CRYBB2 Research Articles

Study of The Molecular Nature of Congenital Cataracts in Patients from The Volga–Ural Region

Hereditary cataracts are characterized by significant clinical and genetic heterogeneity, which can pose challenges for early DNA diagnosis. To comprehensively address this problem, it is essential to investigate the epidemiology of the disease, perform population studies to determine the spectrum and frequencies of mutations in the responsible genes, and examine clinical and genetic correlations. Based on modern concepts, non-syndromic hereditary cataracts are predominantly caused by genetic disease forms associated with mutations in crystallin and connexin genes. Therefore, a comprehensive approach to studying hereditary cataracts is necessary for early diagnosis and improved treatment outcomes. The crystallin (CRYAA, CRYAB, CRYGC, CRYGD, and CRYBA1) and connexin (GJA8, GJA3) genes were analyzed in 45 unrelated families from the Volga–Ural Region (VUR) with hereditary congenital cataracts. Pathogenic and probably pathogenic nucleotide variants were identified in ten unrelated families, nine of which had cataracts in an autosomal dominant pattern of inheritance. Two previously undescribed likely pathogenic missense variants were identified in the CRYAA gene: c.253C > T (p.L85F) in one family and c.291C > G (p.H97Q) in two families. The known mutation c.272_274delGAG (p.G91del) was found in the CRYBA1 gene in one family, while no pathogenic variants were found in the CRYAB, CRYGC, or CRYGD genes in the examined patients. In the GJA8 gene, the known mutation c.68G > C (p.R23T) was found in two families, and previously undescribed variants were identified in two other families: a c.133_142del deletion (p.W45Sfs*72) and a missense variant, c.179G > A (p.G60D). In one patient with a recessive form of cataract, two compound-heterozygous variants were identified—a previously undescribed likely pathogenic missense variant, c.143A > G (p.E48G), and a known variant with uncertain pathogenetic significance, c.741T > G (p.I24M). Additionally, a previously undescribed deletion, c.del1126_1139 (p.D376Qfs*69), was identified in the GJA3 gene in one family. In all families where mutations were identified, cataracts were diagnosed either immediately after birth or during the first year of life. The clinical presentation of the cataracts varied depending on the type of lens opacity, resulting in various clinical forms. This information emphasizes the importance of early diagnosis and genetic testing for hereditary congenital cataracts to guide appropriate management and improve outcomes.

Identification of the interactions between specific genetic polymorphisms and nutrient intake associated with general and abdominal obesity in middle-aged adults

Comprehensive understanding of gene-diet interactions is necessary to establish proper dietary guidelines to prevent and manage general and abdominal obesity. We investigated the role of genetic variants and their interactions with general and abdominal obesity-associated nutrients using a largescale genome-wide association study of Korean adults. A total of 50,808 participants from a Korean genome and epidemiology study were included. Dietary intake was assessed using a food frequency questionnaire. Obesity was defined as a body mass index ≥25kg/m2. Abdominal obesity (AO) was defined as waist circumference ≥90cm and 80cm in males and females, respectively. Dietary nutrient intake was classified based on Korean Dietary Reference Intakes (DRIs). Odds ratios and 95% confidence intervals were calculated after adjusting for age, sex, exercise, smoking, alcohol drinking, total energy consumption, PC1, and PC2. Among the individuals consuming fat (%) above DRI, carriers of Ca binding protein 39 (CAB39)- rs6722579 minor allele (A) have a higher risk of AO than those not carrying the SNP (odds ration [OR]=3.73, p-value=2.05e-07; interaction p-value=1.80e-07). Among the individuals consuming vitamin C above DRI, carriers of carboxypeptidase Q (CPQ)- rs59465035 minor allele (T) have a lower risk of AO than those without that SNP (OR=0.89, p-value=1.44e-08; interaction p-value=9.50e-06). The genetic association with obesity was stronger among individuals with a genetic variant rs4130113 near GHR gene region in those consume folate above DRI and with a genetic variant rs5760920 near CRYBB2 gene region in those consume vitamin B2 above DRI. Our study results suggested that interactions of specific polymorphisms at loci and certain nutrients may influence obesity and abdominal obesity.

