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A CRYGC gene mutation associated with autosomal dominant pulverulent cataract
Gene 529 (2013) 181–185
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Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
A CRYGC gene mutation associated with autosomal dominant pulverulent cataract Luz Ma. González-Huerta a, Olga Messina-Baas b, Héctor Urueta a, Jaime Toral-López c, Sergio A. Cuevas-Covarrubias a,⁎ a b c
Departamento de Genética, Hospital General de México, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F. México, Mexico Departamento de Oftalmología, Hospital General de México, D.F. México, Mexico Departamento de Genética, Centro Médico Ecatepec, ISSEMYM. Edo. México, Mexico
a r t i c l e
i n f o
Article history: Accepted 8 July 2013 Available online 14 August 2013 Keywords: Pulverulent congenital cataract CRYGC gene Gamma-crystallins Clinical heterogeneity
a b s t r a c t Purpose: To describe at molecular level a family with pulverulent congenital cataract associated with a CRYGC gene mutation. Methods: One family with several affected members with pulverulent congenital cataract and 230 healthy controls were examined. Genomic DNA from leukocytes was isolated to analyze the CRYGA-D cluster, CX46, CX50 and MIP genes through high-resolution melting curve and DNA sequencing. Results: DNA sequencing in the affected members revealed the c.143GNA mutation (p.R48H) in exon 2 of the CRYGC gene; 230 healthy controls and ten healthy relatives were also analyzed and none of them showed the c.143GNA mutation. No other polymorphisms or mutations were found to be present. Conclusion: In the present study, we described a family with pulverulent congenital cataract that segregated the c.143GNA mutation (p.R48H) in the CRYGC gene. A few mutations have been described in the CRYGC gene in autosomal dominant cataract, none of them with pulverulent cataract making clear the clinical heterogeneity of congenital cataract. This mutation has been associated with the phenotype of congenital cataract but also is considered an SNP in the NCBI data base. Our data and previous report suggest that p.R48H could be a diseasecausing mutation and not an SNP. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Crystallins are important structural components of the vertebrate eye lens and represent more than 90% of the water-soluble total lens proteins; they play critical roles in maintaining the transparency and refraction function of the lens (Mackay et al, 2004; Wistow and Piatigorsky, 1988). The lens is responsible for the variable refractive power of focusing and one-third of the stationary refractive power. Disruption of the crystallin structure results in the formation of congenital cataract (Ionides et al., 1999; Lambert and Drack, 1996). Congenital cataract represents 10% of the cases of childhood blindness (Gilbert
Abbreviations: CRYAA, αA-crystallin (CRYAA); CRYBA1, βA1-crystallin (CRYBA1); CRYBB1, βB1-crystallin (CRYBB1); CRYBB2, βB2-crystallin (CRYBB2); CRYGC, γC-crystallin (CRYGC); CRYGD, γD-crystallin (CRYGD); CX46, connexin 46 (CX46); CX50, connexin 50 (CX50); MIP, major intrinsic protein (MIP); DNA, deoxyribonucleic acid; G, guanine; A, adenine; PCR, polymerase chain reaction; RE, right eye; LE, left eye; R, arginine; H, histidine; Glu, glutamic acid; Tyr, tyrosine; Cys, cysteine; Gln, glutamine; Arg, arginine; His, histidine; W, tryptophan; C, cysteine; T, threonine; P, proline; G, glycine. ⁎ Corresponding author at: Servicio de Genética, Hospital General de México, Dr. Balmis 148 Col. Doctores C.P. 06726 México D.F., Mexico. Tel./fax: +52 55 53412821. E-mail addresses: [email protected], [email protected] (S.A. Cuevas-Covarrubias). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.07.044
et al., 1993; Ionides et al., 1999; Reddy et al., 2004). This disorder is visible within the first year of life; nevertheless, the age of onset is not necessarily related to the cause of cataract (Francois, 1982; Merin and Crawford, 1971). Cataract is the most common treatable cause of visual disability in infancy or early childhood. 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. About 40% of congenital cataract cases is inherited either in isolation or as part of an ocular syndrome or systemic abnormalities; cataracts that are the result of genetic factors must be distinguished from those that occur as a consequence of systemic diseases (Ionides et al., 1999; Lambert and Drack, 1996). Most familial cases of congenital cataracts show an autosomal dominant pattern. Cataracts are clinically and genetically heterogeneous, similar phenotypes map to different loci and different phenotypes map to the same locus. More than 40 loci have been mapped in primary congenital cataracts (Gilbert et al., 1993; Ionides et al., 1999; Reddy et al., 2004). Candidate genes for hereditary cataracts are highly expressed in the lens and include αA-crystallin (CRYAA), βA1-crystallin (CRYBA1), βB1crystallin (CRYBB1), βB2-crystallin (CRYBB2), γC-crystallin (CRYGC), γD-crystallin (CRYGD), connexin 46 (CX46), connexin 50 (CX50), and major intrinsic protein (MIP) (Blundell et al., 1981; Graw, 1997; Hejtmancik, 2008; Reddy et al., 2004; Wang et al., 2011). Mutations in
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the CRYG gene cluster, located on 2q33–35, are among the most frequent cause of autosomal dominant congenital cataract. A high clinical spectrum in congenital cataract has been observed in patients with CRYG gene mutations. There have been a few cases of congenital cataract due to mutations in the CRYGC gene (NM_020989). To our knowledge, there have been nine cases of congenital cataracts due to seven different mutations associated with the CRYGC gene (Gonzalez-Huerta et al., 2007; Guo et al., 2012; Héon, 1999; Kumar et al., 2011; Li et al., 2012; Ren et al., 2000; Santhiya et al., 2002; Yao et al., 2008; Zhang et al., 2009a,b) (Table 1). In the present study, we describe a family with autosomal dominant pulverulent cataract and the missense mutation c.143GNA (p.R48H) in the exon 2 of CRYGC gene. 2. Material and methods Considering the genes that are highly associated with primary congenital cataract, mutation screening was performed in nine candidate genes, specifically