Molecular Vision 2007; 13:1333-1338 <http://www.molvis.org/molvis/v13/a146/> Received 23 March 2007 | Accepted 25 July 2007 | Published 26 July 2007

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A family with autosomal dominant primary congenital cataract associated with a CRYGC mutation: evidence of clinical heterogeneity

Luz M Gonzalez-Huerta,¹ Olga M Messina-Baas,² Sergio A Cuevas-Covarrubias¹
 
 

¹Department of Genetics, General Hospital of Mexico, Faculty of Medicine, Universidad Nacional Autónoma de México, and ²Department of Ophthalmology, General Hospital of Mexico, Mexico City, Mexico

Correspondence to: Dr. Sergio Cuevas-Covarrubias, Servicio de Genética, Hospital General de México, Dr. Balmis 148 Col. Doctores C.P. 06726, México D.F., México; Phone: (52) 55 27892000; FAX: (52) 55 27892000; email: sergioa@servidor.unam.mx

Abstract

Purpose: To describe a family with primary congenital cataract associated with a CRYGC mutation.

Methods: One family with several affected members with primary congenital cataract and 170 healthy controls were examined. DNA from leukocytes was isolated to analyze the CRYGA-D gene cluster.

Results: DNA sequencing analysis of the CRYGA-D gene cluster of the affected members showed the heterozygous missense mutation c.502C>T in the CRYGC gene. This transition mutation resulted in the substitution of Arg at position 168 by Trp. Analysis of the healthy members of the family and 170 unrelated controls showed a normal sequence of the CRYGA-D gene cluster.

Conclusions: In the present study, we described a family with nuclear congenital cataract that segregated the CRYGC missense mutation c.502C>T. This mutation has been associated with the phenotype of lamellar cataract but is also considered a single nucleotide polymorphism (SNP) in the NCBI database. Our data and previous report support that R168W is the actual disease-causing mutation and should no longer be considered a SNP. This is the first case of phenotypic heterogeneity in the primary congenital cataract specifically associated with the R168W mutation in the CRYGC gene.

Introduction

Lens crystallins represent more than 90% of soluble proteins and are critical in the transparency and refraction function of the lens [1]. The lens provides the variable refractive power of focusing and one-third of the stationary refractive power. At least 13 crystallin genes have been characterized in the human genome and of these 13, two α-crystallins and nine β/γcrystallins have been identified in the human lens. α-Crystallin and β/γ-crystallins belong to a superfamily of proteins; whereas α-crystallins are heat shock proteins, β/γ-crystallins are included in the microbial stress proteins. The three types of crystallins are β-pleated sheets. All β/γ-crystallins are composed of two domains that are formed by two Greek motifs and assemble into monomers, dimers, and oligomers [2,3]. The γ-crystallins are monomeric and comprise about 40% of total proteins in mouse lens and 25% of total crystallin proteins in human lens [1,4,5]. Long terminal extensions of oligomeric β-crystallins are the principal difference with respect to γ-crystallins; truncation of these extensions results in loss of solubility and cataract in rodent models [6]. The γ-crystallins gene cluster includes the six genes, CRYGA-F; only CRYGC and CRYGD encode abundant lens γ-crystallins in humans [7,8]. CRYGD is expressed at high concentrations in the cells of the embryonic human lens. The unexpressed CRYGE and CRYGF pseudogenes are due to insertions of premature stop codons. The γ-crystallin genes encompass three exons; the first one encodes three amino acids and the second and third each encode for two Greek motifs. The increase of refractive index from the periphery to the center of lens depends on the concentration and composition of crystallins [9]. Disruption of the crystallin structure results in the formation of congenital cataract [10,11], large amounts of high-weight protein aggregates result in lens opacity [12].

Ten percent of blindness in children is attributed to congenital cataracts [13], a frequent cause of hereditary visual loss in infants [14]. With no prompt treatment, congenital cataract can result in irreversible visual loss. About one-third of congenital cataracts are hereditary; most of them show an autosomal dominant pattern [10,11]. Cataracts are clinically and genetically heterogeneous, similar phenotypes map to different loci and different phenotypes map to the same locus [15-17]. A high clinical spectrum in congenital cataract is observed in patients with CRYG gene mutations [13,16,18-20]. There are a few cases of congenital cataract due to mutations in the CRYGC gene (NM_020989). In the present study, we describe a family with primary congenital cataract and evidence of the clinical heterogeneity observed in the R168W mutation in the CRYGC gene.

