. The absence of endogenous γD-crystallin expression in COS-7 cells as well as the expression of both recombinant γD-crystallin proteins was confirmed by western blotting with anti-γD-crystallin antibody (data not shown). When extracted by 0.5% Tx, wildtype and G165fs γD-crystallin exhibited a different solubility (Figure 4). Band densitometry and normalization with housekeeping proteins (GAPDH for Tx-soluble fractions and α-tubulin for Tx-insoluble fractions) revealed a significant reduction of Tx-solubility for G165fs γD-crystallin. About 97% wildtype protein remained in the soluble compartment compared to 14% of mutant (Figure 4). Most of the mutant protein was detected in the Tx-insoluble fraction, which was dissolved by 9 M urea (Figure 4). The experiments were conducted in triplicate.

Mislocalization of G165fs γD-crystallin to the nuclear envelope

When expressed in COS-7 cells, FLAG-tagged wildtype γD-crystallin was localized both in nuclear and cytoplasmic regions (Figure 5A,B) whereas FLAG-tagged G165fs was redistributed as a ring-shaped structure on the nuclear periphery (Figure 5C,D). Minimal staining in the cytoplasm was also observed. By confocal double immunofluorescence, mutant γD-crystallin was found colocalized with lamin A/C, indicating its association with the nuclear envelope (Figure 5E-G). Weak staining of mutant protein was observed in the inner nuclear region. The results obtained with anti-γD-crystallin antibody, which recognizes the very COOH-tail region of γD-crystallin also confirmed that the wildtype γD-crystallin was distributed in both nucleus and cytoplasm while the mutant γD-crystallin showed negative staining due to the altered COOH-tail of the protein (Figure 5A,C).


Discussion

In the present study, we have identified a novel deletion mutation (c.494delG) in CRYGD, which caused a truncation of the γD-crystallin polypeptide (G165fs). Our expression studies demonstrated that the mutant was highly insoluble when extracted with Tx detergent and relocated to nuclear envelope. We hypothesize that these changes in the mutant γD-crystallin affect nuclear degradation during lens fiber differentiation thus interfering with lens transparency.

Lens transparency is maintained via short-range ordering of native water-soluble cytoplasmic proteins in fiber cells. Crystallins, consisting of α-, β-, and γ-crystallin families, account for more than 90% of the water soluble proteins in the human lens. Functional changes and alteration of crystallin molecular properties could cause the breakdown of the lens microstructure and result in changes in the refractive index and increased light scattering. γD-crystallin is one of these important crystallins and functions as a structural protein in lens. Various in vitro studies have suggested that γD-crystallin contributes to lens transparency, and impaired γD-crystallin expression can be responsible for cataract formation.

Together with our newly identified mutation, seven missense variations, including R14C, P23T, R36S, R58H, E107A, W156X, and G165fs, of CRYGD have been reported to cause ADCC (Table 3) [7-18]. G165fs is the only deletion mutation of CRYGD so far identified whereas others are all single base substitutions. G165fs resulted in a frameshift and a premature termination codon. Normally in eukaryotes, mRNAs containing the premature termination condon can be detected and destructed by a mRNA surveillance mechanism called nonsense-mediated decay (NMD). However, it usually only happens on the condition that the nonsense codon locates more than approximately 50 nucleotides upstream of the last exon-exon junction [19,20]. G165fs is expected to escape from NMD due to its location in the last exon of CRYGD, leading to translation of a truncated protein. This speculation was verified by total protein expression analysis in vitro in which the expression of mutant protein is similar to that of the wildtype. Meanwhile, wildtype γD-crystallin is known to exist as monomeric protein but mutants such as R14C and P23T are likely to form aggregates with larger molecular sizes in the in vitro situation. Whether our novel mutation has the similar effect will be further investigated.

