Congenital cataract is a clinically and genetically heterogeneous lens disorder that typically appears as a sight-threatening trait in childhood and accounts for one-tenth of the cases of childhood blindness [1]. The prevalence of congenital cataracts is estimated to vary from 0.6 to 6 per 10,000 live births with an incidence of 2.2-2.49 per 10,000 live births [2]. Approximately half of all congenital cataract cases are inherited either in isolation or as part of a syndrome of ocular or systemic abnormalities [3]. All three classical forms of Mendelian inheritance have been associated with non-syndromic cataracts. However, the majority of families with a history of congenital cataracts show an autosomal dominant transmission pattern.

There are currently 34 loci implicated in nonsyndromic congenital cataract, and of these, over 20 have been associated with mutations in specific genes [4]. These genes can be considered in five functional groups: (1) 10 genes that encode crystallines, CRYAA [5,6], CRYAB [7,8], CRYBB1 [9,10], CRYBB2 [11-14], CRYBB3 [15], CRYBA1/A3 [16,17], CRYBA4 [18], CRYGC [19-23], CRYGD [19,21,24-26], and CRYGS [27]; (2) two genes that encode the gap junction proteins, GJA8 [28,29], and GJA3 [30,31]; (3) two genes that encode membrane proteins, MIP [32] and LIM2 [33,34]; (4) two genes that encode beaded filament proteins, BFSP1 [35] and BFSP2 [36-38]; and (5) seven genes that encode growth and transcription factors, PITX3 [39], MAF [40,41], HSF4 [42], FOXE3 [43], EYA1 [44], CHX10 [45], and PAX6 [46].

Crystallins are the dominant structural components of the vertebrate eye lens. At least 11 crystallin genes encode over 95% of the water-soluble structural proteins present in the crystallin lens, representing more than 30% of its mass and accounting for its optical transparency and high refractive index [47]. Therefore, mutations in the crystallin genes are strong candidates for certain hereditary forms of congenital cataracts.

In this study, we performed linkage analysis of a four-generation Chinese family with autosomal dominant nuclear cataract and microcornea, and we identified a novel nonsense mutation in CRYGC on 2q.

The association of developmental cataract and microcornea (congenital cataract-microcornea syndrome, microcornea-cataract [CCMC], OMIM 116150) makes up a distinct phenotype within the group of autosomal dominant congenital cataracts. CCMC has been considered a rare phenotype. The frequency of CCMC may be underestimated because the focus is often on the sight-threatening cataract and corneal diameters may pass unnoticed. Hansen et al. [48] identified five novel mutations by screening 10 CCMC families in nine already known cataract genes. The numbers of known mutations in CCMC are, however, still limited, which hampers evaluation of whether the appearance of CCMC phenotypes depends on specific mutations or closely linked modifying elements.

Herein, we report the identification of a novel nonsense mutation (W157X) in processed CRYGC that cosegregated with autosomal dominant nuclear cataracts and microcornea in a four-generation Chinese family. In addition to the novel W157X mutation, there are four other mutations in CRYGC that have been associated with cataracts including Coppock-like cataracts (T5P) [19], variable zonular pulverulent cataracts (5 bp duplication) [20], lamellar cataracts (R168W) [21], nuclear cataracts (R168W) [22], and nuclear cataracts (C109X) [23]. Therefore, in a manner similar to the nuclear opacities associated with the W157X mutation, these other CRYGC-related opacities involve the nucleus of the lens. This clinical manifestation is in agreement with the function of CRYGC, which is abundantly expressed at an early developmental stage in elongating fiber cells. CRYGC is expressed primarily in the lens nucleus, which is consistent with the location of the opacity in the nucleus.

Crystallins constitute the major proteins of the vertebrate eye lens, and they maintain the transparency and refractive index of the lens. Because lens central fiber cells lose their nuclei during development, these crystallins are made and then retained throughout life, requiring them to be extremely stable proteins. Mammalian lens crystallins are divided into α-, β-, and γ-crystallin families. β- and γ-crystallins are also considered a superfamily. Seven protein regions exist in β-crystallins: four Greek key motifs, a connecting peptide, NH₂-terminal extensions, and COOH-terminal extensions. γ-Crystallins are a homogeneous group of highly symmetric, monomeric proteins. They are differentially regulated after early development. Four γ-crystallin genes (CRYGA, CRYGB, CRYGC, and CRYGD) and two pseudogenes (CRYGE and CRYGF) are tandemly organized in a genomic segment as a gene cluster.

Based on the crystal structure of human CRYGC, the W157X mutation lies in the COOH-terminal domain at the end of the fourth Greek key motif (Figure 3C). However, the deleterious effects of the W157X mutation on CRYGC folding, stability, and solubility are unclear. Interestingly, the T5P mutation, which is associated with Coppock-like cataracts, not only causes changes in conformation (partial unfolding) but also reduces protein solubility and stability [49]. Furthermore, the T5P mutation decreases interactions with wild type γC-crystallin and with αA-, αB-, and βB2-crystallins [50]. How the 5 bp duplication and R168W mutations, which are associated with variable zonular pulverulent cataracts and lamellar cataracts, respectively, exert their unique effects remains unknown. Further biophysical studies of these mutant proteins will be required to provide insights regarding the molecular pathology of CRYGC-associated cataracts.

Alignment of the CRYGC protein sequence from three different species with three human γ-crystallins revealed that the tryptophan residue at position 157 is highly conserved (Figure 3D). To further examine the structural change induced by the W157X mutation, we used the three-dimensional structure of γ-crystallin, which is characterized by the presence of four Greek key motifs. Motifs 1 and 2 are located in the NH₂-terminal domain, and motifs 3 and 4 are located in the COOH-terminal domain [51,52]. The W157X mutation results in an in-frame stop codon at nucleotide 470 that leads to the truncation of 17 amino acids from the COOH-terminus of γC-crystallin. The corresponding alteration affects not only the length of the COOH-terminal arms but also the formation of the fourth Greek-key motif in γC-crystallin, which in turn disrupts the highly symmetric structure of γC-crystallin. The W157X mutation affects the Greek key motifs, and it is predicted to change the folding properties of γC-crystallin and/or affect its interactions with other crystallins. Furthermore, the resulting changes in the three-dimensional structure may ultimately lead to a decrease in the stability and/or solubility of γC-crystallin.

It is noteworthy that an autosomal dominant nuclear and radial cataract caused by a 6 bp deletion in the CRYGC has also been reported in the mouse [53]. A 6-bp deletion in exon 3 of the mouse γC-crystallin encoding gene (Crygc) is causative for the cataract phenotype. The mutation is therefore designated CrygcChl3. The deletion of bases 420−425 leads to a loss of two amino acid residues, Gly and Arg, in the fourth Greek key motif.

In summary, the present study has identified a novel nonsense W157X mutation in CRYGC associated with autosomal dominant cataracts and microcornea in a four-generation Chinese family. The association between microcornea and congenital cataracts in all affected individuals indicate that mutations disrupting lens biochemistry and physiology early in development can result in microphakia and subsequent microcornea as a secondary effect of damage to the lens.