Genetic Analysis in a Swiss Cohort of Bilateral Congenital Cataract

Identification of geographic population-based differences in genotype and phenotype heterogeneity are important for targeted and patient-specific diagnosis and treatment, counseling, and screening strategies. To report disease-causing variants and their detailed phenotype in patients with bilateral congenital cataract from a single center in Switzerland and thereby draw a genetic map and perform a genotype-phenotype comparison of this cohort. This clinical and molecular-genetic cohort study took place through the collaboration of the Department of Ophthalmology at the University Hospital Zurich and the Institute of Medical Molecular Genetics, University of Zurich, Schlieren, Switzerland. Thirty-seven patients from 25 families with different types of bilateral congenital cataract were included. All participating family members received a comprehensive eye examination. Whole exome sequencing was performed in the index patients, followed by a filtering process to detect possible disease-associated variants in genes previously described in association with congenital cataract. Probable disease-causing variants were confirmed by Sanger sequencing in available family members. All data were collected from January 2018 to June 2020, and the molecular-genetic analyses were performed from January 2019 to July 2020. Identification of the underlying genetic causes of bilateral congenital cataract, including novel disease-causing variants and phenotype correlation. Among the 37 patients (18 [49%] male and 19 [51%] female; mean [SD] age, 17.3 [15.9] years) from 25 families, pathogenic variants were detected in 20 families (80% detection rate), which included 13 novel variants in the following genes: BCOR, COL4A1, CRYBA2, CRYBB2, CRYGC, CRYGS, GJA3, MAF, NHS, and WFS1. Putative disease-causing variants were identified in 14 of 20 families (70%) as isolated cases and in 6 of 20 families (30%) with syndromic cases. A recessive variant in the CRYBB2 gene in a consanguineous family with 2 affected siblings showing a nuclear and sutural cataract was reported in contrast to previously published reports. In addition, the effect on splicing in a minigene assay of a novel splice site variant in the NHS gene (c.[719-2A>G]) supported the pathogenicity of this variant. This study emphasizes the importance of genetic testing of congenital cataracts. Known dominant genes need to be considered for recessive inheritance patterns. Syndromic types of cataract may be underdiagnosed in patients with mild systemic features.

A novel missense mutation of CRYBA1 in a northern Chinese family with inherited coronary cataract with blue punctate opacities.

To demonstrate the underlying genetic defect that contribute to inherited cataract in a northern Chinese pedigree. The study recruited a family pedigree with a diagnosis of bilateral coronary cataract with blue punctate opacities. Fourteen family members and 100 healthy volunteers were enrolled. DNA sample of the proband in this family were analyzed by high-throughput sequencing, which was then demonstrated by Sanger sequencing in the remained people in the family and 100 controls. The functional effect of mutant genes was investigated via bioinformatics analysis, including Polymorphism Phenotyping version2 (PolyPhen-2), Protein Variation Effect Analyzer (PROVEAN v1.1.3) Scale-Invariant Feature Transform (SIFT), and Mutation Taster. In this three-generation family, a novel heterozygous mutation was found in the kinase domain of CRYBA1 gene (c.340C > T, p.R114C), which was only detected in patients in the family with inherited cataract and were not detected in the remained people in the family nor in normal people. The pathogenic effect of the mutation was verified via bioinformatics analysis. Our study presented the molecular experiments to confirm that a novel missense mutation of c.340 C > T located in exon 4 of CRYBA1 gene results in a bilateral coronary cataract with blue punctate opacities, which enriches the mutation spectrum of CRYBA1 gene in inherited cataract and deepens the understanding of the pathogenesis of inherited cataract.

Clinical Spectrum and Genetic Diagnosis of 54 Consecutive Patients Aged 0-25 with Bilateral Cataracts.