Methods

The family was referred to the General Hospital of Mexico by presenting primary congenital cataract. Protocol was approved by the Ethics Committee of the General Hospital of Mexico. Patients gave informed consent to the study. We analyzed a three-generation Mexican family, segregating autosomal dominant cataract with no systemic anomalies. The family included nine affected patients and 13 unaffected subjects. There was no history of consanguinity (Figure 1). Photographs of the lens opacities of the most affected members of the family were not available. Ophthalmic records showed that the onset of cataract was in infancy; in all cases, cataract was described only as "congenital cataract". Since the number of mutations in the CRYGA-D genes in dominant cataracts is high in humans, this gene cluster was analyzed as a candidate. To perform the molecular analysis of the CRYGA-D gene cluster, we obtained genomic DNA from peripheral blood with conventional methods. Conditions to amplify exons through polymerase chain reaction (PCR) were as follows: DNA (500 ng), primers (0.4 μM), dNTP's (0.08 mM), MgCl₂ (1.5 mM), buffer (1X), Taq Pol (1.5 U), in a total volume of 50 μl. PCR was performed with an initial denaturation step at 95 °C for one min, followed by 30 cycles of 94 °C then denaturation for another one min, annealing at 60 °C for one min, and extension at 72 °C for two min. Exon primers are described elsewhere [13]. PCR products were purified with a PCR purification kit (Qiaex II, Qiagen, Hilden, Germany). DNA sequencing analysis was performed in ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA) according to the supplier's conditions. To identify disease haplotype, microsatellite markers D2S325 and D2S2382 were amplified through PCR under the supplier's conditions (Applied Biosystems, Foster City, CA). All assays were performed two times with a normal control included. Clinical characteristics of lens opacities were analyzed through slit lamp. The method for assessing length was "A scan technique" with an OcuScan Biophysic Alcon Biometer (3.02 version; Alcon, Ft. Worth, TX). SRK-T formula was used to calculate intraocular lens power.

Results

The propositus was a three-year-old Mexican male weighting 3,300 g and was the product of an apparently uncomplicated term pregnancy with normal spontaneous vaginal delivery. The pedigree is shown in Figure 1. Onset of ocular symptoms was at the age of nine months with nystagmus and photophobia. On clinical examination, the patient showed nystagmus, peripupillary iris atrophy, and cataract. The morphology of cataract is shown in Figure 2. His antero-posterior diameters were: Right eye (RE) 20.21 mm and left eye (LE) 19.9 mm. No other ocular findings were found to be present. The rest of the general examination was normal. His father was affected with congenital cataract (no morphology description of cataract was obtained) and underwent surgery in childhood.

The brother (IV-1) of the proband was an 11-year-old male. Onset of symptoms was at the age of one year with photophobia; a diagnosis of nuclear congenital cataract was made. He presented myopia with the following antero-posterior diameters: RE 25.11 mm, LE 25.69 mm. The cycloplegic refraction showed: LE: -3.00 spherical equivalent and RE: -2.5 spherical equivalent. After initial diagnosis the patient underwent surgery. Ophthalmic records indicated his lens opacities as "nuclear congenital cataract". At this moment, on clinical examination, the patient achieves visual acuity (VA) of 20/20 in both eyes. No other ocular findings were found to be present. The rest of the general examination was normal. After carefully examining two additional siblings, we found no ocular affliction. They have VA of 20/20 in both eyes. In the ophthalmic records, the morphology of cataract of affected members in the family was referred only as "congenital cataract". All affected patients of the family underwent surgery.

