|Molecular Vision 2004;
Received 17 July 2003 | Accepted 24 March 2004 | Published 29 March 2004
Special fasciculiform cataract caused by a mutation in the γD-crystallin gene
(The first two authors contributed equally to this publication)
1Eye Center, Affiliated Second Hospital, College of Medicine and 2Institute of Ophthalmology, Zhejiang University, Hangzhou, China; 3Cancer Institute of Zhejiang University, Hangzhou, China; 4Huada Gene Institute, Hangzhou, China; 5Department of Cell Biology, The Scripps Research Institute, La Jolla, CA
Correspondence to: Ke Yao, Eye Center, Affiliated Second Hospital College of Medicine, Zhejiang University & Institute of Ophthalmology, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China; Phone: 0086-571-87783897; FAX: 0086-571-87783897; email: email@example.com
Purpose: To identify ultrastructure changes and genetic defects associated with a special fasciculiform autosomal dominant congenital cataract (ADCC) affecting a large Chinese family.
Methods: Members of the Chinese family affected by this special fasciculiform ADCC were studied. Clinical examinations, light and transmission electron microscopy of the removed lens tissue, linkage analyses using polymorphisms of microsatellite markers, and mutational analyses of candidate genes by direct sequencing were performed.
Results: The thirteen affected individuals in the family showed no variability between one another. The lens fiber cells showed focal degeneration, dense globular intracellular deposits, and an enlarged intercellular space. This ADCC was associated with a locus on chromosome 2q33-35 (maximum lod score [Zmax]=3.34; θ=0.05). Mutational analyses of the candidate genes (γ-crystallin; CRYG), identified a C->A heterozygous transversion at nucleotide position 70 in the γD-crystallin gene (CRYGD) exon2, which co-segregated with the presence of ADCCs and was not observed in 100 unrelated controls.
Conclusions: This study identified ultrastructure changes and genetic defects associated with a special fasciculiform ADCC affecting a large Chinese family. It appeared to be caused by a missense mutation in the CRYGD gene, further supporting the notion that alterations to CRYG play an important factor in human cataract formation.
Congenital cataracts may be caused by intrauterine embryopathies, single gene defects, or chromosomal rearrangements. They are a common abnormality of the eye and account for approximately one third of all pediatric visual loss . Because these congenital cataracts result in cloudy imaging onto the retina, the abnormal development of visual cortical synaptic connections results in amblyopia, which causes affected children irreversible visual loss without suitable intervention. About 25% of all congenital cataracts are inherited [2,3], with the most common being nonsyndromal autosomal dominant congenital cataracts (ADCC).
In general, congenital cataracts are bilateral and characterized by the location and structure of the opacities, which include shape, size, color, and the presence or absence of refractivity. However, phenotypic variability has been documented among and within families [4-6], whereby identical mutations can result in different phenotypes [7,8]. These identical cataracts have also been mapped to different loci [9,10]. Although congenital cataracts are phenotypically and genetially heterogeneous, more than twenty loci have been shown to be linked with congenital cataracts and the disease associated mutations have been identified in 19 genes, including αA-crystallin , αB-crystallin , βA1-crystallin , βB1-crystallin , βB2-crystallin , γC-crystallin , γD-crystallin , γE-crystallin , beaded filament structural protein 2 , heat shock transcription factor 4 , gap junction protein alpha-3 , gap junction protein alpha-8 , paired-like homeodomain transcription factor-3 , ferritin , galactokinase 1 , glucosaminyl (N-acetyl) transferase 2 , major intrinsic protein of lens fiber (MIP) , lens intrinsic membrane protein 2 (LIM2) , and paired box gene 6 .
In this report we studied a large, four-generation Chinese family containing members affected by special fasciculiform ADCC. Using linkage analysis, we mapped an associated locus to 2q33-35, which contains the γ-crystallin genes (CRYG). The CRYG gene family has six members including γA-crystallin (CRYGA), γB-crystallin (CRYGB), γC-crystallin (CRYGC), γD-crystallin (CRYGD), γE-crystallin (CRYGE), and γF-crystallin (CRYGF). We identified a mutation within exon 2 of the γD-crystallin gene (CRYGD), which was present in all affected family members.
The family was ascertained through the Eye Center of Affiliated Second Hospital College of Medicine, Zhejiang University, Hangzhou, China. Informed consent in accordance with the Declaration of Helsinki and the Zhejiang Institutional Review Board approval was obtained from all participants. Informed consent was also obtained from the subjects after explanation of the nature and possible consequences of the study. Thirty-seven individuals participated in the study, 13 affected individuals and 24 unaffected individuals of whom 12 were spouses (Figure 1). Affected status was determined by a history of cataract extraction or ophthalmologic examination, which included slit lamp examination with dilated pupils, visual acuity testing, intraocular pressure measurement, and fundus examination. The cataract phenotype of the patients who previously had cataract extraction was obtained from history records.