Childhood cataract affects 2.5–3.5 per 10,000 children in the UK, with a genetic mutation identified in 50–90% of bilateral cases. However, cataracts can also manifest in adolescence and early adulthood in isolation, as part of a complex ocular phenotype or with systemic features making accurate diagnosis more challenging. We investigate our real-world experience through a retrospective review of consecutive bilateral cataract patients (0–25 years) presenting to the ocular genetics service at Moorfields Eye Hospital between 2017 and 2020. Fifty-four patients from 44 unrelated families were identified, with a median age of 13.5 years (range 1 to 68 years) and a median age at diagnosis of 43.9 months IQR (1.7–140.3 months); 40.7% were female and 46.3% were Caucasian. Overall, 37 patients from 27 families (61.4%) were genetically solved (50%) or likely solved (additional 11.4%), with 26 disease-causing variants (8 were novel) in 21 genes; the most common were crystallin genes, in 8 (29.6%) families, with half occurring in the CRYBB2 gene. There was no significant difference in the molecular diagnostic rates between sporadic and familial inheritance (P = 0.287). Associated clinical diagnoses were retinal dystrophies in five (18.5%) and aniridia in three (11.1%) families. Bilateral cataracts were the presenting feature in 27.3% (6/22) of either complex or syndromic cases, and isolated cataract patients were 11.5 years younger (rank-sum Z = 3.668, P = 0.0002). Prompt genetic investigation with comprehensive panel testing can aid with diagnosis and optimise management of cataract patients.

A novel CRYBB2 mutation causes autosomal dominant cataract: A report from a Chinese family.

This study aimed to examine pathogenic mutation within one Chinese family of five-generations suffering from autosomal dominant cataract. Next-generation sequencing and Sanger sequencing were used to find the pathogenic variants. A rare mutation, c.563G > A, in CRYBB2 gene was found in the proband that showed symptom of non-syndromic congenital autosomal dominant cataract. This mutation had been found in all affected individuals and in one healthy infant, but it did not exist between two individuals who did not develop such disease in that family, as well as in 100 healthy subjects who showed no relation with that family. Cataracts in this family varied with different severity of lens opacities and elongation of axial length. One missense mutation c.563G > A is reported in the CRYBB2 gene among one Chinese family suffering from early-onset cataract, and associated novel phenotypes are the elongation of axial length and the types of cataract. Our results expand the spectrum of associated phenotypes of CRYBB2 mutation.

Polymorphisms in CRYBB2 encoding \u03b2B2-crystallin are associated with antisaccade performance and memory function

βB2-crystallin (gene symbol: Crybb2/CRYBB2) was first described as a structural protein of the ocular lens before it was detected in various brain regions of the mouse, including the hippocampus and the cerebral cortex. Mutations in the mouse Crybb2 gene lead to alterations of sensorimotor gating measured as prepulse inhibition (PPI) and reduced hippocampal size, combined with an altered number of parvalbumin-positive GABAergic interneurons. Decreased PPI and alterations of parvalbumin-positive interneurons are also endophenotypes that typically occur in schizophrenia. To verify the results found in mice, we genotyped 27 single nucleotide polymorphisms (SNPs) within the CRYBB2 gene and its flanking regions and investigated different schizophrenia typical endophenotypes in a sample of 510 schizophrenia patients and 1322 healthy controls. In the case-control study, no association with schizophrenia was found. However, 3 of the 4 investigated haplotype blocks indicated a decreased CRYBB2 mRNA expression. Two of these blocks were associated with poorer antisaccade task performance and altered working memory-linked functional magnetic resonance imaging signals. For the two haplotypes associated with antisaccade performance, suggestive evidence was found with visual memory and in addition, haplotype block 4 showed a nominally significant association with reduced sensorimotor gating, measured as P50 ratio. These results were not schizophrenia-specific, but could be detected in a combined sample of patients and healthy controls. This is the first study to demonstrate the importance of βB2-crystallin for antisaccade performance and memory function in humans and therefore provides implications for βB2-crystallin function in the human brain.

