Mutation analysis of CRYBB2 in family A. A, Pedigree of family A. B, DNA sequence chromatograms show that a single transition is observed at position 436 (G>A) as a G/A double peak (arrow). F indicates forward strand. C, A multiple sequence alignment of the amino acid sequence of βB2-crystallin with different species. The R146 residue is indicated by an arrow. D, The secondary structure prediction shows that the mutation leads to the replacement of an original β-strand by an α-helix. The target sequences are indicated by red circles. Blue represents helix; yellow, strand; and green, turn.
Mutation analysis of CRYBB2 in family B. A, Pedigree of family B. B, Slitlamp photograph of individual III-2 shows the nuclear autosomal dominant congential cataract. C, DNA sequence chromatograms show a heterozygous T > A transversion at position 62 that replaces Ile by Asn (arrow). F indicates forward strand. D, A multiple sequence alignment of the amino acid sequence of βB2-crystallin with different species. The I21 residue is indicated by an arrow. E, The secondary structure prediction shows that the mutation leads to the replacement of an original β-strand by an α-helix. The target sequences are indicated by red circles. Violet represents helix; orange, strand; and green, turn.
Mutation analysis of CRYBB1 in family C. A, Pedigree of family C. B, Slitlamp photograph of individual II-1 shows the nuclear cataract. C, DNA sequence chromatograms show a single transition is observed at position 698 (G>A) as a G/A double peak (arrow). F indicates forward strand. D, A multiple sequence alignment of the amino acid sequence of βB1-crystallin with different species. The R233 residue is indicated by an arrow.
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Wang KJ, Wang BB, Zhang F, Zhao Y, Ma X, Zhu SQ. Novel β-Crystallin Gene Mutations in Chinese Families With Nuclear Cataracts. Arch Ophthalmol. 2011;129(3):337–343. doi:10.1001/archophthalmol.2011.11
Janey L.WiggsMD, PhD
To investigate the molecular genetic background in families with nuclear congenital cataract.
Family history and clinical data were recorded. Ten candidate genes were screened for causative mutations. Direct sequencing was performed to analyze the cosegregation of the genotype with the disease phenotype. Effects of amino acid changes on the structure and function of protein were predicted by bioinformatics analysis.
Analyses of 20 Chinese families with hereditary nuclear congenital cataract revealed 3 novel mutations. Two of these mutations (V146M and I21N) affected βB2-crystallin (CRYBB2). One mutation (R233H) was detected in βB1-crystallin (CRYBB1). These mutations cosegregated with all affected individuals and were not observed in unaffected family members or the 150 healthy unrelated individuals.
The CRYBB2 gene was shown to be another causative gene associated with congenital cataract and microcornea. Three novel mutations in β-crystallin genes (CRYB) were detected in Chinese families with nuclear autosomal dominant congenital cataracts, which underscores the genetic heterogeneity of this condition.
Studying the genetics of nuclear cataracts is helpful for better understanding the pathophysiologic mechanisms that underlie this phenotype and for better disease management. This study helps expand the genotype of nuclear cataract and microcornea.
Congenital cataract is a leading cause of visual disability in children. Globally, its prevalence is 0.6 to 6.0 in 10 000 live births, causing approximately 10% of childhood blindness worldwide.1 Inherited isolated (nonsyndromic) cataracts represent one-third of cases, and recently many causative genetic mutations have been identified. To date, more than 30 loci and 18 genes on different chromosomes have been associated with autosomal dominant congenital cataract (ADCC).2 Of these mutations, approximately half involve crystallins, one-quarter involve connexins, and the remaining one-quarter involve the other genes.3
Phenotypes are described mainly based on the physical appearance and site of occurrence of the opacity. Phenotypic variabilities among and within families have been documented, including nuclear, anterior polar, posterior polar, coralliform, cerulean (blue-dot), pulverulent, cortical, zonular, and sutural cataracts.1 Of the already characterized phenotypes, nuclear congenital cataracts are the most common type of hereditary congenital cataract.4 Despite attempts to categorize hereditary cataracts clinically, correlation of phenotypes with genetic locus and specific mutation is limited. The clinical and genetic heterogeneity of congenital cataracts is well substantiated. Conversely, congenital cataracts with similar or identical clinical phenotypes can result from mutations in completely different genes. Several genes have been associated with nuclear cataract to date (CRYAA, CRYBB1, CRYBB2, CRYBA3, CRYGC, CRYGD, GJA8, and GJA3).3 Therefore, it is appropriate to consider these genes as the top list of functional candidates in hereditary congenital cataracts. In this study, we screened 20 Chinese families with nuclear ADCC for mutations in 8 crystallin and 2 connexin genes.
