CRYBB2 Research Articles

A Missense Mutation in CRYBB2 Leads to Progressive Congenital Membranous Cataract by Impacting the Solubility and Function of βB2-Crystallin

Congenital cataract is a major cause of visual impairment and childhood blindness. The solubility and stability of crystallin proteins play critical roles in maintaining the optical transparency of the lens during the life span. Previous studies have shown that approximately 8.3%∼25% of congenital cataracts are inherited, and mutations in crystallins are the most common. In this study, we attempted to identify the genetic defect in a four-generation family affected with congenital cataracts. The congenital cataract phenotype of this four-generation family was identified as membranous cataract by slit-lamp photography. Mutation screening of the candidate genes detected a heterozygous c.465G→C change in the exon6 of the βB2-crystallin gene (CRYBB2) in all family members affected with cataracts, resulting in the substitution of a highly conserved Tryptophan to Cystine (p.W151C). The mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis and found that the transition resulted in the absence of a BslI restriction site in the affected members of the pedigree. The outcome of PolyPhen-2 and SIFT analysis predicted that this W151C mutation would probably damage to the structure and function of βB2-crystallin. Wild type (wt) and W151C mutant βB2-crystallin were expressed in human lens epithelial cells (HLECs), and the fluorescence results showed that Wt-βB2-crystallin was evenly distributed throughout the cells, whereas approximately 34.7% of cells transfected with the W151C mutant βB2-crystallin formed intracellular aggregates. Taken together, these data suggest that the missense mutation in CRYBB2 gene leads to progressive congenital membranous cataract by impacting the solubility and function of βB2-crystallin.

Crybb2 coding for βB2-crystallin affects sensorimotor gating and hippocampal function

βB2-crystallin (gene symbol: Crybb2/CRYBB2) was first described as a structural protein of the ocular lens. This gene, however, is also expressed in several regions of the mammalian brain, although its function in this organ remains entirely unknown. To unravel some aspects of its function in the brain, we combined behavioral, neuroanatomical, and physiological analyses in a novel Crybb2 mouse mutant, O377. Behavioral tests with male O377 mutants revealed altered sensorimotor gating, suggesting modified neuronal functions. Since these mouse mutants also displayed reduced hippocampal size, we concentrated further investigations on the hippocampus. Free intracellular Ca2+ levels were increased and apoptosis was enhanced in the hippocampus of O377 mutants. Moreover, the expression of the gene encoding calpain 3 (gene symbol Capn3) was elevated and the expression of genes coding for the NMDA receptor subunits was downregulated. Additionally, the number of parvalbumin-positive interneurons was decreased in the hippocampus but not in the cortex of the mutants. High-speed voltage-sensitive dye imaging demonstrated an increased translation of input-to-output neuronal activity in the dentate gyrus of this Crybb2 mutant. These results point to an important function of βB2-crystallin in the hippocampal network. They indicate pleiotropic effects of mutations in the Crybb2 gene, which previously had been considered to be specific to the ocular lens. Moreover, our results are the first to demonstrate that βB2-crystallin has a role in hippocampal function and behavioral phenotypes. This model can now be further explored by future experiments.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-013-9478-7) contains supplementary material, which is available to authorized users.

Mutation analysis of CRYBB1 gene and prenatal diagnosis for a Chinese kindred featuring autosomal dominant congenital nuclear cataract

To perform mutation screening and prenatal diagnosis for a five-generation Chinese pedigree with autosomal dominant congenital nuclear cataract from Henan province by DNA sequencing. Blood samples were taken from the family members. Four candidate genes (CRYBA1/A3, CRYBB1, CRYBB2 and CRYGD) were screened for mutations using direct sequencing. Prenatal genetic diagnosis was provided for a fetus at early gestation through chorionic villus sampling. A missense mutation, c.387C to A, was detected in exon 4 of the CRYBB1 gene in all of the patients. The mutation has resulted in a p.S129R transversion. The same mutation was not found in the fetus of the proband, who was confirmed to be healthy by one-year follow-up. A missense mutation p.S129R of the CRYBB1 gene probably underlies the autosomal dominant congenital nuclear cataract in this pedigree. Detection of the mutation also facilitated prenatal genetic testing for the family.