A novel missense mutation of CRYBB1 causes congenital cataract in a Chinese family.

To identify the pathogenic gene and mutation site of a Chinese family with congenital cataract. Eight family members and 100 controls were employed, and targeted exome sequencing was used to identify the genetically pathogenic factor of the proband. Targeted next-generation sequencing identified a novel missense mutation c.209A>C (p.Q70P) of CRYBB1 gene in the family. Sanger sequencing results showed that this heterozygous mutation was a causative mutation, which was not found in unaffected family members and healthy controls. Bioinformatics predicts that the effect of this mutation on protein function is probably harmful. We demonstrate that c.209A>C of CRYBB1 gene is a pathogenic mutation in the family of congenital nuclear cataract in this study. This is the first report that this mutation leads to congenital nuclear cataract, which broadens the mutation spectrum of CRYBB1 gene in congenital nuclear cataract.

Study on iTRAQ-based differential expression protein analysis in nuclear cataract lens with different axial length

Objective To identify and verify differentially expressed proteins in lens of nuclear cataract eyes with different axial lengths. Methods Forty-eight nuclear cataract patients with the hardness grade Ⅲ to Ⅳ were enrolled from January 2016 to December 2017 in the east area of Xining city.The lens nucleus was obtained during extracapsular cataract extraction (ECCE) surgery.The samples were divided into six groups according to age and axial length, with 8 samples in each group.Seven normal transparent human lens nuclei were obtained from donors during corneal transplantation surgery and served as normal control.Water-soluble, water-insoluble and water-insoluble-urea-soluble protein fractions were extracted separately and merged to analyze by using 8-plex isobaric tags for relative and absolute quantitation (iTRAQ) labeling combined with 2D-LC/MS/MS.The data were processed with ProteinPilot software.Two technical replicates of iTRAQ analyses were carried out.Up-regulated and down-regulated proteins were selected in two replicates as candidate biomarkers.Twenty lens nuclei from 20 nulear cataract patients were divided into long axis young age group, normal axis young age group, long axis old age group and normal axis old age group.Western blot was used to confirm the most different proteins.This study protocol was approved by the Ethics Committee of TangDu Hospital of Air Force Medical University (TDLL2015046). Written consent was obtained from each participant. Results Eighty proteins were identified.Six proteins were selected as differentially-expressed proteins.Sorbitol dehydrogenase (SDH) was elevated whereas beta-crystallin B2 (CRYBB2) was decreased in the long axis groups.The relative expression of SDH protein in the long axis low age group was 1.204±0.142, which was significantly higher than 0.711±0.072 in the normal axis low age group, the relative expression level of SDH in the long axis old age group was 1.092±0.334, which was significantly higher than 0.617±0.089 in the normal axis old age group, the difference was statistically significant (all at P<0.05). Conclusions The expression levels of SDH in the nuclear cataract were elevated with long axial length.SDH may be involved in the pathogenesis of axial myopia-associated nuclear cataract. Key words: Axial lengths, eye; Human lens, crystalline; Nuclear cataract; Isotope labeling; Proteomics

A Novel Mutation p.S93R in CRYBB1 Associated with Dominant Congenital Cataract and Microphthalmia

ABSTRACTPurpose: To identify the pathogenetic mutations in a four-generation Chinese family with dominant congenital cataracts and microphthalmia.Methods: A four-generation Chinese family with dominant congenital cataracts were recruited. Genomic DNAs were collected from their peripheral blood leukocytes and subjected to whole exome sequencing. The genetic mutations were identified by bioinformatic analyses and verified by Sanger sequencing.Results: Whole exome sequencing revealed a c.279C>G point mutation in the CRYBB1 gene which was further verified by Sanger sequencing. The nucleotide replacement results in a novel mutation p.S93R in a conserved residue of βB1 crystallin which is predicted to disrupt normal βB1 structure and function.Conclusions: We identified a novel missense mutation p.S93R in CRYBB1 in a Chinese family with autosomal dominant congenital cataracts and microphthalmia. This serine residue is extremely conserved evolutionarily in more than 50 βγ-crystallins of many species. These data will be very helpful to further understand the structural and functional features of crystallins.