CRYAA (GenBank NM_000394.2), CRYBA1 (GenBank NM_005208.4), CRYBB1 (GenBank NM_001887.3), CRYBB2 (GenBank NM_000496.2), CRYGC (GenBank NM_020989.3), CRYGD (GenBank NM_006891.3), CX46 (GenBank NM_021954.3), CX50 (GenBank NM_005267.4), and MIP (GenBank NM_012064.3). All coding exons and splice sites were amplified by polymerase chain reactions (PCR) with the primers previously described (Wang et al., 2011). PCR products obtained from the proband and affected/unaffected members of the family were sequenced on an ABI 3730 Automated Sequencer (PE Biosystems, Foster City, CA). Two hundred and thirty ethnically matched controls were analyzed to confirm the mutation identified in the CRYGC gene. The family was referred to the General Hospital of Mexico due to primary congenital cataract. The protocol was approved by the Ethics Committee of the General Hospital of Mexico. Patients gave informed consent to participate in the study. We analyzed a four-generation Mexican family, segregating autosomal dominant cataract with no systemic anomalies. The family included four affected and 10 unaffected members. There was no history of consanguinity. Photographs of the lens opacities of affected members of the family were not available. Ophthalmic records showed that the onset of cataract occurred in infancy; in all cases, cataract was described only as pulverulent congenital cataract. 2.1. High resolution melting analysis After detecting the CRYGC gene mutation, high-resolution melting analysis was performed in all affected members with cataract, 10 healthy relatives and 230 ethnically matched controls. Oligonucleotide primers were obtained commercially. PCR was performed in 10 μl vol with a 1× LightCycler FastStart DNA Master HybProbe (Roche Applied Systems), with 0.5 μM of each CRYGC primers, 0.06 μM of lowtemperature correction control and 0.08 μM of high-temperature correction control, 3.5 mM of MgCl2 (including 1 mM MgCl2 contributed
by the LightCycler FastStart DNA Master HybProbe solution), 0.01 of U/reaction heat-labile uracil-DNA glycosylase (Roche Applied Systems), 1× LCGreen Plus (Idaho Technology), and 20 ng of template DNA. All oligonucleotides were mixed together and stored as a 20× stock solution. PCR and high-resolution melting were done on the LS-32 (Idaho Technology). PCR included an initial hold of 95 °C for 10 min, followed by 15 cycles of 95 °C for 2 s, 56 °C for 1 s, and 72 °C for 1 s, and 25 cycles of 95 °C for 2 s, 58 °C for 1 s, and 72 °C for 4 s. During amplification, no fluorescence acquisition was performed to avoid prolonging the temperature cycles. All heating and cooling steps during PCR were carried out with ramp rates programmed at 20 °C/s. After PCR was performed, samples were cooled (10 °C/s) from 95 °C to 40 °C and melting curves were generated with continuous fluorescence acquisition from 55 °C to 95 °C at 0.3 °C/s. Data processing included normalization of fluorescence, exponential background removal, and display of derivative melting curves. The melting curves were adjusted by identifying the maxima of the temperature correction control peaks and aligning curves through shifting and linear-scaling using custom software. Heterozygotes were identified by melting-peak width and shape. Homozygous and heterozygous genotypes were assigned by visual inspection based on Tm (melting-peak maxima). Predicted Tms based on nearestneighbor parameters were calculated as described previously (Liew et al., 2004). 2.2. CRYGC three-dimensional protein prediction Models were viewed in a Swiss-Pdb Viewer, versión 4.0, and RasMol version 2.7.4. Homology modeling was done by SWISS-MODEL (Arnold et al., 2006; Guex and Peitsch, 1997; Schwede et al., 2003). 3. Results The proband (III-2), a 20-year-old Mexican female, was the product of an apparently uncomplicated term pregnancy with normal spontaneous vaginal delivery. The pedigree is shown in Fig. 1. Onset of ocular symptoms occurred at the age of one year. The patient underwent phacoemulsification surgery with intraocular lens implant in both eyes after diagnosis. No complications were observed. The diagnosis of pulverulent congenital cataract was obtained from ophthalmic records. The antero-posterior diameters were right eye (RE) 20.21 mm and left eye (LE) 19.9 mm. No other ocular findings were observed. The rest of the general examination was normal. The patient's mother (I-2) was affected with congenital cataract (no morphological description of cataract was obtained) and underwent phacoemulsification surgery with intraocular lens implant in both eyes in childhood. No complications were observed. Ophthalmic registers of patient I-2 were not available. Sib III-4 of the proband was a 17-year-old female. Onset of symptoms occurred at the age of one year with photophobia; a diagnosis of pulverulent congenital cataract was made. The patient presented myopia with the following antero-posterior diameters: RE 25.11 mm
Table 1 The CRYGC gene mutations. Mutation
Amino acid change
Protein domain
Phenotype
Ref
c.13ANC c.123–128insGCGGC c.502CNT c.502CNT c.327CNA c.470GNA c.385GNT c.471GNA C.181GNA
p.T5P p.G41insfsX62 R168W R168W C109X W157X G129C W157X p.R48H
GKM1 GKM2 GKM4 GKM4 GKM3 GKM4 GKM4 GKM4 GKM2
Coppock-like Zonular pulverulent Lamellar Nuclear Nuclear Nuclear + microcornea Nuclear Nuclear + microcornea Zonular and nuclear
[Héon, 1999] [Ren et al., 2000] [Santhiya et al., 2002] [Gonzalez-Huerta et al., 2007] [Yao et al., 2008] [Zhang et al., 2009a, 2009b] [Li et al., 2012] [Guo et al., 2012] [Kumar et al., 2011]
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Fig. 1. Pedigree of the family.
and LE 25.69 mm. Cycloplegic refraction showed: the following values LE: −3.00 spherical equivalent and RE: −2.5 spherical equivalent. After diagnosis the patient underwent phacoemulsification surgery with intraocular lens implant in both eyes. No complications were observed. The diagnosis of pulverulent congenital cataract was described in ophthalmic records. At the time of clinical examination, the patient achieved a visual acuity (VA) of 20/20 in both eyes. No other ocular findings were observed. The rest of the general examination was normal. After carefully examining two additional siblings, we found no ocular affliction. They had VA of 20/20 in both eyes.