The vast phenotypic variations of lens opacities resulting from mutations in CRYGD could be due to differences in their biologic effects. In this study, the G165fs mutant exhibited dramatically reduced solubility in Triton X-100 extraction. This could be resulted from the loss of amino acids at the COOH-terminus that contributes to the conformational stability of γD-crystallin. In the mature human lens, γD-crystallin exists as a monomeric protein with a highly symmetric structure containing four Greek key motifs organized into two highly homologous β-sheet domains. Protein analyses have indicated that the NH2-terminal domain (NH2-td) and COOH-terminal domain (COOH-td) are covalently connected by a six-residue linker and interact non-covalently through the side chains of 10 amino acids across the domain interface. With these two conserved regions and a central hydrophobic domain interface, γD-crystallin exhibits high intrinsic stability [21,22]. A kinetic study has revealed that COOH-td is important to the stability of the whole γD-crystallin protein. Local disturbance in COOH-td will thus shift the protein toward a partially folded status or even unfolding [21]. During the refolding process of γD-crystallin in vitro, COOH-td was found to fold first [23] and residues located in the hydrophobic domain interface of the refolded COOH-td acted as a nucleating center for the subsequent folding of the NH2-td [21]. The G165fs truncation mutant removed the last β-strand of the fourth Greek key motifs and deleted Val170, a crucial interdomain residue expected to influence the intrinsic stability [21]. We suggest that G165fs not only affects the COOH-td folding but also alters the overall protein conformation and eventually leads to protein precipitation. Another mutation in γD-crystallin, W156X, may operate with a similar mechanism, and this might explain its association with a similar nuclear cataract. How the insoluble G165fs mutant associates with cataractogenesis remains to be investigated.

Instead of affecting protein conformation and stability, other reported mutations in γD-crystallin only cause local alterations such as changes of regional hydrophobicity or polarity, which could result in abnormal oligomerization or aggregation. For example, in the R14C mutant, the newly formed reactive cysteine at the protein surface led to the formation of disulfide cross-linked aggregates of the mutant protein [24]. The P23T mutant appears to aggregate due to an attractive peptide stretch at the mutation site [25,26]. Spontaneous crystallization has been shown in mutant proteins of R36S and R58H in which a highly polar and charged residue was replaced by less polar residues [27,28]. These mutants unlike G165fs did not exhibit any global changes of protein structure.

Unlike wildtype γD-crystallin, which exists in both the nucleus and cytoplasm [29], the G165fs mutant was associated with the nuclear envelope as shown by its codistribution with lamin A/C by confocal double immunofluorescence. This mislocalization together with the reduced solubility could be linked to nuclear changes during lens development, in particular lens fiber maturation. During lens development, lens fiber cells arisen from lens epithelial cells will elongate the cell body and lose the cell nucleus and organelles to form the maturated lens fibers. Principally, two mechanisms have been proposed for nuclear loss. Modak et al. [30] reported a programmed cell death-associated nuclear change with the formation of pyknotic nuclei. On the other hand. Kuwabara et al. [31] showed that the nuclei faded away during lens fiber cell differentiation with no nucleus degeneration being observed. Although the mechanism of denucleation is still unclear, there is no doubt that the nucleus would eventually disappear in this differentiation process. In this study, G165fs mutant γD-crystallin is highly localized to the nuclear envelope. We speculate the G165fs protein could influence the nuclear stability or vulnerability during nuclear degradation. As a matter of fact, γ-crystallins but not α- or β-crystallins characterize lens fiber differentiation in a lens regeneration model of adult newt [32]. Transgenic animal model expressing various γD-crystallin mutants should be established to further elucidate the role of γD-crystallin in nuclear degradation and lens fiber differentiation.

In conclusion, we identified a novel deletion mutation, c.494delG, in CRYGD in a Chinese family associated with the nuclear type of congenital cataract. The truncated mutant protein had greatly reduced solubility and was relocalized to the nuclear envelope. Hence, this new genotype, leading to altered protein features, is the cause of lens opacification.


Acknowledgements

The authors thank all participants of this cataract family for their cooperation in the clinical data collection and blood samples donation, and the authors also thank Mr. Ricky Y.K. Lai for his endeavors in patient recruitment.


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