Novel mutations in CRYBB1/CRYBB2 identified by targeted exome sequencing in Chinese families with congenital cataract.

To summarize the phenotypes and identify the underlying genetic cause of the CRYBB1 and CRYBB2 gene responsible for congenital cataract in two Chinese families. Detailed family histories and clinical data were collected from patients during an ophthalmologic examination. Of 523 inheritable genetic vision system-related genes were captured and sequenced by targeted next-generation sequencing, and the results were confirmed by Sanger sequencing. The possible functional impacts of an amino acid substitution were performed with PolyPhen-2 and SIFT predictions. The patients in the two families were affected with congenital cataract. Sixty-five (FAMILY-1) and sixty-two (FAMILY-2) single-nucleotide polymorphisms and indels were selected by recommended filtering criteria. Segregation was then analyzed by applying Sanger sequencing with the family members. A heterozygous CRYBB1 mutation in exon 4 (c.347T>C, p.L116P) was identified in sixteen patients in FAMILY-1. A heterozygous CRYBB2 mutation in exon 5 (c.355G>A, p.G119R) was identified in three patients in FAMILY-2. Each mutation co-segregated with the affected individuals and did not exist in unaffected family members and 200 unrelated normal controls. The mutation was predicted to be highly conservative and to be deleterious by both PolyPhen-2 and SIFT. The CRYBB1 mutation (c.347T>C) and CRYBB2 mutation (c.355G>A) are novel in patients with congenital cataract. We summarize the variable phenotypes among the patients, which expanded the phenotypic spectrum of congenital cataract in a different ethnic background.

Crybb2 Mutations Consistently Affect Schizophrenia Endophenotypes in Mice.

As part of the βγ-superfamily, βB2-crystallin (CRYBB2) is an ocular structural protein in the lens, and mutation of the corresponding gene can cause cataracts. CRYBB2 also is expressed in non-lens tissue such as the adult mouse brain and is associated with neuropsychiatric disorders such as schizophrenia. Nevertheless, the robustness of this association as well as how CRYBB2 may contribute to disease-relevant phenotypes is unknown. To add further clarity to this issue, we performed a comprehensive analysis of behavioral and neurohistological alterations in mice with an allelic series of mutations in the C-terminal end of the Crybb2 gene. Behavioral phenotyping of these three βB2-mutant lines Crybb2O377, Crybb2Philly, and Crybb2Aey2 included assessment of exploratory activity and anxiety-related behavior in the open field, sensorimotor gating measured by prepulse inhibition (PPI) of the acoustic startle reflex, cognitive performance measured by social discrimination, and spontaneous alternation in the Y-maze. In each mutant line, we also quantified the number of parvalbumin-positive (PV+) GABAergic interneurons in selected brain regions that express CRYBB2. While there were allele-specific differences in individual behaviors and affected brain areas, all three mutant lines exhibited consistent alterations in PPI that paralleled alterations in the PV+ cell number in the thalamic reticular nucleus (TRN). The direction of the PPI change mirrored that of the TRN PV+ cell number thereby suggesting a role for TRN PV+ cell number in modulating PPI. Moreover, as both altered PPI and PV+ cell number are schizophrenia-associated endophenotypes, our result implicates mutated Crybb2 in the development of this neuropsychiatric disorder.