3.1. Sequencing analysis DNA analysis of the proband (III-2) and affected members of the family (I-2, II-2, and III-4) showed the c.143GNA missense heterozygous mutation within exon 2 of the CRYGC gene (Fig. 2); this transition mutation leads to a substitution of arginine at position 48 by histidine (p.R48H). Analysis of unaffected members of the family showed a normal sequence of the CRYGA-D gene cluster. No other nucleotide variations or polymorphisms were found in the rest of the analyzed genes. This amino acid (arginine) is found in a region that is conserved in the different CRYGs (A to F) and is invariant through various species
Fig. 2. Electropherogram of a partial sequence of the CRYGC gene. Arrow shows the heterozygous change c.143GNA.
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Fig. 3. Amino acid sequence alignment of CRYGC protein between several species. Conserved amino acid at position 48 is marked in black.
(Fig. 3) (Héon, 1999). The genotype–phenotype association between the presence of congenital cataract and CRYGC gene mutation showed a statistical significance (p b 0.001, Fisher's exact test). 3.2. High resolution melting analysis After detecting the c.143GNA mutation, melting curve analysis of the four affected members with cataract showed heterozygous curves, suggesting the presence of a mutation; this was confirmed through DNA sequencing analysis. Melting curve analysis of the 10 healthy relatives and 230 ethically matched normal healthy controls showed homozygous curves (Fig. 4). Mutation detection sensitivity was 100%, with 4 of 4 mutations being identified in the patients with cataract. 3.3. Modeling on CRYGC protein Structural change predictions of the mutant form of γC-crystallin (p.R48H) with SWISS MODEL revealed that the H48 mutant retains strong bonds with Ser78 y Glu52. H48 also breaks the weak bond with Glu52 losing the link and causing a lateral displacement towards left. Besides, Tyr51 and Cys79 gain a link producing a lateral displacement
Fig. 4. Melting curve analysis of the affected patients and some controls.
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Fig. 5. Protein-structure prediction of mutated human γC-crystallin. The p.R48H mutation (B) and surrounding region are shown (SWISS-MODEL). The R groups involved with putative hydrogen bonding are displayed; dashed green lines denote strong bonds, and dashed white lines denote weak bonds. A) Wild-type γC-crystallin showing Arg48 and the putative hydrogen bond between its R group and the oxygen of the carbonyl and carboxyl group of Gln52. B) Mutated His48 showing the putative loss of hydrogen bonds between its R group and Gln52, the putative loss of hydrogen bonds between Gln52 and Tyr51, the new hydrogen bond between Tyr51 and Cys79 and the new spatial conformation of Arg77, Tyr51 and His48.
of Tyr51 to the left (Fig. 5). Computed analysis (GROMOS96 implementation of Swiss-PdbViewer) of the alignment of CRYGC amino acids with an electron density or electrostatic potential maps showed that Arg48 represented 223.16 kJ/mol, whereas His48 represented 10.79 kJ/mol. In addition, a previous study showed that the hydrophobicity of the mutant CRYGC increased around R48H (Kumar et al., 2011). These data suggest that H48 decreases the surface area of interaction with solvents, thereby hampering the solubility and stability of the mutant form. Thus, H48 could be considered a mutant that causes the phenotype of pulverulent cataract in this family (Fig. 6). 4. Discussion CRYGC protein has a two-domain beta-structure, folded into four very similar Greek key motifs (Blundell et al., 1981). CRYGC belongs to
the β/γ-crystallin family (microbial stress proteins), located as a cluster of six closely related genes (CRYGA–F) on mouse chromosome 1 and on human chromosome 2; in man, the CRYGE and CRYGF genes are pseudogenes. The seventh CRYG gene is mapped on mouse chromosome 16 and human chromosome 3 (Graw, 1997). The γ-crystallin genes encompass three exons on the reverse strand; the first one encodes three amino acids and the second and third each encodes two Greek key motifs. CRYGC has a molecular mass of 21 kDa with a length of 173 amino acids; it comprises about 40% of the total proteins in the mouse lens and 25% in the human lens (Graw, 1997; Wistow and Piatigorsky, 1988). A previous study showed a duplication of 5 bp in exon 2 of the CRYGC gene mutation causing pulverulent cataract; this mutation was present in the first Greek key domain (Ren et al., 2000). In addition, seven different mutations with various phenotypes have been associated with
Fig. 6. A) Arg48 (green) shows the electrostatic charge (blue) indicated by white arrow. B) His48 (red) has no electrostatic charge. Upper pictures show changes in the electrostatic charge of R48 vs H48.