This study adhered to the tenets of the Declaration of Helsinki and was approved by the ethics committee for medical research at Capital Medical University. Twenty families affected by nuclear ADCC were recruited at the Beijing Tongren Eye Center, Beijing Tongren Hospital (Capital Medical University). Affected and unaffected individuals underwent detailed ophthalmic examinations, including visual acuity and corrected visual acuity in addition to slitlamp and fundus examinations, corneal diameter measurement, ultrasonography, and intraocular pressure measurement using applanation tonometry. There was no evidence of systemic abnormalities associated with congenital cataract in the probands. Control subjects who matched the ethnic background of the probands were also recruited. They were given the same complete ophthalmologic examinations as the study individuals of the families with ADCC cataract and did not have eye diseases except mild myopia and age-related cataracts. Blood samples were obtained from the probands and their available family members after providing informed consent.
Polymerase chain reaction was used to amplify all the exons and intron/exon boundaries of the candidate genes: CRYAA (GenBank NM_000394), CRYAB (GenBank NM_001885), CRYBA1 (GenBank NM_005208), CRYBB1 (GenBank NM_001887), CRYBB2 (GenBank NM_000496), CRYGC (GenBank NM_020989), CRYGD (GenBank NM_006891), CRYGS (GenBank NM_017541), GJA3 (GenBank NM_021954), and GJA8 (GenBank NM_005267). Polymerase chain reaction was performed on genomic DNA samples using the primer pairs listed in the Table. Polymerase chain reaction products were sequenced using an automated sequencer (model ABI 3730, Automated Sequencer; PE Biosystems, Foster City, California). Direct sequencing was also performed to analyze the cosegregation of the genotype with the disease phenotype.
Computational methods were used to determine whether a specific amino acid substitution in a protein sequence might lead to altered protein function and possibly contribute to the disease. The possible functional impact of an amino acid change was predicted by using the PolyPhen (Polymorphism Phenotyping) program (http://genetics.bwh.harvard.edu/pph/). The prediction is based on the position-specific independent counts score derived from multiple sequence alignments of observations. PolyPhen scores greater than 2.0 indicate that the polymorphism is probably damaging to protein function, scores of 1.5 to 2.0 are possibly damaging, and scores less than 1.5 are likely benign. The secondary structure of mutant and wild-type amino acid sequences were analyzed using Antheprot 2000 version 6.0 software (IBCP, Lyon, France).
Twenty families with nuclear ADCC were identified. Cataract phenotypes showed some variability among families, but all involved the embryonal or fetal nucleus to a variable extent. Nystagmus was present in some families and absent in others, depending primarily on the degree of visual impairment during the first months of life. Three mutations were observed in 3 families (15%): 2 resided in CRYBB2 and 1 in CRYBB1.
Four members (2 affected and 2 unaffected) participated in the study (Figure 1A). The proband was a 3-year-old boy who had had cataract extraction performed at age 6 months. He was recorded as having nuclear lens opacification and microcornea. The corneal diameter was 9 mm in both eyes, and the axial length was 23.82 mm in the right eye and 23.74 mm in the left eye. The 2 affected individuals had similar poor visual acuity measured using the decimal system of 0.02 to 0.05, and they also had nystagmus and amblyopia. There was no evidence of other ocular or systemic abnormalities.
Mutation analysis revealed a c.436G>A transversion in exon 5 of CRYBB2 that led to the replacement of valine at position 146 by methionine (V146M) (Figure 1B). The mutation cosegregated with the disease in the family (II-1 and III-1) and was not observed in unaffected family members or in control individuals of Chinese descent.
The Val at position 146 of human βB2-crystallin was located in a phylogenetically conserved region by multiple sequence alignment (Figure 1C). The PolyPhen score from PolyPhen analysis was 2.046, which meant that this V146M CRYBB2 was predicted to be “probably damaging.” The secondary structure prediction showed that the mutation V146M led to the replacement of an original β-strand by an α-helix, a significant difference in coding position 146 of the secondary structure of βB2-crystallin protein (Figure 1D).