Pathology and ultrastructure
Anterior lens capsules with attached lens epithelial cells were obtained by capslotomy and the lens material was aspirated by double cannula during cataract surgery. Part of the extracted lens material was fixed in formalin overnight and embedded in paraffin. Paraffin embedded sections (5 μm) were then subjected to HE staining. The remaining lens material was fixed in 2.5% glutaraldehyde for 30 min and postfixed in 1% osmic acid for 1 h at 4 °C. After dehydration with a graded series of ethanol concentrations, specimens were embedded in epon resin. Ultrathin sections (60-90 nm thickness) were stained with lead citrate and uranyl acetate and then observed with transmission electron microscopy (JEM-1200, Tokyo, Japan) at 60 Kv.
Genotyping and linkage analysis
Blood specimens (5 ml) were collected in EDTA and leukocyte genomic DNA was extracted. Microsatellite markers and their distances were obtained from the Cooperative Human Linkage Center (CHLC) and Genethon . Microsatellites were amplified by polymerase chain reaction (PCR) with fluorescent labeled primers (Shenyou Bi-technology LTD, Shanghai, China) following standard methods. PCR products were denatured for 3 min at 90 °C and immediately placed on ice, then resolved on a 5% sequencing gel (FMC BioProducts, Copenhagen, Denmark) using a DNA sequencer (ABI PrismTM 377; Perkin-Elmer Applied Biosystems, Foster city, CA). Data were collected and analyzed using the followinmg computer programs; ABI PrismTM 377-18 Collection (Genescan) and Genescan analysis® 2.1 (Perkin-Elmer Applied Biosystems). For autosomal dominant inheritance, a disease-gene frequency of 0.0001 and full penetrance was assumed. Two point lod scores between the cataract locus and markers were calculated by the MLINK program of LINKAGE package (version 5.1) for a full range of θ values.
Mutational analysis of CRYG
We screened all exons of the candidate gene, CRYG, by direct sequencing for mutations. The genomic sequence of CRYG was obtained from the National Center for Biotechnology Information (CRYGA, OMIM 123660; CRYGB, OMIM 123670; CRYGC, OMIM 123680, CRYGD, OMIM 123690). Nine gene specific PCR primer pairs (Sangon Biological Engineering Technology and Service Co., LTD, Shanghai, China) were designed to amplify the 12 exons and flanking intron sequences of CRYGA, CRYGB, CRYGC, and CRYGD (Table 1). PCR products were subsequently purified using QIAquick Gel Extraction kit and were sequenced using Big Dye Primer Cycle Sequencing kit (Perkin-Elmer Applied Biosystems) and a DNA sequencer (ABI PrismTM 377, Perkin-Elmer Applied Biosystems). Data were collected and analyzed by the following computer programs; the ABI PrismTM 377-18 Collection (sequence run) and Sequencing analysis 3.3 (Perkin-Elmer Applied Biosystems). For screening, two unaffected and two affected individuals were compared. After detecting a probable mutation, all members of the family and 100 unrelated controls, who had undergone full ophthalmic examinations to be sure of free of eye diseases, were screened for the proposed disease associated mutation.
All affected family members showed a special fasciculiform cataract characterized by corroliform opacification, which included embryonic and fetal nuclei, and a fiberglasslike opacity projecting in different directions from the central zone with a radially oriented metal-like refractivity (Figure 2A,B). This special fasciculiform cataract was a congenital cataract and there was no progressive development of lens opacities since birth. The degree of opacification of the cataract showed no variability among the affected family members (Figure 2C). Also, there was no family history of other ocular or systemic abnormalities aside from age related disorders. Based on the presence of affected individuals in each of the four generations, equal numbers of affected males and females, and male to male transmission, autosomal dominant inheritance of the cataract was demonstrated.
There were no detectable abnormal alterations to the lens epithelial cells by HE staining. However, the lens fiber cells showed manifestations of degeneration, including focal mucus-like degeneration, reticular change, crystal precipitation, and irregular refracility (Figure 3).
There were no characteristic or consistent alterations to the lens epithelial cells. However, lens fiber cells displayed abnormal intercellular and intracellular alterations. Normally, there are tight contacts between the lens fiber cells and a uniformly fine granular appearance within the cytoplasm. However, in affected samples there were foci of dense, globular intracellular deposits 0.05 to 0.5 μM in diameter. They were either surrounded by a single membrane or deposited freely in the cytoplasm (Figure 4A). The cell borders were highly irregular and there were enlarged spaces (0.1 to 0.5 μM in width) between the cells (Figure 4B).
Linkage and mutation analysis
Thirty members of the affected family, including 13 affected individuals, 9 unaffected individuals, and 8 spouses were genotyped and studied by microsatallite marker linkage analysis. For chromosome 2q33-35, around the γ-crystallin locus, seven locus genotypes and inferred haplotypes were showed (Figure 5) and two point lod scores were summarized (Table 2). Significant linkage was found with markers D2S2208 ([Zmax]=3.34; θ=0.05).