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autosomal dominant cataract in the CRYGC gene, denoting the clinical and molecular heterogeneity. In our family, the heterozygous missense mutation c.143GNA in exon 2 of the CRYGC gene, is associated with autosomal dominant pulverulent cataract. This transition changes arginine (R), a basic, large amino acid, by histidine (H), a medium, less polar amino acid with an imidazole ring. The p.R48H change is located in loop 3 within the second Greek key motif of the CRYGC protein (http://www.uniprot.org/uniprot/P07315). Nucleotide change c.143GNA (p.R48H) was found to be nonpathogenic on software analysis (PANTHER and SIFT software). Nevertheless, a previous study associated this type of mutation with primary congenital cataract and the fact that this change is on a highly conserved domain strongly supports the idea that this change may affect protein function. Moreover, the nucleotide change was not detected in the unaffected members of the family or in the 230 ethnically matched controls. Prediction of structural changes using the SWISS MODEL revealed that mutated p.R48H showed a putative loss of hydrogen bonds between the R group and Gln52, a putative loss of hydrogen bonds between Gln52 and Tyr51, a new hydrogen bond between Tyr51 and Cys79 and a new spatial conformation of Arg77, Tyr51 and His48 (Fig. 4). The p.R48H mutation presented an increase of hydrophobicity around His48; it is likely, the case His48 substitution decreases the interaction of the surface area with solvents, thereby hampering the solubility and stability of the p.R48H mutant (Kumar et al., 2011). On the CRYGC gene, a change of arginine was involved in p.R168W and showed two phenotypes, a lamellar cataract in an Indian family (Santhiya et al., 2002) and a nuclear cataract in a Mexican family (Gonzalez-Huerta et al., 2007). In both cases, the pathogenesis was considered to be due to reduction in solubility of the protein. Arginine has also been implicated in five missense mutations within the CRYGD gene: p.R15C (Gu et al., 2006; Stephan, 2006), p.R15S (Zhang et al., 2009a,b), p.R37S (Gu et al., 2005; Kmoch et al., 2000), p.R58H (Héon, 1999), p.R77S (Roshan et al., 2010) and a mutation truncation (p.R140X) (Devi et al., 2008). However, none of these cases had pulverulent cataract. Among all mutations involving arginine, p.R58H in CRYGD has an aculeiforme phenotype (Héon, 1999). In this study the mutation induced a change in the ionic charges and introduced hydrogen bonds in neighboring amino acids altering the properties of protein folding, rigidity and stability (Basak, 2003). In a previous study that reported on the p.R48H mutation in the CRYGC gene, the authors found a similar non-pathogenic effect of this mutation after analysis with PANTHER and SIFT software. Nevertheless, using the MODELER 9.2 program available in Discovery Studio (DS) 2.0, the authors detected that this mutation alters the characteristics of this residue in terms of both its nature and length; this is reflected in the difference in its interactions with neighboring amino acid residues. The mutant, results in the modification of the relative orientation of the loop 3 comprising residues 47 to 54 (Kumar et al., 2011). The authors also proposed that the deleterious effect of p.R48H mutation could be a consequence of anomalies in the interaction between γC-crystallin and other crystallins. The pulverulent nuclear cataract was considered the same as Coppock-like cataract (Lubsen et al., 1987), which is phenotypically similar to the cataract in some members of the family described by Scott et al. (1994). In these individuals, with a severe cataract in one eye and no detectable cataract in the other one, the authors suggested a random variation in the phenotype, since the effects of modifying genes and environment should be similar for both eyes. However, whereas it remains a formal possibility, this hypothesis is difficult to test. In conclusion, we described an autosomal dominant primary congenital cataract, recognizable by several phenotypes, associated with a p.R48H CRYGC gene mutation. This mutation not only has been linked to the phenotype of congenital cataract but also is considered a singlenucleotide polymorphism (SNP) in the NCBI data base. Our data and previous report suggest that p.R48H is probably a disease-causing mutation. The possible influence of this mutation on the structure and the function of γC-crystallin requires further investigation.
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Conflict of interest None. References Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006, 195–201. Basak, A., 2003. High-resolution X-ray crystal structures of human gammaD crystallin (1.25 A) and the R58H mutant (1.15 A) associated with aculeiform cataract. J. Mol. Biol. 328, 1137–1147. Blundell, T., et al., 1981. The molecular structure and stability of the eye lens: x-ray analysis of gamma-crystallin II. Nature 289, 771–777. Devi, R.R., Yao, W., Vijayalakshmi, P., Sergeev, Y.V., Sundaresan, P., Hejtmancik, J.F., 2008. Crystallin gene mutations in Indian families with inherited pediatric cataract. Mol. Vis. 14, 1157–1170. Francois, J., 1982. Genetics of cataract. Ophthalmologica 184, 61–71. Gilbert, C.E., Canovas, R., Hagan, M., Rao, S., Foster, A., 1993. Causes of childhood blindness: results from West Africa, South India and Chile. Eye 7, 184–188. Gonzalez-Huerta, L.M., Messina-Baas, O.M., Cuevas-Covarrubias, S.A., 2007. A family with autosomal dominant primary congenital cataract associated with a CRYGC mutation: evidence of clinical heterogeneity. Mol. Vis. 13, 1333–1338. Graw, J., 1997. The crystallins: genes, proteins and diseases. Biol. Chem. 378, 1331–1348. Gu, J., et al., 2005. A new congenital nuclear cataract caused by a missense mutation in the gammD-crystallin gene (CRYGD) in a Chinese family. Mol. Vis. 11, 971–976. Gu, F., Li, R., Ma, X.X., Shi, L.S., Huang, S.Z., Ma, X., 2006. A missense mutation in the gammaD-crystallin gene CRYGD associated with autosomal dominant congenital cataract in a Chinese family. Mol. Vis. 12, 26–31. Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723. Guo, Y., et al., 2012. A nonsense mutation of CRYGC associated with autosomal dominant congenital nuclear cataracts and microcornea in a Chinese pedigree. Mol. Vis. 18, 1874–1880. Hejtmancik, J.F., 2008. Congenital cataracts and their molecular genetics. Semin. Cell Dev. Biol. 19, 134–149. Héon, E., 1999. The gamma-crystallins and human cataracts: a puzzle made clearer. Am. J. Hum. Genet. 65, 1261–1267. Ionides, A., et al., 1999. Clinical and genetic heterogeneity in autosomal dominant cataract. Br. J. Ophthalmol. 83, 802–808. Kmoch, S., et al., 2000. Link between phenotype explained by protein crystallography. Hum. Mol. Genet. 9, 1779–1786. Kumar, M., et al., 2011. Mutation screening and genotype phenotype correlation of αcrystallin, γ-crystallin and GJA8 gene in congenital cataract. Mol. Vis. 11, 693–707. Lambert, S.R., Drack, A.V., 1996. Infantile cataracts. Surv. Ophthalmol. 40, 427–458. Li, X.Q., et al., 2012. A novel mutation impairing the tertiary structure and stability of γCcrystallin (CRYGC) leads to cataract formation in humans and zebrafish lens. Hum. Mutat. 33, 391–401. Liew, M., et al., 2004. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin. Chem. 50, 1156–1164. Lubsen, N.H., Renwick, J.H., Tsui, L.C., Breitman, M.L., Schoenmakers, J.G., 1987. A locus for a human hereditary cataract is closely linked to the gamma-crystallin gene family. Proc. Natl. Acad. Sci. U. S. A. 84, 489–492. Mackay, D.S., Andley, U.P., Shiels, A., 2004. A missense mutation in the gammaD crystallin gene (CRYGD) associated with autosomal dominant “coral-like” cataract linked to chromosome 2q. Mol. Vis. 10, 155–162. Merin, S., Crawford, J.S., 1971. The etiology of congenital cataracts. A survey of 386 cases. Can. J. Ophthalmol. 6, 178–182. Reddy, M.A., Francis, P.J., Berry, V., Bhattacharya, S.S., Moore, A.T., 2004. Molecular genetic basis of inherited cataract and associated phenotypes. Surv. Ophthalmol. 49, 300–315. Ren, Z., et al., 2000. A 5-base insertion in the gammaC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract. Hum. Genet. 106, 531–537. Roshan, M., et al., 2010. A novel human CRYGD mutation in a juvenile autosomal dominant cataract. Mol. Vis. 16, 887–896. Santhiya, S.T., et al., 2002. Novel mutations in the gamma-crystallin genes cause autosomal dominant congenital cataracts. J. Med. Genet. 39, 352–358. Schwede, T., Kopp, J., Guex, N., Peitsch, M.C., 2003. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385. Scott, M.H., Hejtmancik, J.F., Wozencraft, L.A., Reuter, L.M., Parks, M.M., Kaiser-Kupfer, M.I., 1994. Autosomal dominant congenital cataract: interocular phenotypic heterogeneity. Ophthalmology 101, 866–871. Stephan, D.A., 2006. Progressive juvenile-onset punctuate cataracts caused by mutation of the gD-crystallin gene. Proc. Natl. Acad. Sci. U. S. A. 96, 1008–1012. Wang, K.J., Li, S.S., Yun, B., Ma, W.X., Jiang, T.G., Zhu, S.Q., 2011. A novel mutation in MIP associated with congenital nuclear cataract in a Chinese family. Mol. Vis. 17, 70–77. Wistow, G.J., Piatigorsky, J., 1988. Lens crystallins: the evolution and expression of proteins for a highly specialized tissue. Annu. Rev. Biochem. 57, 479–504. Yao, K., et al., 2008. A nonsense mutation in CRYGC associated with autosomal dominant congenital nuclear cataract in a Chinese family. Mol. Vis. 14, 1272–1276. Zhang, L., Fu, S., Ou, Y., Zhao, T., Su, Y., Liu, P., 2009a. A novel nonsense mutation in CRYGC is associated with autosomal dominant congenital nuclear cataracts and microcornea. Mol. Vis. 2009 (15), 276–282. Zhang, L.Y., et al., 2009b. 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Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
A CRYGC gene mutation associated with autosomal dominant pulverulent cataract Luz Ma. González-Huerta a, Olga Messina-Baas b, Héctor Urueta a, Jaime Toral-López c, Sergio A. Cuevas-Covarrubias a,⁎ a b c
Departamento de Genética, Hospital General de México, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F. México, Mexico Departamento de Oftalmología, Hospital General de México, D.F. México, Mexico Departamento de Genética, Centro Médico Ecatepec, ISSEMYM. Edo. México, Mexico
a r t i c l e
i n f o
Article history: Accepted 8 July 2013 Available online 14 August 2013 Keywords: Pulverulent congenital cataract CRYGC gene Gamma-crystallins Clinical heterogeneity
a b s t r a c t Purpose: To describe at molecular level a family with pulverulent congenital cataract associated with a CRYGC gene mutation. Methods: One family with several affected members with pulverulent congenital cataract and 230 healthy controls were examined. Genomic DNA from leukocytes was isolated to analyze the CRYGA-D cluster, CX46, CX50 and MIP genes through high-resolution melting curve and DNA sequencing. Results: DNA sequencing in the affected members revealed the c.143GNA mutation (p.R48H) in exon 2 of the CRYGC gene; 230 healthy controls and ten healthy relatives were also analyzed and none of them showed the c.143GNA mutation. No other polymorphisms or mutations were found to be present. Conclusion: In the present study, we described a family with pulverulent congenital cataract that segregated the c.143GNA mutation (p.R48H) in the CRYGC gene. A few mutations have been described in the CRYGC gene in autosomal dominant cataract, none of them with pulverulent cataract making clear the clinical heterogeneity of congenital cataract. This mutation has been associated with the phenotype of congenital cataract but also is considered an SNP in the NCBI data base. Our data and previous report suggest that p.R48H could be a diseasecausing mutation and not an SNP. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Crystallins are important structural components of the vertebrate eye lens and represent more than 90% of the water-soluble total lens proteins; they play critical roles in maintaining the transparency and refraction function of the lens (Mackay et al, 2004; Wistow and Piatigorsky, 1988). The lens is responsible for the variable refractive power of focusing and one-third of the stationary refractive power. Disruption of the crystallin structure results in the formation of congenital cataract (Ionides et al., 1999; Lambert and Drack, 1996). Congenital cataract represents 10% of the cases of childhood blindness (Gilbert