Decreased levels of Ca2+-calmodulin-dependent protein kinase IV in the testis as a contributing factor to reduced fertility in male Crybb2−/− mice

βB2-crystallin (Crybb2), a member of the βγ-crystallin superfamily, in conjunction with α-crystallin, constitute the major proteins of the mammalian eye lens. Crybb2 is also expressed outside the lens, and certain related functions in these tissues have been reported. In the present study, in order to define the physiological role of Crybb2, we generated mice with a targeted deletion of the Crybb2 gene. Surprisingly, fertility was markedly reduced in male homozygous knockout mice compared to wild-type (WT) mice. Further experiments were performed to explore the underlying mechanism of subfertility in male Crybb2⁻/⁻ mice. Our results showed that Crybb2 was mainly expressed in the spermatogonia from the testes of mice with the WT C57BL/C genetic background. The testes of 4-week-old Crybb2⁻/⁻ mice were significantly hyperplastic, and no significant difference was found within 3 weeks postpartum. Additionally, there was a marked increase in the proliferation and apoptosis of germ cells, and the biological defects of these cells correlated with the decreased Bcl-2 levels, which correlated with the reduction of Ca²⁺-calmodulin-dependent protein kinase IV (CaMKIV) in the testis. These results suggest that the reduced fertility of Crybb2⁻/⁻ male mice may result from the disordered proliferation and apoptosis of germ cells in the testis, possibly due to reduced CaMKIV from the loss of Crybb2.

Characterization of the EMT response in Crybb2Phil mutants

The most abundant protein in the adult lens fiber cells is βB2‐crystallin (Crybb2). Mutations in the Crybb2 gene cause cataracts in mice and humans. A twelve nucleotide deletion in mouse lens epithelial cells (LEC), known as the Crybb2Phil mutation, leads to cataracts associated with lens fiber cell defects as well as epithelial to mesenchymal transition (EMT) of the LEC in adulthood. However, the mechanism behind this phenotype is poorly understood. The aim of this project is to characterize the EMT response caused by the Crybb2Phil mutation.Tissues obtained from Crybb2Phil mutants were immunostained for alpha and beta integrin subunits, phospo‐SMAD3 (p‐SMAD3), transforming growth factor β‐induced (BIGH3), and EMT the markers E‐cadherin, vimentin, follistatin, and α‐smooth muscle actin (α‐SMA).Compared to the wildtype lens, β1, αV, and α5 integrins, vimentin, α‐SMA, and p‐SMAD3 were upregulated in the Crybb2Phil mice. No changes were seen in α2, α3, and α6 integrin subunits or follistatin. Consistent with EMT, E‐cadherin was downregulated in the Crybb2Phil mice.Upregulation of α‐SMA signifies that Crybb2Phil mice undergo EMT around two months of age. Furthermore, over expression of β1 and αV integrins along with p‐SMAD3 in the Crybb2Phil mutant suggests that the EMT seen in Crybb2Phil mice may be occurring via the TGFβ‐SMAD interacting pathway. This project is funded by National Eye Institute grant EY015279.

βA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for βA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express βA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant βA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that βA3/A1-crystallin is essential for normal astrocyte function in the retina.

Investigation of Four Genes Responsible for Autosomal Recessive Congenital Cataract and Highly Expressed in the Brain in Four Unrelated Tunisian Families

Purpose: To identify the causative gene for phenotypes associating autosomal recessive congenital cataract, mental retardation and congenital cataract, mental retardation and microcephaly in four unrelated Tunisian families. Methods: Four genes (EPHA2, GALK1, GCNT2, and CRYBB1) were selected based on expression in human brain and their known or putative function. Linkage analyses were performed for the four genes in multiple affected and unaffected families’ members and results were explored by the GeneMapper ID v3.2 software. Results: No linkage was identified for the four studied genes in the four families. Affected members of each family did not share common haplotypes in corresponding candidate regions containing selected gene. Conclusion: Although the four studied genes were reported responsible for autosomal recessive congenital cataract and highly expressed in the human brain, we report no linkage for EPHA2, GALK1, GCNT2, and CRYBB1 genes in four families with congenital cataract, mental retardation and congenital cataract, mental retardation and microcephaly.

βA3/A1-Crystallin controls anoikis-mediated cell death in astrocytes by modulating PI3K/AKT/mTOR and ERK survival pathways through the PKD/Bit1-signaling axis

