Childhood cataract is the most common form of treatable blindness in children [1]. It accounts for more than 1 million blind children in Asia and about 10% of childhood blindness worldwide [2]. Non-syndromic congenital cataracts have an estimated incidence of 1–6 per 10,000 live births [3,4]. The prevalence of childhood cataract varies between different population, geographic area, socioeconomic condition and other factors. Etiologic factors include genetic, metabolic, systemic disorders, intrauterine infections, and trauma. Approximately 50% of the childhood cataracts are genetic and at least one-third may have the familial basis. It is both clinically and genetically diverse as demonstrated by several investigators suggesting involvement of possible modifying factors [2]. Hereditary cataracts typically follow Mendelian inheritance with majority being autosomal dominant type with complete penetrance and may be either static or progressive. Cataracts are classified based on morphology, location, or time of onset. Childhood cataracts have been mapped to 36 genetic loci and mutations in 22 genes have been identified toward the pathogenesis of various forms of congenital and developmental cataract including 12 loci for autosomal recessive cataract [5-8].

Biochemically up to 90% of the water soluble proteins in the vertebrate lens belong to α-, β-, and γ-crystallins. Being a part of small heat shock protein the α-crystallins form high-molecular aggregates and function as molecular chaperones. The β/γ-crystallin superfamily exhibits a characteristic Greek key motif, in a quadruple organization showing two in the NH_2- and two in the COOH-terminal domain. Evolutionary analysis has demonstrated the relationship of crystallins to other stress proteins and is expressed in other tissues of the body as well [9].

The human gamma-crystallins (CRYG) gene cluster comprise six genes including two pseudogenes. The spectrum of mutations in CRYG gene leading to diverse cataract phenotypes is on the increase [10,11]. The aim of the present study therefore was to screen families with inherited cataracts to document known as well as novel mutations if any in human genes coding for alpha-A crystallin (CRYAA), beta-B2 crystallin (CRYBB2), gamma-crystallins (CRYG), gap junction alpha-3 and alpha-8 (GJA3, GJA8), and transcription factor paired box gene 6 (PAX6).

Cases of childhood cataract were registered through Kasturba Hospital (KH), Manipal, India. Clinical details of the proband were recorded in all the cases. Ophthalmic investigation included slit lamp examination with dilated pupils, visual acuity testing, intraocular pressure measurement, and fundus examination done by a senior ophthalmologist (V.P.). In familial cases, identification of cataract phenotype and detailed examination of the affected as well as available unaffected family members were performed. A detailed pedigree of the kindred was ascertained by interviewing the parents or any available family member. Clinical details of the patients who previously had cataract extraction were obtained through medical records. Cases presenting conditions such as unilateral, congenital rubella, systemic disorders, traumatic, syndromic, and other known causes were excluded for further study. Seeking of informed consent from all participants and parents of the probands was in accordance with the Declaration of Helsinki and Institutional Ethical Committee of Manipal University. Blood samples were collected from available affected/unaffected members of the family.

Genes reported to cause cataract-specific mutations include those of the crystallins, cytoskeletal proteins, membrane proteins, transcription factors, glucosaminyl transferase 2 chromatin modifying protein-4B, and transmembrane protein 114 (TMEM114) [19]. Crystallin specific mutations account for 16.6% of inherited pediatric cataract in south India [20]. Among the reported genes, crystallins are of special interest because it encodes the major proportion of water soluble structural proteins of the lens fiber cells. In vertebrates, the α-, β-, and γ- crystallins are ubiquitous lens proteins. γ- Crystallin amounts for 25% of the total crystallins in the human lens. Both CRYGC and CRYGD are expressed at high concentrations in the fiber cells of the human embryonic lens which subsequently form the lens nucleus. The precise cellular micro-architecture and homeostasis are critical in maintaining the transparency of the lens. Alterations that impairs the proper solubility of lens proteins can lead to progressive protein aggregation that might act as light scattering centers in the lens [11].

