Molecular Vision 2007; 13:1333-8 ©2007 Molecular Vision Received 23 March 2007 | Accepted 25 July 2007 | Published 26 July 2007

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

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 con- sidered 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.

Lens crystallins represent more than 90% of soluble pro- one encodes three amino acids and the second and third each teins and are critical in the transparency and refraction func- encode for two Greek motifs. The increase of refractive index tion of the lens [1]. The lens provides the variable refractive from the periphery to the center of lens depends on the con- power of focusing and one-third of the stationary refractive centration and composition of crystallins [9]. Disruption of power. At least 13 crystallin genes have been characterized in the crystallin structure results in the formation of congenital the human genome and of these 13, two α-crystallins and nine cataract [10,11], large amounts of high-weight protein aggre- β/γcrystallins have been identified in the human lens. α-Crys- gates result in lens opacity [12]. tallin and β/γ-crystallins belong to a superfamily of proteins; Ten percent of blindness in children is attributed to con- whereas α-crystallins are heat shock proteins, β/γ-crystallins genital cataracts [13], a frequent cause of hereditary visual are included in the microbial stress proteins. The three types loss in infants [14]. With no prompt treatment, congenital cata- of crystallins are β-pleated sheets. All β/γ-crystallins are com- ract can result in irreversible visual loss. About one-third of posed of two domains that are formed by two Greek motifs congenital cataracts are hereditary; most of them show an au- and assemble into monomers, dimers, and oligomers [2,3]. tosomal dominant pattern [10,11]. Cataracts are clinically and The γ-crystallins are monomeric and comprise about 40% of genetically heterogeneous, similar phenotypes map to differ- total proteins in mouse lens and 25% of total crystallin pro- ent loci and different phenotypes map to the same locus [15- teins in human lens [1,4,5]. Long terminal extensions of oli- 17]. A high clinical spectrum in congenital cataract is observed gomeric β-crystallins are the principal difference with respect in patients with CRYG gene mutations [13,16,18-20]. There to γ-crystallins; truncation of these extensions results in loss are a few cases of congenital cataract due to mutations in the of solubility and cataract in rodent models [6]. The γ-crystallins CRYGC gene (NM_020989). In the present study, we describe gene cluster includes the six genes, CRYGA-F; only CRYGC a family with primary congenital cataract and evidence of the and CRYGD encode abundant lens γ-crystallins in humans clinical heterogeneity observed in the R168W mutation in the [7,8]. CRYGD is expressed at high concentrations in the cells CRYGC gene. of the embryonic human lens. The unexpressed CRYGE and CRYGF pseudogenes are due to insertions of premature stop METHODS codons. The γ-crystallin genes encompass three exons; the first The family was referred to the General Hospital of Mexico by presenting primary congenital cataract. Protocol was ap- Correspondence to: Dr. Sergio Cuevas-Covarrubias, Servicio de proved by the Ethics Committee of the General Hospital of Genética, Hospital General de México, Dr. Balmis 148 Col. Doctores Mexico. Patients gave informed consent to the study. We ana- C.P. 06726, México D.F., México; Phone: (52) 55 27892000; FAX: lyzed a three-generation Mexican family, segregating autoso- (52) 55 27892000; email: [email protected] mal dominant cataract with no systemic anomalies. The fam- 1333 Molecular Vision 2007; 13:1333-8 ©2007 Molecular Vision ily included nine affected patients and 13 unaffected subjects. microsatellite markers D2S325 and D2S2382 were amplified There was no history of consanguinity (Figure 1). Photographs through PCR under the supplier’s conditions (Applied of the lens opacities of the most affected members of the fam- Biosystems, Foster City, CA). All assays were performed two ily were not available. Ophthalmic records showed that the times with a normal control included. Clinical characteristics onset of cataract was in infancy; in all cases, cataract was de- of lens opacities were analyzed through slit lamp. The method scribed only as “congenital cataract”. Since the number of for assessing length was “A scan technique” with an OcuScan mutations in the CRYGA-D genes in dominant cataracts is high Biophysic Alcon Biometer (3.02 version; Alcon, Ft. Worth, in humans, this gene cluster was analyzed as a candidate. To TX). SRK-T formula was used to calculate intraocular lens perform the molecular analysis of the CRYGA-D gene cluster, power. we obtained genomic DNA from peripheral blood with con- ventional methods. Conditions to amplify exons through poly- RESULTS merase chain reaction (PCR) were as follows: DNA (500 ng), The propositus was a three-year-old Mexican male weight- primers (0.4 µM), dNTP’s (0.08 mM), MgCl2 (1.5 mM), buffer ing 3,300 g and was the product of an apparently uncompli- (1X), Taq Pol (1.5 U), in a total volume of 50 µl. PCR was cated term pregnancy with normal spontaneous vaginal deliv- performed with an initial denaturation step at 95 °C for one ery. The pedigree is shown in Figure 1. Onset of ocular symp- min, followed by 30 cycles of 94 °C then denaturation for toms was at the age of nine months with nystagmus and pho- another one min, annealing at 60 °C for one min, and exten- tophobia. On clinical examination, the patient showed nys- sion at 72 °C for two min. Exon primers are described else- tagmus, peripupillary iris atrophy, and cataract. The morphol- where [13]. PCR products were purified with a PCR purifica- ogy of cataract is shown in Figure 2. His antero-posterior di- tion kit (Qiaex II, Qiagen, Hilden, Germany). DNA sequenc- ameters were: Right eye (RE) 20.21 mm and left eye (LE) ing analysis was performed in ABI PRISM 310 genetic ana- 19.9 mm. No other ocular findings were found to be present. lyzer (Applied Biosystems, Foster City, CA) according to the The rest of the general examination was normal. His father supplier’s conditions. To identify disease haplotype, was affected with congenital cataract (no morphology descrip-