Mutation analysis of the CRYG genes in the affected family detected two heterozygous transversions. Direct sequencing of the amplified exons of CRYGA and CRYGC failed to document any sequence change. CRYGB showed a A->C heterozygous transversion at nucleotide position 331 (I110L) in exon 3, which was an SNP (dbSNP:796287) loci and not disease specific. This polymorphic change did not co-segregate with cataract in the family. The change was observed in some (not all) affected family members and some unaffected family members. Sequencing of CRYGD revealed a C->A heterozygous transversion at nucleotide position 70 in exon 2 (Figure 6), which was predicted to substitute the conserved amino acid proline for a threonine (P23T). This sequence change was observed in all affected family members and was not observed in all unaffected family members, spouses in the family, and 100 unrelated control individuals.
Cataracts are a leading cause of blindness worldwide  and the number of cases are increasing due to an increased aging population. Unfortunately, the mechanism of lens opacification remains unknown. The study of congenital cataracts, which are usually the result of autosomal dominant inheritance, is useful in attempting to ascertain cataract related genetic defects. Genes that have been previously reported to be associated with ADCC are crystallins [11-18], connexins [21,22], cytoskeletal structure protein , and homeobox gene .
Among the reported genes, the CRYG genes were of great interest in this study. Although congenital cataracts are phenotypially and genetically heterogeneous, the position and degree of the opacity provide clues to the time and cause of the cataract. The balance of proteins and water in a regular array of cells is critical in maintaining the transparency of the lens. Modifications that reduce the effective solubility of lens proteins can lead to progressive protein aggregation and the formation of light scattering centers in the lens [32,33]. Crystallins constitute more than 90% of the soluble proteins in the human lens. They are made up of α-crystallin (40%), β-crystallin (35%), and γ-crystallin (25%) .The congenital cataracts in this study were characterized by opacification of the lens nucleus, which was made up of enucleated lens fiber cells. The opacification was denser in the center than in the periphery. Also, there were many structural alterations to the lens fiber cells at the microscopic level suggesting that a special protein expressed only in the lens fiber cells was the most probable cause of the pathologic events leading to the formation of high molecular weight aggregates and deposition.
The CRYG genes encode γ-crystallin, a major component of the lens structural protein. In addition, it is only expressed in differentiated fiber cells of the human lens . Also, γ-crystallin is associated with high protein concentrations and high refractive indices in the human lens. Thus, it plays a key role in maintaining the development and transparency of the lens [36-38]. The improper folding of γ-crystallin would cause protein aggregation and deposition leading to cataract formation. The CRYG genes are associated with cataract formation in human. In fact, Coppock-like cataracts  and cataracts with a dust-like opacity , an aculeiform phenotype , or a deposition of crystals  have been determined to be due to mutations in the CRYG genes. Interestingly, the crystal precipitations we found in the lens cortices of our affected family members were very similar to the prismatic crystals found by Kmoch et al. .
The CRYG gene family has six members; CRYGA, CRYGB, CRYGC, CRYGD, CRYGE, and CRYGF. CRYGE and CRYGF are considered to be pseudogenes, by virtue of an in-frame stop codon in an exon . In combination with linkage and gene mutational analysis, we identified a mutation in CRYGD, a C->A heterozygous transversion at nucleotide position 70 in exon 2, which was predicted to substitute the conserved amino acid proline for a threonine (P23T) in the γD-crystallin protein. This mutation co-segregated with the cataract in the affected family and was not observed in 100 unrelated controls, implying that it was associated with ADCC rather than a rare polymorphism. It was noteworthy that the same mutation had recently been suggested to cause the lamellar cataract in an Indian family  and the cerulean cataract in a large Moroccan family . Both the lamellar cataract in an Indian family, which was a perinuclear shaped cataract with a transparent embryonic nucleus, and the cerulean cataract in a large Moroccan family, which was a cerulean blue dot cataract and characterized by a very early onset of lens opacities and a rapid progression towards blindness, were clinically different from our special fasciculiform cataract, which was not progressive in development of lens opacities and showed a special corroliform opacification including embryonic and fetal nuclei and a fiberglasslike opacity projecting in different directions from the central zone with the radially oriented metal-like refractivity. Interestingly, during galley processing of this article, Mackay et al.  reported that an autosomal dominant form of coral-shaped cataract segregating in a three generation Caucasian pedigree was also caused by the P23T mutation in GRYGD. As no clinical photographs of this coral-shaped cataract were presented and it was only "...described as coralliform or axial, having long trumpet-shaped opacities grouped toward the center of the lens and projecting radially forward resembling a piece of coral" , it is not clear that this coral-shaped cataract is the same as, or even similar to, our special fasciculiform cataract. An explanation for the observation of these different phenotypes with the same mutation may be related to the influence of an unidentified modifier gene or to sequence variations that may influence CRYGD expression. The phenotypic heterogeneity of congenital cataracts has been reported before .
In summary, we have identified a missense mutation (P23T) in exon 2 of the CRYGD gene which is linked and associated with a special fasciculiform ADCC affecting a large Chinese family. This is the first time to report a mutation in CRYGD resulting in special fasciculiform ADCC. Future studies will include crystallization of the mutant γD-crystallin and the generation of a transgenic animal model of this mutation. These studies will help us to better understand cataract formation and will provide a useful system for testing strategies for cataract prevention.
This work was financed by the key project of the Natural Science foundation of Zhejiang Province of china.
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