DNA analysis of the propositus (IV-4) and affected members of the family (II-2, II-4, III-2, III-5, IV-1) showed a c.502C>T heterozygous missense mutation within exon 3 of the CRYGC gene (Figure 3); this transition mutation leads to a substitution of an Arg at position 168 by Trp (R168W). Analysis of nonaffected members of the family (III-1, III-4, III-8, IV-2, and IV-3) and 170 normal controls showed a normal sequence of the CRYGA-D gene cluster. No other nucleotide variations or polymorphisms in the CRYGA-D gene cluster were found to be present. Haplotype analysis indicated that the affected patients shared a haplotype with the markers, D2S325 166 bp long and D2S2382 316 bp long; a region where the CRYG family is located.

Discussion

Cataractogenesis is a complex mechanism associated with the breakdown of the lens microarchitecture [13,21]. Cataracts that are the result of genetic factors must be distinguished from those that occur as a consequence of systemic diseases. Congenital cataract is visible within the first year of life and may be hereditary or secondary to an intrauterine event. Nevertheless, the age of onset is not necessarily related to the cause of cataract. Inherited cataracts occur between 8.3% and 25% of congenital cataracts [22,23], they may be isolated or associated with additional findings. Cataracts are characterized by the location and structure of opacities. Several classification systems of human inherited cataracts has been developed based on the anatomic location or morphology of the opacity; however, classification has been difficult due to wide phenotypic variability [24,25].

Mutations in the CRYG gene cluster, located on 2q33-35, are the most frequent cause of autosomal dominant congenital cataract. All mutations identified in the human CRYGC gene are shown in Table 1. In three previous reports, two missense mutations and one insertion in the CRYGC gene cosegregated with the cataract phenotype: coppok-like cataract that presents dustlike opacity of the fetal nucleus with involvement of the zonular lens [16]; zonular pulverulent cataract that involves the larger fetal nucleus with more opacification in the periphery [26]; and lamellar cataract, also called zonular, perinuclear, or polymorphic cataract with different degree of opacification [27]. The latter is associated with a missense mutation in the CRYGC gene [13] and results in the substitution of Arg at position 168 by Trp in the fourth Greek key motif, a highly conserved position present in γ-crystallins of several species (Figure 4). This mutation is identical to the mutation observed in our family. Trp is a hydrophobic amino acid (MW 204.23) while Arg is a hydrophilic amino acid (MW 174.20) with a positive charge. Residue R168 is within an extended strand on the surface of the molecule interacting with water. Change of the solvation property of an amino acid residue predicted to be on the surface of the γ-crystallin protein molecule diminishes the protein solubility [28]. The R168W mutant differs in its ability to aggregate and scatter light [29]. In the study, we propose that the R168W mutation in the CRYGC gene results in a reduction in protein solubility and subsequently results in the genesis of cataract.

On the other hand, although R168W mutation has been associated with the phenotype of lamellar cataract [13], R168W is also considered a SNP in the NCBI database. There are several explanations for the molecular finding in our family and in the one previously described with lamellar cataract. One is that the segregation is by chance, another is that R168W is a marker for a mutation that cannot be detected by sequencing, and finally, that R168W is the actual disease-causing mutation and should no longer be considered a SNP. The mutation reported in this paper and the one previously associated with lamellar cataract [13] support that R168W actually is the disease-causing mutation. We used software SIFT to predict the phenotypic effect of the amino acid substitution R168W in CRYGC [30]. Substitution at position 168 from R to W is predicted to affect protein function with a score of 0.00 (probabilities less than 0.05 are predicted to be deleterious). Besides, cataract phenotypes are the result of the same molecular defect [16-18,31,32]. CRYGC gene mutations present different phenotypes; Thr5Pro and 123-128insGCGGC phenotypes are characterized by dust-like (or pulverulent) opacities in the fetal nucleus with involvement of the zonular lens [16,26]; Arg168Trp, previously described, is characterized by clear lens in the inner fetal nucleus surrounded by an opacified shell [27]. In this study, Arg168Trp phenotype differs by a dense nuclear cataract confined to the fetal nucleus of the lens (Figure 2).

Finally, this is the first case of phenotypic heterogeneity in the congenital cataract specifically associated with the R168W mutation in the CRYGC gene. This phenotypic variability excludes the genotype-phenotype correlation and remarks on the influence of environmental factors and/or modifier loci in the process of cataractogenesis.

Acknowledgements

This project was supported by CONACyT-SaLUD, contract grant number 2002-C01-8038.

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