A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration

PurposeThe RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that βA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells.MethodsRPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of βA3/A1-crystallin.ResultsβA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of βA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and vimentin, downregulation of E-cadherin, and increased cell migration. βA3/A1-crystallin binds to cortactin, and loss of βA3/A1-crystallin resulted in increased P-cortactinY421. The RPE from AMD samples had increased Snail and vimentin, and decreased E-cadherin, compared to age-matched controls.ConclusionsWe introduced a novel concept of dry AMD initiation induced by lysosomal clearance defects in the RPE and subsequent attempts by RPE cells to avoid the resulting stress by undergoing EMT. We demonstrate that βA3/A1-crystallin is a potential therapeutic target for AMD through rejuvenation of lysosomal dysfunction and potentially, reversal of EMT.

The Hirosaki small-eye rat: A novel recessive model animal of lens and retinal degeneration with loss of betaA3/A1-crystallin

We established the Hirosaki small-eye rat (HiSER), which have small, opaque eyes transmitted in an autosomal recessive manner, from our stock of Sprague-Dawley rats (SDRs). HiSERs show massive and progressive disorganization of the eye structures, including disruption and involution of the lens and detachment and aggregation of the retina. Rudimentary ciliary muscle and inflammatory cell infiltration in the vitreous are also observed. In HiSERs, the last three exons (exons 4-6) of the Cryba1 gene, encoding betaA3/A1-crystallin on chromosome 10, is deleted. As a consequence, the Nufip2 gene, located downstream of the Cryba1 gene in the opposite direction, is juxtaposed to exon 3 of the Cryba1 gene. In HiSER eyes, a chimeric transcript is expressed containing exons 1-3 of the Cryba1 gene and a sequence derived from the 3’UTR of the Nufip2 gene. Irrespective of the presence of the chimeric transcript, neither mutant nor normal betaA3/A1-crystallin proteins are detected in HiSER eyes. On the other hand, the expression level of the Nufip2 gene is decreased in HiSER eyes. Because it is known that betaA3/A1-crystallin is expressed in the lens and retina, and the Nufip2 gene is expressed in the central nervous system, it is hypothesized that the disappearance of betaA3/A1-crystallin and, in addition, downregulation of the Nufip2 gene are causal reasons of the HiSER phenotype. So far, several animals with functional disruption of betaA3/A1-crystallin and, in humans, cataract patients with mutation of the Cryba1 gene, have been reported. In addition to these familiar phenotypes observed in both animal models and patients, HiSERs exhibit unique characteristics presumably due to its distinct genotype. HiSERs may provide a good opportunity for investigation of the functional roles of Cryba1 and Nufip2 in ocular development, and may become a useful animal model of lens and retinal degenerative disorders.

A CRYBB2 mutation in a Taiwanese family with autosomal dominant cataract.

To identify the underlying genetic cause of a Taiwanese family with autosomal dominant cerulean cataract. A three-generation cerulean cataract family with 13 affected and 13 normal was identified. Whole exome sequencing, whole genome single nucleotide polymorphism genotyping and haplotype analysis, and fine mapping using polymorphic short tandem repeat markers were used to identify the causative gene mutation. Whole genome single nucleotide polymorphism genotyping and haplotype analysis mapped the candidate disease loci to chromosome 18 and chromosome 22. Polymorphic short tandem repeat markers further narrowed down the disease interval to chromosome 22 between markers D22S1174 and D22S1163. Whole exome sequencing was performed on selected individuals. Polymorphisms detected were filtered based on their genomic positions, allele frequency (<1%), and segregation within the pedigree. Affected individuals were found to be heterozygous carrying a C to T mutation on exon 6 of the CRYBB2 gene (with SNP ID: rs74315489). The mutation was predicted to produce a premature stop mutation Q155X. The mutation is co-segregation across the pedigree and the disease "T" allele was not detected in healthy members of the family and in additional 50 normal controls (100 chromosomes). Phylogenic protein alignment was also performed for the CRYBB2 gene across 68 species ranging from fishes, Sauropsida, Placentalia, carnivores, rodents, and primates with total 56 orthologous genes. The Q155 residue is 100% conserved across the evolutionary tree, indicating its crucial function. Here we identify the first Taiwanese cerulean cataract family carrying a CRYBB2_Q155X mutation.