During eye development, apoptosis is vital to the maturation of highly specialized structures such as the lens and retina. Several forms of apoptosis have been described, including anoikis, a form of apoptosis triggered by inadequate or inappropriate cell–matrix contacts. The anoikis regulators, Bit1 (Bcl-2 inhibitor of transcription-1) and protein kinase-D (PKD), are expressed in developing lens when the organelles are present in lens fibers, but are downregulated as active denucleation is initiated. We have previously shown that in rats with a spontaneous mutation in the Cryba1 gene, coding for βA3/A1-crystallin, normal denucleation of lens fibers is inhibited. In rats with this mutation (Nuc1), both Bit1 and PKD remain abnormally high in lens fiber cells. To determine whether βA3/A1-crystallin has a role in anoikis, we induced anoikis in vitro and conducted mechanistic studies on astrocytes, cells known to express βA3/A1-crystallin. The expression pattern of Bit1 in retina correlates temporally with the development of astrocytes. Our data also indicate that loss of βA3/A1-crystallin in astrocytes results in a failure of Bit1 to be trafficked to the Golgi, thereby suppressing anoikis. This loss of βA3/A1-crystallin also induces insulin-like growth factor-II, which increases cell survival and growth by modulating the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR and extracellular signal-regulated kinase pathways. We propose that βA3/A1-crystallin is a novel regulator of both life and death decisions in ocular astrocytes.

Linkage analysis of one Chinese family with autosomal dominant congenital cataract

Background About one third of congenital cataract is associated with inheriting factor.The inherited heterogeneity has been found in congenital cataract.To seek the pathogenic gene is essential for the gene therapy. Objective Present study was to map and identify the causal gene for autosomal dominant congenital cataract (ADCC) in a Chinese family. Methods The clinical features of all affected members in this family were examined.Blood samples were collected from eleven family members for genetic linkage analysis.Polymorphic microsatellite markers were selected from the regions which harbor all known loci linked with ADCC.Universal fluorescent-labeled M13 primer was used in linkage analysis.Direct genomic sequencing was used to evaluate the candidate gene for example CRYBB2 gene.This study followed Helsinki Declaration and was proved by Tianjin City Ethic Committee.Written informed consent was obtained from each SUbject before any medical procees. Results The maximum two-point LOD score of 1.20 was obtained for marker D22S315 (θ=0).The LOD score of 0.6 was obtained for marker D16S3068.No mutation in all exons of CRYBB2 gene was found in the family. Conclusion CRYBB2 gene associated with ADCC was excluded from the family.A genome-wide linkage screening should be conducted.Genotyping with microsatellite markers using Universal fluorescent-labeled M13 primer can decrease the cost and obtain the same result. Key words: congenital cataract; Linkage analysis; fluorescent labeling; universal M13

Mutation in the βB2‐crystallin gene leads to cataracts, epithelial mesenchymal transition, and upregulation of integrins

The most abundant protein in the lens fiber cells is βB2‐crystallin (Crybb2). Mutations in the Crybb2 gene have been shown to cause cataracts in both mice and humans. A twelve nucleotide deletion in the lens epithelial cells, known as the Crybb2Phil mutation, leads to epithelial mesenchymal transition (EMT) during development. However, the mechanism behind this phenotype is poorly understood. The aim of this project is to understand the causes and effects of mutations in βB2‐crystallin. Tissues obtained from Crybb2Phil mutant adult mice were immunostained with αV, α2, α3, α5, α6, β1 integrin subunits, and phospo‐SMAD3. Compared to the wild type lens, β1, αV, and α5 were upregulated in the Crybb2Phil mutant. However, no changes were seen in α2, α3, and α6 integrin subunits. Phospho‐SMAD3 and smooth muscle actin (SMA) levels were also upregulated in Crybb2Philmice. Upregulation of SMA signifies that the Crybb2phil mice undergo EMT during development. Furthermore, over expression of β1 and αV integrin along with SMAD3 in these mutants suggest that the TGFβ‐SMAD interacting pathway may be involved. This project is funded by National Eye Institute grant EY015279. VR is a Charles Peter White Scholar.

Novel β-Crystallin Gene Mutations in Chinese Families With Nuclear Cataracts

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.

Mutation screen of β-crystallin genes in 274 patients with age-related macular degeneration

Purpose: The crystallin family of proteins comprise the main structural proteins of the vertebrate lens and have been classified into α-, β-, and γ- families. Several of the β-crystallin proteins have been detected in the retina where they are each localized to different compartments of rod and cone photoreceptors. Functionally, β-crystallins have been implicated in the protection of the retina from intense light exposure. Two members of the β-crystallins, CRYBB1 and CRYBB2, have been identified in drusen preparations isolated from the retina of donor eyes of patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population of developed countries. We therefore investigated CRYBB1 and CRYBB2 as candidate genes for AMD in 274 unrelated patients.Results: A mutation screen of the entire coding region of the CRYBB1gene uncovered eight sequence variations, including three missense changes, two intronic changes and three isocoding changes. A mutation screen of the entire coding region of the CRYBB2 gene uncovered three sequence variations, one isocoding change and two intronic changes.Conclusions: Although variant alleles of the CRYBB1 and CRYBB2 genes were found, none are considered pathogenic.