Eight members (3 affected and 5 unaffected) participated in the study (Figure 2A). The proband was a 2-year-old boy with dense white opacities distributed throughout the embryonic and fetal nuclei (Figure 2B). His mother (II-3) had had cataract extraction performed at age 5 years and was recorded as having nuclear cataract. There was no evidence of other ocular or systemic abnormalities.
The mutation in this family was identified as a c.62T>A transition in CRYBB2 that led to a missense mutation where a highly conserved isoleucine was replaced by asparagine (I21N) (Figure 2C). The mutation cosegregated with the disease in the family and was not observed in unaffected family members or in controls of Chinese descent.
The Ile at position 21 of human βB2-crystallin was located in a phylogenetically conserved region by multiple sequence alignment (Figure 2D). The PolyPhen score from PolyPhen analysis was 1.155, which meant that this I21N CRYBB2 was predicted to be “probably damaging.” The secondary structure prediction showed that the mutation led to the replacement of an original β-strand by an α-helix (Figure 2E).
Eight members (2 affected and 6 unaffected) participated in the study (Figure 3A). The proband was 2 years old and had had cataract surgery performed at age 3 months. Hospital records confirmed that the cataract was present at birth. On clinical examination, affected individual II-1 displayed a nuclear cataract phenotype (Figure 3B). He had nystagmus and a poor visual acuity measured using the decimal system of 0.1. There were no other ocular or systemic abnormalities.
This family was identified as having a single base alteration c.698G>A in exon 6 of CRYBB1, which resulted in a substitution of Arg to His at codon 233 (R233H) (Figure 3C). The mutation cosegregated with the disease in the family (II-1 and III-1) and was not observed in unaffected family members or in controls of Chinese descent.
The Arg at position 233 of human βB1-crystallin was located in a phylogenetically conserved region by multiple sequence alignment (Figure 3D). The R233H mutation caused no evident secondary structural change to the protein by Antheprot 2000 prediction (data not shown). However, The PolyPhen score from PolyPhen analysis was 2.002, which meant that this R233H CRYBB1 was predicted to be “probably damaging.”
In 17 additional families (85%), no mutations in the 8 crystallin and 2 connexin genes were observed. The phenotypes in all affected members were nuclear opacities to a variable extent. Twelve families also revealed other associated ocular disorders, such as nystagmus, strabismus, and microcornea. Because no mutation was cosegregated with these 10 genes, these families need to be further investigated for the causative mutations.
In this study, we identified the causative mutations in 3 families with nuclear ADCC. Among them, 1 family showed a nuclear cataract and microcornea phenotype. These mutations are observed only in affected individuals and not in unaffected family members or the 150 controls. We, therefore, assume that these variations are the disease-causing mutations rather than polymorphisms. To understand how the single amino acid exchange might affect the protein structure and function, several computational methods are undertaken.
β-Crystallins make up approximately 35% of total crystallin protein and are recognized as a member of the β/γ-crystallin superfamily, which contains 4 Greek key motifs encoded by separate exons. The β-crystallin family contains 5 protein chains (3 CRYBB and 4 CRYBA). CRYBB1, BB2, BB3, and BA4 all map to chromosome 22q11.2-13.1 with a similar gene structure. Different β-crystallin proteins are found in prenatal and postnatal developing lens, and their interactions with each other and with other lens proteins are postulated to be critical for lens transparency.5
The CRYBB2 gene consists of 6 exons: the first exon is not translated, the second exon encodes the NH2-terminal extension, and the subsequent 4 exons are responsible for 1 Greek key motif each.6 Two CRYBB2 mutations were identified in the present study. The mutation V146M in family A is located in the highly conserved valine residues in a major functional domain of the βB2-crystallin, the third Greek key motif of the protein and a region crucial to the correct formation of the tertiary structure. The secondary structure prediction shows that the mutation leads to the replacement of an original β-strand by an α-helix, which may affect the Greek key motifs and change the folding properties of βB2-crystallin. Loss of valine would lead to a change in intrapeptide or interpeptide bonding and folding. The phenotype of cataract is presumed to be caused by the formation of a heavy molecular weight fraction6 or a decrease in the stability of βB2-crystallin. The associated microcornea might be due to the inductive effects from the abnormally formed lens on the cornea during embryogenesis or the change in its steric coordinations with other proteins in lens.