Mutation in the Crybb1 gene encoding beta‐B1‐crystallin leads to recessive cataracts in the mouse

PurposeCongenital cataracts are caused by mutations in various genes, and CRYBB1 is one of them. Aim of the study was to create a corresponding mouse model for a cataract‐causing mutation in Crybb1.MethodsThe ENU‐sperm archive of F1 mouse mutants (https://www.helmholtz-muenchen.de/ieg/services/scientific-resources/index.html) was searched for mutations in Crybb1. After selection of an appropriate mutation the corresponding mouse mutant was recovered after in‐vitro fertilization.ResultsWe recently identified a mutation in exon 6 of CRYBB1 (c.613delC; p.Q205Sfs4*) as causative for congenital dominant cataracts in an Indian family. Correspondingly, we established a mouse mutant line, which affects the exon 5 of the Crybb1 gene (c.500A–&gt;T; p.D167V). The mouse mutation affects the 3rd Greek Key motif; correspondingly, the size of the alpha‐helical region and the size of several beta‐sheets might be modified in the mutant protein. PolyPhen‐2 predicted this mutation as “possibly damaging” with a score of 0.731. This mutation leads to a recessive form of cataracts with clear lenses in heterozygotes, although Crybb1 is expressed in mouse lenses from E12.5 onwards. Similar to other beta‐crystallins, Crybb1 is also expressed in the brain, which might lead to additional neuronal changes and might explain the involvement of Crybb1 in association with schizophrenia and stress (Spadaro PA et al., Biol. Psychiatr. 2015;78: 848‐859).ConclusionsIn humans, several cataract‐causing mutations have been identified ‐ most of them leading to dominant cataracts, and only a few of these mutations have been shown to cause a recessive way of inheritance. Here we present the first Crybb1 mouse mutant line suffering from a recessive congenital cataract.

Establishment of a recessive mutant small-eye rat with lens involution and retinal detachment associated with partial deletion and rearrangement of the Cryba1 gene.

From our stock of SDRs (Sprague-Dawley rats), we established a mutant strain having small opaque eyes and named it HiSER (Hirosaki small-eye rat). The HiSER phenotype is progressive and autosomal recessive. In HiSER eyes, disruption and involution of the lens, thickening of the inner nuclear layer, detachment and aggregation of the retina, rudimentary muscle in the ciliary body and cell infiltration in the vitreous humour were observed. Genetic linkage analysis using crossing with Brown Norway rat suggested that the causative gene(s) is located on chromosome 10. Microarray analysis showed that the expression level of the Cryba1 gene encoding βA3/A1-crystallin on chromosome 10 was markedly decreased in HiSER eyes. Genomic PCR revealed deletion of a 3.6-kb DNA region encompassing exons 4-6 of the gene in HiSERs. In HiSER eyes, a chimaeric transcript of the gene containing exons 1-3 and an approximately 250-bp sequence originating from the 3'-UTR of the Nufip2 gene, located downstream of the breakpoint in the opposite direction, was present. Whereas the chimaeric transcript was expressed in HiSER eyes, neither normal nor chimaeric βA3/A1-crystallin proteins were detected by Western blot analysis. Real-time RT (reverse transcription)-PCR analysis revealed that expression level of the Nufip2 gene in the HiSER eye was 40% of that in the SDR eye. These results suggest that the disappearance of the βA3/A1-crystallin protein and, in addition, down-regulation of the Nufip2 gene as a consequence of gene rearrangement causes the HiSER phenotype.