Abbreviations: CRYAA, αA-crystallin (CRYAA); CRYBA1, βA1-crystallin (CRYBA1); CRYBB1, βB1-crystallin (CRYBB1); CRYBB2, βB2-crystallin (CRYBB2); CRYGC, γC-crystallin (CRYGC); CRYGD, γD-crystallin (CRYGD); CX46, connexin 46 (CX46); CX50, connexin 50 (CX50); MIP, major intrinsic protein (MIP); DNA, deoxyribonucleic acid; G, guanine; A, adenine; PCR, polymerase chain reaction; RE, right eye; LE, left eye; R, arginine; H, histidine; Glu, glutamic acid; Tyr, tyrosine; Cys, cysteine; Gln, glutamine; Arg, arginine; His, histidine; W, tryptophan; C, cysteine; T, threonine; P, proline; G, glycine. ⁎ Corresponding author at: Servicio de Genética, Hospital General de México, Dr. Balmis 148 Col. Doctores C.P. 06726 México D.F., Mexico. Tel./fax: +52 55 53412821. E-mail addresses: [email protected], [email protected] (S.A. Cuevas-Covarrubias). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.07.044
et al., 1993; Ionides et al., 1999; Reddy et al., 2004). This disorder is visible within the first year of life; nevertheless, the age of onset is not necessarily related to the cause of cataract (Francois, 1982; Merin and Crawford, 1971). Cataract is the most common treatable cause of visual disability in infancy or early childhood. 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. About 40% of congenital cataract cases is inherited either in isolation or as part of an ocular syndrome or systemic abnormalities; cataracts that are the result of genetic factors must be distinguished from those that occur as a consequence of systemic diseases (Ionides et al., 1999; Lambert and Drack, 1996). Most familial cases of congenital cataracts show an autosomal dominant pattern. Cataracts are clinically and genetically heterogeneous, similar phenotypes map to different loci and different phenotypes map to the same locus. More than 40 loci have been mapped in primary congenital cataracts (Gilbert et al., 1993; Ionides et al., 1999; Reddy et al., 2004). Candidate genes for hereditary cataracts are highly expressed in the lens and include αA-crystallin (CRYAA), βA1-crystallin (CRYBA1), βB1crystallin (CRYBB1), βB2-crystallin (CRYBB2), γC-crystallin (CRYGC), γD-crystallin (CRYGD), connexin 46 (CX46), connexin 50 (CX50), and major intrinsic protein (MIP) (Blundell et al., 1981; Graw, 1997; Hejtmancik, 2008; Reddy et al., 2004; Wang et al., 2011). Mutations in
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the CRYG gene cluster, located on 2q33–35, are among the most frequent cause of autosomal dominant congenital cataract. A high clinical spectrum in congenital cataract has been observed in patients with CRYG gene mutations. There have been a few cases of congenital cataract due to mutations in the CRYGC gene (NM_020989). To our knowledge, there have been nine cases of congenital cataracts due to seven different mutations associated with the CRYGC gene (Gonzalez-Huerta et al., 2007; Guo et al., 2012; Héon, 1999; Kumar et al., 2011; Li et al., 2012; Ren et al., 2000; Santhiya et al., 2002; Yao et al., 2008; Zhang et al., 2009a,b) (Table 1). In the present study, we describe a family with autosomal dominant pulverulent cataract and the missense mutation c.143GNA (p.R48H) in the exon 2 of CRYGC gene. 2. Material and methods Considering the genes that are highly associated with primary congenital cataract, mutation screening was performed in nine candidate genes, specifically CRYAA (GenBank NM_000394.2), CRYBA1 (GenBank NM_005208.4), CRYBB1 (GenBank NM_001887.3), CRYBB2 (GenBank NM_000496.2), CRYGC (GenBank NM_020989.3), CRYGD (GenBank NM_006891.3), CX46 (GenBank NM_021954.3), CX50 (GenBank NM_005267.4), and MIP (GenBank NM_012064.3). All coding exons and splice sites were amplified by polymerase chain reactions (PCR) with the primers previously described (Wang et al., 2011). PCR products obtained from the proband and affected/unaffected members of the family were sequenced on an ABI 3730 Automated Sequencer (PE Biosystems, Foster City, CA). Two hundred and thirty ethnically matched controls were analyzed to confirm the mutation identified in the CRYGC gene. The family was referred to the General Hospital of Mexico due to primary congenital cataract. The protocol was approved by the Ethics Committee of the General Hospital of Mexico. Patients gave informed consent to participate in the study. We analyzed a four-generation Mexican family, segregating autosomal dominant cataract with no systemic anomalies. The family included four affected and 10 unaffected members. There was no history of consanguinity. Photographs of the lens opacities of affected members of the family were not available. Ophthalmic records showed that the onset of cataract occurred in infancy; in all cases, cataract was described only as pulverulent congenital cataract. 2.1. High resolution melting analysis After detecting the CRYGC gene mutation, high-resolution melting analysis was performed in all affected members with cataract, 10 healthy relatives and 230 ethnically matched controls. Oligonucleotide primers were obtained commercially. PCR was performed in 10 μl vol with a 1× LightCycler FastStart DNA Master HybProbe (Roche Applied Systems), with 0.5 μM of each CRYGC primers, 0.06 μM of lowtemperature correction control and 0.08 μM of high-temperature correction control, 3.5 mM of MgCl2 (including 1 mM MgCl2 contributed