Progress in pathogenic genes and their functions of congenital cataract

Congenital cataract is the common cause of visual disability in children. Inherited isolated (non-syndromic) cataract represents one third of cases. Currently, at least 22 specific genes associated with isolated inherited cataract have been identified: ten crystallin genes: CRYAA, CRYAB, CRYBA1/A3, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, CRYGS; 4 membrane protein genes: GJA3, GJA8, MIP, LIM2; three growth and transcription factor genes: PITX3, MAF, HSF4; two cytoskeletal protein gene: BSFP1, BSFP2; chromatin modifying protein-4B gene: CHMP4B, EPHA2 and NHS, it is likely that more genes remain to be discovered. Some of the genes have been studied for their function by expression in cells or/and by knock-out animal models. The increasing availability of more detailed information about their functions makes it possible to understand the pathophysiology of congenital cataracts.

Novel Allele ofCrybb2in the Mouse and Its Expression in the Brain

O377 was identified as a new dominant cataract mutation in mice after radiation experiments. The purpose of this study was to genetically characterize the mutation and to analyze its biological consequences. Linkage analysis of the O377 mouse mutant was performed; candidate genes including Crybb2 were sequenced. The authors analyzed eyes and brains of the mutants by histology and the expression domains of Crybb2 by in situ hybridization and immunohistochemistry. RNA was isolated from whole brains of heterozygous and homozygous O377 mutants, and differential expression arrays were performed. All studies were compared with age- and strain-matched wild-type mice. The mutation was mapped to chromosome 5 and characterized as an A-->T substitution at the end of intron 5 of the Crybb2 gene. It led to alternative splicing with a 57-bp insertion in the mRNA and to 19 additional amino acids in the protein. In the brain, betaB2-crystallin was expressed in the cerebellum, olfactory bulb, cerebral cortex, and hippocampus. The only morphologic difference in the brain is the increased number of Purkinje cells in the cerebellum of homozygous strain-matched mutants. Differential expression analysis revealed the upregulation of calpain-3 in the brain of homozygous mutants, which was confirmed by quantitative real-time PCR. These results confirm the third allele of Crybb2 in the mouse that also affected exon 6 and the fourth Greek key motif. Moreover, expression analysis of Crybb2 identified for the first time distinct regions of expression in the brain, and the differential expression analysis points to the participation of Ca2+ in the corresponding pathologic processes.

Molecular cloning, sequence identification, and tissue expression profile analysis of three novel porcine genes: SDHB, SNRPA and CRYBB1

The complete coding sequences of three porcine genes-SDHB, SNRPA and CRYBB1 were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved coding sequence information of the mouse or other mammals and highly homologous pig ESTs. These three genes were then deposited into GenBank database and assigned to GeneID: 100125544, 768109 and 780429. The phylogenetic tree analysis revealed that the swine SDHB and SNRPA have closer genetic relationships with the human SDHB and SNRPA, but swine CRYBB1 has a closer genetic relationship with the bovine CRYBB1.The tissue expression analysis indicated that that swine SDHB, SNRPA and CRYBB1 gene were differentially expressed in tissues including fat, lung, muscle, small intestine, kidney, large intestine, spleen and liver. Our experiment is the first to establish the primary foundation for further research on these three swine genes.

A missense mutation S228P in the CRYBB1 gene causes autosomal dominant congenital cataract

Congenital cataract is a highly heterogeneous disorder at both the genetic and phenotypic levels. This study was conducted to identify disease locus for autosomal dominant congenital cataracts in a four generation Chinese family. Family history and clinical data were recorded. All the members were genotyped with microsatellite markers which are close to the known genetic loci for autosomal congenital cataracts. Two-point Lod scores were obtained using the MLINK of the LINKAGE program package (ver 5.1). Candidate genes were amplified by polymerase chain reaction (PCR) and direct cycle sequencing. The maximum Lod score of Zmax-2.11 was obtained with three microsatellite markers D22S258, D22S315, and D22S1163 at recombination fraction theta=0. Haplotype analysis showed that the disease gene was localized to a 18.5 Mbp region on chromosome 22 flanked by markers D22S1174 and D22S270, spanning the beta-crystallin gene cluster. A c.752T-->C mutation in exon 6 of CRYBB1 gene, which resulted in a heterozygous S228P mutation in predicted protein, was found to cosegregate with cataract in the family. This study identified a novel mutation in CRYBB1 gene in a Chinese family with autosomal dominant congenital cataract. These results provide strong evidence that CRYBB1 is a pathogenic gene for congenital cataract.