Human γD-crystallin is a monomeric eye lens protein that must remain soluble throughout life for lens transparency. It is composed of two highly homologous beta-sheet domains which interact through interdomain side chain contacts forming two structurally distinct regions, a central hydrophobic cluster and peripheral residues. The specificity of domain interface interactions is likely important for preventing incorrect associations in the high protein concentrations of the lens nucleus [21]. Features of the interface between the two domains conserved among γ-crystallins are a central six-residue hydrophobic cluster, and two pairs of interacting residues flanking the cluster. In human γD-crystallin these pairs are Gln54/Gln143 and Arg79/Met147. It is suggested that these residues stabilize the native state by shielding the central hydrophobic cluster from solvent. In aged and cataractous lenses, glutamine and methionine side chains are among the residues covalently damaged. Such damage may generate partially unfolded, aggregation-prone conformations of human γD-crystallin that could be significant in cataract [22].

Many of the identified mutations in CRYGD seems to have been associated with autosomal dominant cataract phenotypes of congenital or juvenile nature. The eleven mutations identified so far occur as missense [10,15,23-34], nonsense [15,34-36], and as frameshift [37] to have been associated with diverse phenotypes (Table 3). Three of the truncation mutations and a frameshift mutation seem to be associated exclusively with nuclear phenotype and all are congenital [15,34-36]. The phenotype described here (C-35) is an anterior polar and coronary and the age at onset being within the first decade of life. As this mutation (R77S) involves the substitution of a highly basic and polar charged Arg for a less polar Ser residue it probably may not cause any major conformational change, as suggested earlier [32]. Along with the R77S mutation, four other variations as SNPs haplogroup show the transmission of T-G-A-G-C block from affected father to two affected children. The significance of this mutation, along with the prevalence of any specific haplotype, remains to be confirmed through analysis from a large number of familial cataracts. Predictions on structural changes in the mutant form of γD-crystallin using PyMOL reveals the shorter Ser side chain to establish weaker bonding with Glu46 that might result in unfolding. Furthermore, substitution with serine reduces the surface area of interaction with solvents thus hampering solubility of the mutant form as also revealed by increase in hydrophobicity (Figure 6). This may therefore be considered as the causative mutation underlying the cataract phenotype in the family (C-35) investigated. Interestingly four of the CRYGD mutant alleles reported earlier also involves the residue Arg at different codon positions viz., 15, 37, 59, and 140 . In our study, by sequence alignment, the observed variation involves an arginine residue at position 77 which is highly conserved in γD-crystallin across various other species. Among cataract specific CRYGD mutations, the Arg residue has been replaced either by cysteine [10,23], serine [28,29,32], Histidine [38], or for a stop codon [36]. Similar to the substitution in this study, the R37S mutation has been reported earlier to result in phenotypes with protein crystals in a Czech [28] and in a Chinese study [29]. Recently, in a Chinese family a corolliform cataract was found to be associated with an R15S allele of CRYGD [32]. It has been suggested that an increase in hydrophobicity and a putative phosphorylation site-mediated protein aggregation as the probable cause of opacification [32]. Both R15S and R37S results in congenital cataracts and fall within the I Greek key motif, while R77S falls within II Greek key motif and in association with a juvenile form of cataract in the C-35 family. Cataract-specific mutations involving the Arg residue has also been frequently reported in CRYAA [34,35]. This suggests that Arg residues are more critical toward maintaining the structural and functional integrity of proteins.

In the 16 other families studied no putative mutation could be observed in the candidate genes screened which therefore makes it rather unlikely that the selected genes are involved in the cataract-forming process in these families. It prompts screening of other known candidate genes. This demonstrates that cataract need not arise only through point mutations but might be influenced also by many other factors, which may include unidentified modifier genes and other sequence variations. Detailed information about such factors and their precise role should enable one to understand the pathophysiology of cataracts and the biology of the lens in general.