by the LightCycler FastStart DNA Master HybProbe solution), 0.01 of U/reaction heat-labile uracil-DNA glycosylase (Roche Applied Systems), 1× LCGreen Plus (Idaho Technology), and 20 ng of template DNA. All oligonucleotides were mixed together and stored as a 20× stock solution. PCR and high-resolution melting were done on the LS-32 (Idaho Technology). PCR included an initial hold of 95 °C for 10 min, followed by 15 cycles of 95 °C for 2 s, 56 °C for 1 s, and 72 °C for 1 s, and 25 cycles of 95 °C for 2 s, 58 °C for 1 s, and 72 °C for 4 s. During amplification, no fluorescence acquisition was performed to avoid prolonging the temperature cycles. All heating and cooling steps during PCR were carried out with ramp rates programmed at 20 °C/s. After PCR was performed, samples were cooled (10 °C/s) from 95 °C to 40 °C and melting curves were generated with continuous fluorescence acquisition from 55 °C to 95 °C at 0.3 °C/s. Data processing included normalization of fluorescence, exponential background removal, and display of derivative melting curves. The melting curves were adjusted by identifying the maxima of the temperature correction control peaks and aligning curves through shifting and linear-scaling using custom software. Heterozygotes were identified by melting-peak width and shape. Homozygous and heterozygous genotypes were assigned by visual inspection based on Tm (melting-peak maxima). Predicted Tms based on nearestneighbor parameters were calculated as described previously (Liew et al., 2004). 2.2. CRYGC three-dimensional protein prediction Models were viewed in a Swiss-Pdb Viewer, versión 4.0, and RasMol version 2.7.4. Homology modeling was done by SWISS-MODEL (Arnold et al., 2006; Guex and Peitsch, 1997; Schwede et al., 2003). 3. Results The proband (III-2), a 20-year-old Mexican female, was the product of an apparently uncomplicated term pregnancy with normal spontaneous vaginal delivery. The pedigree is shown in Fig. 1. Onset of ocular symptoms occurred at the age of one year. The patient underwent phacoemulsification surgery with intraocular lens implant in both eyes after diagnosis. No complications were observed. The diagnosis of pulverulent congenital cataract was obtained from ophthalmic records. The antero-posterior diameters were right eye (RE) 20.21 mm and left eye (LE) 19.9 mm. No other ocular findings were observed. The rest of the general examination was normal. The patient's mother (I-2) was affected with congenital cataract (no morphological description of cataract was obtained) and underwent phacoemulsification surgery with intraocular lens implant in both eyes in childhood. No complications were observed. Ophthalmic registers of patient I-2 were not available. Sib III-4 of the proband was a 17-year-old female. Onset of symptoms occurred at the age of one year with photophobia; a diagnosis of pulverulent congenital cataract was made. The patient presented myopia with the following antero-posterior diameters: RE 25.11 mm
Table 1 The CRYGC gene mutations. Mutation
Amino acid change
Protein domain
Phenotype
Ref
c.13ANC c.123–128insGCGGC c.502CNT c.502CNT c.327CNA c.470GNA c.385GNT c.471GNA C.181GNA
p.T5P p.G41insfsX62 R168W R168W C109X W157X G129C W157X p.R48H
GKM1 GKM2 GKM4 GKM4 GKM3 GKM4 GKM4 GKM4 GKM2
Coppock-like Zonular pulverulent Lamellar Nuclear Nuclear Nuclear + microcornea Nuclear Nuclear + microcornea Zonular and nuclear
[Héon, 1999] [Ren et al., 2000] [Santhiya et al., 2002] [Gonzalez-Huerta et al., 2007] [Yao et al., 2008] [Zhang et al., 2009a, 2009b] [Li et al., 2012] [Guo et al., 2012] [Kumar et al., 2011]
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Fig. 1. Pedigree of the family.
and LE 25.69 mm. Cycloplegic refraction showed: the following values LE: −3.00 spherical equivalent and RE: −2.5 spherical equivalent. After diagnosis the patient underwent phacoemulsification surgery with intraocular lens implant in both eyes. No complications were observed. The diagnosis of pulverulent congenital cataract was described in ophthalmic records. At the time of clinical examination, the patient achieved a visual acuity (VA) of 20/20 in both eyes. No other ocular findings were observed. The rest of the general examination was normal. After carefully examining two additional siblings, we found no ocular affliction. They had VA of 20/20 in both eyes.
3.1. Sequencing analysis DNA analysis of the proband (III-2) and affected members of the family (I-2, II-2, and III-4) showed the c.143GNA missense heterozygous mutation within exon 2 of the CRYGC gene (Fig. 2); this transition mutation leads to a substitution of arginine at position 48 by histidine (p.R48H). Analysis of unaffected members of the family showed a normal sequence of the CRYGA-D gene cluster. No other nucleotide variations or polymorphisms were found in the rest of the analyzed genes. This amino acid (arginine) is found in a region that is conserved in the different CRYGs (A to F) and is invariant through various species
Fig. 2. Electropherogram of a partial sequence of the CRYGC gene. Arrow shows the heterozygous change c.143GNA.
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Fig. 3. Amino acid sequence alignment of CRYGC protein between several species. Conserved amino acid at position 48 is marked in black.
(Fig. 3) (Héon, 1999). The genotype–phenotype association between the presence of congenital cataract and CRYGC gene mutation showed a statistical significance (p b 0.001, Fisher's exact test). 3.2. High resolution melting analysis After detecting the c.143GNA mutation, melting curve analysis of the four affected members with cataract showed heterozygous curves, suggesting the presence of a mutation; this was confirmed through DNA sequencing analysis. Melting curve analysis of the 10 healthy relatives and 230 ethically matched normal healthy controls showed homozygous curves (Fig. 4). Mutation detection sensitivity was 100%, with 4 of 4 mutations being identified in the patients with cataract. 3.3. Modeling on CRYGC protein Structural change predictions of the mutant form of γC-crystallin (p.R48H) with SWISS MODEL revealed that the H48 mutant retains strong bonds with Ser78 y Glu52. H48 also breaks the weak bond with Glu52 losing the link and causing a lateral displacement towards left. Besides, Tyr51 and Cys79 gain a link producing a lateral displacement
Fig. 4. Melting curve analysis of the affected patients and some controls.