Figure 1. Pedigree and haplotype analysis of the cataract. A four-generation pedigree, segregating autosomal dominant nuclear cataract and two microsatellite markers (D2S325 and D2S2382), are shown. Squares and circles symbolize males and females, respectively, and the blackened symbols denote affected patients. Disease haplotype was marker D2S325 of 166 bp long and marker D2S2382 of 316 bp long. Position of markers in physical order from 2p-tel is: D2S325 202.03 Mb, CRYG genes 202.76 Mb, and D2S2382 210.81 Mb. Open squares in red show the disease haplotype. 1334 Molecular Vision 2007; 13:1333-8 ©2007 Molecular Vision tion of cataract was obtained) and underwent surgery in child- DISCUSSION hood. Cataractogenesis is a complex mechanism associated with the The brother (IV-1) of the proband was an 11-year-old male. breakdown of the lens microarchitecture [13,21]. Cataracts that Onset of symptoms was at the age of one year with photopho- are the result of genetic factors must be distinguished from bia; a diagnosis of nuclear congenital cataract was made. He those that occur as a consequence of systemic diseases. Con- presented myopia with the following antero-posterior diam- genital cataract is visible within the first year of life and may eters: RE 25.11 mm, LE 25.69 mm. The cycloplegic refrac- be hereditary or secondary to an intrauterine event. Neverthe- tion showed: LE: -3.00 spherical equivalent and RE: -2.5 spherical equivalent. After initial diagnosis the patient under- went 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 care- fully examining two additional siblings, we found no ocular affliction. They have VA of 20/20 in both eyes. In the oph- thalmic records, the morphology of cataract of affected mem- bers 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 mem- bers 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). Analy- sis 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 Figure 3. Partial forward DNA sequence of exon 3 showing the were found to be present. Haplotype analysis indicated that CRYGC gene mutation within codon 168. The CRYGC gene was the affected patients shared a haplotype with the markers, sequenced and found to contain a C>T nucleotide transition (for- D2S325 166 bp long and D2S2382 316 bp long; a region where ward) at position 502 of exon 3 resulting in an R168W amino acid the CRYG family is located. change.

Figure 2. Preoperative photographs of the right eye of the patient with nuclear cataract. The child was initially examined at the age of three years and was found to have focal opacities in the nuclear and perinuclear areas of both lenses. 1335 Molecular Vision 2007; 13:1333-8 ©2007 Molecular Vision less, the age of onset is not necessarily related to the cause of our family. Trp is a hydrophobic amino acid (MW 204.23) cataract. Inherited cataracts occur between 8.3% and 25% of while Arg is a hydrophilic amino acid (MW 174.20) with a congenital cataracts [22,23], they may be isolated or associ- positive charge. Residue R168 is within an extended strand ated with additional findings. Cataracts are characterized by on the surface of the molecule interacting with water. Change the location and structure of opacities. Several classification of the solvation property of an amino acid residue predicted systems of human inherited cataracts has been developed based to be on the surface of the γ-crystallin protein molecule di- on the anatomic location or morphology of the opacity; how- minishes the protein solubility [28]. The R168W mutant dif- ever, classification has been difficult due to wide phenotypic fers in its ability to aggregate and scatter light [29]. In the variability [24,25]. study, we propose that the R168W mutation in the CRYGC Mutations in the CRYG gene cluster, located on 2q33-35, gene results in a reduction in protein solubility and subse- are the most frequent cause of autosomal dominant congeni- quently results in the genesis of cataract. tal cataract. All mutations identified in the human CRYGC gene On the other hand, although R168W mutation has been are shown in Table 1. In three previous reports, two missense associated with the phenotype of lamellar cataract [13], R168W mutations and one insertion in the CRYGC gene cosegregated is also considered a SNP in the NCBI database. There are sev- with the cataract phenotype: coppok-like cataract that presents eral explanations for the molecular finding in our family and dustlike opacity of the fetal nucleus with involvement of the in the one previously described with lamellar cataract. One is zonular lens [16]; zonular pulverulent cataract that involves that the segregation is by chance, another is that R168W is a the larger fetal nucleus with more opacification in the periph- marker for a mutation that cannot be detected by sequencing, ery [26]; and lamellar cataract, also called zonular, perinuclear, and finally, that R168W is the actual disease-causing muta- or polymorphic cataract with different degree of opacification tion and should no longer be considered a SNP. The mutation [27]. The latter is associated with a missense mutation in the reported in this paper and the one previously associated with CRYGC gene [13] and results in the substitution of Arg at po- lamellar cataract [13] support that R168W actually is the dis- sition 168 by Trp in the fourth Greek key motif, a highly con- ease-causing mutation. We used software SIFT to predict the served position present in γ-crystallins of several species (Fig- phenotypic effect of the amino acid substitution R168W in ure 4). This mutation is identical to the mutation observed in CRYGC [30]. Substitution at position 168 from R to W is pre-