The observed p.I21N substitution is located in the first Greek key motif of the βB2-crystallin protein and replaces the highly conserved nonpolar isoleucine with polar asparagine at position 21, in association with the nuclear cataract in the present study. The I21N mutation is predicted to be possibly damaging by PolyPhen analysis, which highlights the functional importance of this region of the protein. The secondary structure of the mutant protein is predicted, and the β-strand is replaced by an α-helix, which may be the reason for the dysfunction of the mutant protein. We, thus, hypothesize that the novel mutation I21N changes the protein structure around the mutant site that could alter the local binding ability, which would disrupt dimerization of the βB2-crystallin protein or impair binding with other lens-soluble proteins. The phenotype observed shows marked nuclear cataract comparable with the mutation in the first Greek key motif of βB2-crystallin, which is linked with coronary cataract. The result highlights the phenotypic heterogeneity of congenital cataracts.7
There are other mutations in CRYBB2 reported to cause a cataract phenotype in families of different ethnic origins (W151C,8 D128V,9 V187M,10 S31W,11 and Q155X12-17). However, all these families, including family B in the present study, are described as having no microcornea. The mutation V146M reported herein is the first CRYBB2 mutation identified in association with cataract and microcornea, which highlights the functional importance of this region of the protein. It is probable that some lens changes affect cornea development and others do not. Further physicochemical experiments are needed to demonstrate the function of CRYBB2.
βB1-crystallin is another major subunit of the β-crystallins and composes 9% of the total soluble crystallins in the human lens.18 A novel mutation R233H of CRYBB1 was identified in the present study. The proband shows a nuclear cataract phenotype with no ocular or systemic abnormalities. The mutation is located in exon 6, which encodes the Greek key IV and the COOH-terminal domain, and the arginine at position 233 is highly conserved among β-crystallin. The PolyPhen analysis considers the mutation to be possibly damaging to the protein. All the findings indicate that Arg233 is an important residue for the function of βB1-crystallin protein. Although major structural changes are not found by Antheprot 2000 analysis, previous functional studies19,20 have shown that the same amino acid change in different genes causes reduced solubility of the protein, which may contribute to cataract formation. The strongly positively charged guanidino group of arginine is replaced by the alkalescent positively charged imidazole functional group of a histidine, which may result in instability of the molecule. Therefore, a strong positively charged residue may be important for the structural and functional integrity of βB1-crystallin, but its effect on the βB1-crystallin structure should be further investigated.
To our knowledge, there are only 4 previous studies of CRYBB1 mutations in patients with ADCC.21-24 All the sequence changes reported in these families are located in exon 6, encoding the Greek key IV and the COOH-terminal extension. All these changes suggest that disruption of the fourth Greek key motif in βB1-crystallin might result in an abnormal COOH-terminus and production of a mutant protein, which underscores the importance of the fourth Greek key motif.
In conclusion, we reported 3 novel mutations in CRYB genes associated with nuclear ADCC in Chinese families, which strongly underscores the importance of the β-crystallins for lens development and contributes to the evaluation of the phenotype-genotype relationship. In addition, the analysis of the remaining 17 families reported herein excludes possible mutations in those genes, suggesting that other genes or loci could be involved with nuclear congenital cataract.
Correspondence: Si Quan Zhu, MD, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, 1 Dong Jiao Min Xiang, Beijing 100730, China (firstname.lastname@example.org).
Submitted for Publication: March 17, 2010; final revision received June 29, 2010; accepted July 2, 2010.
Author Contributions: Drs K. J. Wang and B. B. Wang contributed equally to the article; Drs Ma and Zhu contributed equally to the project and may be considered co-corresponding authors. Dr K. J. Wang had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Financial Disclosure: None reported.
Funding/Support: This study was supported by grants 2008BAH24B05 from the National Science & Technology Pillar Program of China (Dr Ma), 2009-3-37 from the high-level technical personnel training project of Beijing Municipal health system (Dr Zhu), and 2006DKA21300 from the National Infrastructure Program of Chinese Genetic Resources (Dr Ma).
Additional Contributions: We are grateful to the patients and their families for their enthusiastic participation in this study.