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Fig. 5. Protein-structure prediction of mutated human γC-crystallin. The p.R48H mutation (B) and surrounding region are shown (SWISS-MODEL). The R groups involved with putative hydrogen bonding are displayed; dashed green lines denote strong bonds, and dashed white lines denote weak bonds. A) Wild-type γC-crystallin showing Arg48 and the putative hydrogen bond between its R group and the oxygen of the carbonyl and carboxyl group of Gln52. B) Mutated His48 showing the putative loss of hydrogen bonds between its R group and Gln52, the putative loss of hydrogen bonds between Gln52 and Tyr51, the new hydrogen bond between Tyr51 and Cys79 and the new spatial conformation of Arg77, Tyr51 and His48.
of Tyr51 to the left (Fig. 5). Computed analysis (GROMOS96 implementation of Swiss-PdbViewer) of the alignment of CRYGC amino acids with an electron density or electrostatic potential maps showed that Arg48 represented 223.16 kJ/mol, whereas His48 represented 10.79 kJ/mol. In addition, a previous study showed that the hydrophobicity of the mutant CRYGC increased around R48H (Kumar et al., 2011). These data suggest that H48 decreases the surface area of interaction with solvents, thereby hampering the solubility and stability of the mutant form. Thus, H48 could be considered a mutant that causes the phenotype of pulverulent cataract in this family (Fig. 6). 4. Discussion CRYGC protein has a two-domain beta-structure, folded into four very similar Greek key motifs (Blundell et al., 1981). CRYGC belongs to
the β/γ-crystallin family (microbial stress proteins), located as a cluster of six closely related genes (CRYGA–F) on mouse chromosome 1 and on human chromosome 2; in man, the CRYGE and CRYGF genes are pseudogenes. The seventh CRYG gene is mapped on mouse chromosome 16 and human chromosome 3 (Graw, 1997). The γ-crystallin genes encompass three exons on the reverse strand; the first one encodes three amino acids and the second and third each encodes two Greek key motifs. CRYGC has a molecular mass of 21 kDa with a length of 173 amino acids; it comprises about 40% of the total proteins in the mouse lens and 25% in the human lens (Graw, 1997; Wistow and Piatigorsky, 1988). A previous study showed a duplication of 5 bp in exon 2 of the CRYGC gene mutation causing pulverulent cataract; this mutation was present in the first Greek key domain (Ren et al., 2000). In addition, seven different mutations with various phenotypes have been associated with
Fig. 6. A) Arg48 (green) shows the electrostatic charge (blue) indicated by white arrow. B) His48 (red) has no electrostatic charge. Upper pictures show changes in the electrostatic charge of R48 vs H48.
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autosomal dominant cataract in the CRYGC gene, denoting the clinical and molecular heterogeneity. In our family, the heterozygous missense mutation c.143GNA in exon 2 of the CRYGC gene, is associated with autosomal dominant pulverulent cataract. This transition changes arginine (R), a basic, large amino acid, by histidine (H), a medium, less polar amino acid with an imidazole ring. The p.R48H change is located in loop 3 within the second Greek key motif of the CRYGC protein (http://www.uniprot.org/uniprot/P07315). Nucleotide change c.143GNA (p.R48H) was found to be nonpathogenic on software analysis (PANTHER and SIFT software). Nevertheless, a previous study associated this type of mutation with primary congenital cataract and the fact that this change is on a highly conserved domain strongly supports the idea that this change may affect protein function. Moreover, the nucleotide change was not detected in the unaffected members of the family or in the 230 ethnically matched controls. Prediction of structural changes using the SWISS MODEL revealed that mutated p.R48H showed a putative loss of hydrogen bonds between the R group and Gln52, a putative loss of hydrogen bonds between Gln52 and Tyr51, a new hydrogen bond between Tyr51 and Cys79 and a new spatial conformation of Arg77, Tyr51 and His48 (Fig. 4). The p.R48H mutation presented an increase of hydrophobicity around His48; it is likely, the case His48 substitution decreases the interaction of the surface area with solvents, thereby hampering the solubility and stability of the p.R48H mutant (Kumar et al., 2011). On the CRYGC gene, a change of arginine was involved in p.R168W and showed two phenotypes, a lamellar cataract in an Indian family (Santhiya et al., 2002) and a nuclear cataract in a Mexican family (Gonzalez-Huerta et al., 2007). In both cases, the pathogenesis was considered to be due to reduction in solubility of the protein. Arginine has also been implicated in five missense mutations within the CRYGD gene: p.R15C (Gu et al., 2006; Stephan, 2006), p.R15S (Zhang et al., 2009a,b), p.R37S (Gu et al., 2005; Kmoch et al., 2000), p.R58H (Héon, 1999), p.R77S (Roshan et al., 2010) and a mutation truncation (p.R140X) (Devi et al., 2008). However, none of these cases had pulverulent cataract. Among all mutations involving arginine, p.R58H in CRYGD has an aculeiforme phenotype (Héon, 1999). In this study the mutation induced a change in the ionic charges and introduced hydrogen bonds in neighboring amino acids altering the properties of protein folding, rigidity and stability (Basak, 2003). In a previous study that reported on the p.R48H mutation in the CRYGC gene, the authors found a similar non-pathogenic effect of this mutation after analysis with PANTHER and SIFT software. Nevertheless, using the MODELER 9.2 program available in Discovery Studio (DS) 2.0, the authors detected that this mutation alters the characteristics of this residue in terms of both its nature and length; this is reflected in the difference in its interactions with neighboring amino acid residues. The mutant, results in the modification of the relative orientation of the loop 3 comprising residues 47 to 54 (Kumar et al., 2011). The authors also proposed that the deleterious effect of p.R48H mutation could be a consequence of anomalies in the interaction between γC-crystallin and other crystallins. The pulverulent nuclear cataract was considered the same as Coppock-like cataract (Lubsen et al., 1987), which is phenotypically similar to the cataract in some members of the family described by Scott et al. (1994). In these individuals, with a severe cataract in one eye and no detectable cataract in the other one, the authors suggested a random variation in the phenotype, since the effects of modifying genes and environment should be similar for both eyes. However, whereas it remains a formal possibility, this hypothesis is difficult to test. In conclusion, we described an autosomal dominant primary congenital cataract, recognizable by several phenotypes, associated with a p.R48H CRYGC gene mutation. This mutation not only has been linked to the phenotype of congenital cataract but also is considered a singlenucleotide polymorphism (SNP) in the NCBI data base. Our data and previous report suggest that p.R48H is probably a disease-causing mutation. The possible influence of this mutation on the structure and the function of γC-crystallin requires further investigation.
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