A novel p.Glu75Lys mutation in CRYBB1 with low penetrance in an Indian family with nuclear form of congenital cataract

IntroductionTo screen CRYAA, CRYAB, CRYGC, CRYGD, CRYGS, CRYBA1, CRYBA4, CRYBB1, CRYBB2, CRYBB3, BFSP1, GJA3, GJA8 and HSF4 gene in an Indian family having congenital cataract. MethodsThe congenital cataract family presented at Dr. R.P. Centre for Ophthalmic Sciences, a tertiary research and referral hospital (AIIMS, New Delhi, India). CRYAA, CRYAB, CRYGC, CRYGD, CRYGS, CRYBA1, CRYBA4, CRYBB1, CRYBB2, CRYBB3, BFSP1, GJA3, GJA8 and HSF4 genes were amplified. Protein structure differences analysis was performed using Discovery Studio (DS) 2.0. ResultsA three-generation Indian family with diagnosis of congenital cataract presented with bilateral dense nuclear opacities in the affected individuals. Direct sequencing analysis of 14 genes identified a novel and pathogenic nucleotide variation p.E75K in CRYBB1 gene. The mutation p.Glu75Lys alters the environment and charge on the protein surface, disrupting ionic interaction between Glu75 and Arg60. Thus p.Glu75Lys changes the electrostatic potential of the protein surface which could affect the interactions with other interacting partner proteins. DiscussionsIn this case a novel heterozygous p.Glu75Lys mutation in CRYBB1 was detected in an Indian family with nuclear cataract. The mutation was inherited in autosomal dominant pattern and showed reduced penetrance. Identification and characterization of this mutation further confirm the importance of protein interactions in maintaining lens transparency, and provides insight into the molecular mechanism underlying the pathogenesis of human congenital cataract.

Crybb2 deficiency impairs fertility in female mice

Beta-B2-crystallin (CRYBB2), encoded by Crybb2 gene, is a major protein in the mammalian eye lens that plays an important role in maintaining the transparency of the ocular lens. However, CRYBB2 also plays important roles in many extra-lenticular tissues and organs such as the retina, brain and testis. Our previous studies demonstrated that male Crybb2 deficient (Crybb2−/−) mice have reduced fertility compared with wild-type (WT) mice, while female Crybb2−/− mice exhibited reduced ovary weights and shorter estrous cycle percentages. Here we specifically investigated the role of CRYBB2 in the female reproductive system. Our studies revealed that ovaries from female Crybb2−/− mice exhibited significantly reduced numbers of primordial, secondary and pre-ovulatory follicles when compared with WT mice, while the rate of atretic follicles was also increased. Additionally, fewer eggs were collected from the oviduct of Crybb2−/− female mice after superovulation. Estrogen levels were higher in the metestrus and diestrus cycles of female Crybb2−/− mice, while progesterone levels were lower in diestrus cycles. Furthermore, the expression of survival and cell cycle genes, Bcl-2, Cdk4 and Ccnd2, were significantly decreased in granulosa cells isolated from female Crybb2−/− mice, consistent with the predominant expression of CRYBB2 in ovarian granulosa cells. Our results reveal a critical role for CRYBB2 in female fertility and specific effects on the proliferation and survival status of ovarian granulosa cells.

Tale of lysosome in RPE cells and in the pathogenesis of AMD

AbstractIn RPE cells, the lysosomal system is a crucial regulator of both phagocytosis and autophagy. We have recently shown in the Nuc1 rat (a spontaneous mutation in the Cryba1 gene encoding for betaA3/A1‐crystallin) and in mice lacking betaA3/A1‐crystallin specifically in the RPE, that impaired lysosomal clearance decreases both autophagy and phagocytosis. This leads to the accumulation of lipofuscin within RPE cells, subretinal lesions in the posterior pole, and deposits between the RPE and Bruch’s membrane. It is likely that disturbances in the homeostasis of RPE cells, due to accumulation of undegraded intracellular material in the mice lacking betaA3/A1‐crystallin, trigger a para‐inflammatory response in an attempt to restore normal RPE function. Severe or prolonged impairment of lysosomal‐mediated clearance may also convert para‐inflammation to chronic inflammation, leading to retinal degeneration. Several studies support the hypothesis that the immune system is involved in the pathogenesis of AMD, in concert with, or in addition to, other factors. We have generated a genetically engineered mouse model that exhibits a slowly progressing form of AMD‐like pathology associated with inefficient lysosomal clearance.