TABLE 1. HUMAN CRYGC MUTATIONS ASSOCIATED WITH CONGENITAL CATARACT

Nucleotide defect Codon Phenotype Protein effect Reference ------A13C 5 Coppock-like Thr5Pro [16] 123-128insGCGGC 52 Zonular pulverulent New amino acid [33] C502T 168 Lamellar Arg168Trp [13] C502T 168 Nuclear Arg168Trp This paper

Clinical heterogeneity is evident with the presence of different phenotypes with CRYGC mutations, specially in Arg168Trp.

Figure 4. Multiple protein sequence alignments of CRYG in different species. These sequence alignments of the γ-crystallin protein in different species show a high degree of the mutated amino acid conservation. The mutated Arg168Trp sequence indicates the sequence with the mutation (red letter) detected in our family.Only the last 34 amino acids are shown. 1336 Molecular Vision 2007; 13:1333-8 ©2007 Molecular Vision dicted to affect protein function with a score of 0.00 (prob- light scattering. Curr Eye Res 1987; 6:1421-32. abilities less than 0.05 are predicted to be deleterious). Be- 13. Santhiya ST, Shyam Manohar M, Rawlley D, Vijayalakshmi P, sides, cataract phenotypes are the result of the same molecu- Namperumalsamy P, Gopinath PM, Loster J, Graw J. Novel lar defect [16-18,31,32]. CRYGC gene mutations present dif- mutations in the gamma-crystallin genes cause autosomal domi- nant congenital cataracts. J Med Genet 2002; 39:352-8. ferent phenotypes; Thr5Pro and 123-128insGCGGC pheno- 14. Lund AM, Eiberg H, Rosenberg T, Warburg M. Autosomal domi- types are characterized by dust-like (or pulverulent) opacities nant congenital cataract; linkage relations; clinical and genetic in the fetal nucleus with involvement of the zonular lens heterogeneity. Clin Genet 1992; 41:65-9. [16,26]; Arg168Trp, previously described, is characterized by 15. Litt M, Carrero-Valenzuela R, LaMorticella DM, Schultz DW, clear lens in the inner fetal nucleus surrounded by an opaci- Mitchell TN, Kramer P, Maumenee IH. Autosomal dominant fied shell [27]. In this study, Arg168Trp phenotype differs by cerulean cataract is associated with a chain termination muta- a dense nuclear cataract confined to the fetal nucleus of the tion in the human beta-crystallin gene CRYBB2. Hum Mol Genet lens (Figure 2). 1997; 6:665-8. Finally, this is the first case of phenotypic heterogeneity 16. Heon E, Priston M, Schorderet DF, Billingsley GD, Girard PO, Lubsen N, Munier FL. The gamma-crystallins and human cata- in the congenital cataract specifically associated with the racts: a puzzle made clearer. Am J Hum Genet 1999; 65:1261- R168W mutation in the CRYGC gene. This phenotypic vari- 7. ability excludes the genotype-phenotype correlation and re- 17. Gill D, Klose R, Munier FL, McFadden M, Priston M, Billingsley marks on the influence of environmental factors and/or modi- G, Ducrey N, Schorderet DF, Heon E. Genetic heterogeneity of fier loci in the process of cataractogenesis. the Coppock-like cataract: a mutation in CRYBB2 on chromo- some 22q11.2. Invest Ophthalmol Vis Sci 2000; 41:159-65. ACKNOWLEDGEMENTS 18. Kmoch S, Brynda J, Asfaw B, Bezouska K, Novak P, Rezacova This project was supported by CONACyT-SaLUD, contract P, Ondrova L, Filipec M, Sedlacek J, Elleder M. 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The print version of this article was created on 26 Jul 2007. This reflects all typographical corrections and errata to the article through that date. Details of any changes may be found in the online version of the article. α 1338