Autosomal Dominant Cataract Research Articles

TRPM3-mediated inhibition of active Na-K transport by the ocular lens epithelium

Purpose: The TRPM3 channel is selectively expressed in the lens epithelium, a monolayer at the anterior lens surface, but undetectable in fiber cells that make up the lens bulk. A point mutation in TRPM3 gene (substitution of isoleucine by methionine at codon 65; I65M) causes early onset autosomal dominant cataract in humans and in mice. From studies on transgenic mice, it is apparent the I65M TRPM3 mutation disrupts lens Na-K homeostasis. However, the normal role of lens TRPM3 in the lens is currently unknown. Here, we explore the lens response to TRPM3 activation. Methods: Studies were carried out using intact mouse lenses, cultured mouse lens epithelial cells and a human lens epithelium cell line (HLEB3). Active Na-K transport was studied by using rubidium (Rb) as a tracer for potassium entry. Rb was detected by atomic absorption spectrophotometry. Intracellular calcium was measured in nonconfluent single cells by Fura-2 ratiometric imaging. Results: Rb uptake was measured over a period of 10 min in intact mouse lenses exposed to a TRPM3 agonist CIM0216 (10 μM). CIM0216 reduced the rate of Rb uptake by ~20% (Ctrl 6.4±0.4 vs CIM 4.5±0.4 mmoles/kg lens dry weight/10 min, n=7, p<.01). The concentration dependence of the response to CIM0216 (1 to 50 μM) was examined in primary cultured mouse lens epithelium and Rb uptake was reduced by CIM0216 concentrations > 10 μM. The TRPM3 agonist reduced Rb uptake to a similar extent (~20%) in mouse lens epithelium, HLEB3 cells and HLEB3 cells that had been stably transfected with the I65M mutant TRPM3. However, the mutant cells responded to CIM0216 at a 10-fold lower concentration than WT mouse and HLEB3 cells. The Rb uptake response to CIM0216 was significantly inhibited by a TRPM3 antagonist, primidone (50 μM), and was abolished when cells were bathed in calcium-free solution. To study calcium entry, Fura2-loaded mouse lens epithelial cells were exposed to the TRPM3 agonist pregnenolone (100 μM). Pregnenolone caused an immediate increase in cytoplasmic calcium that was inhibited by primidone (Peak calcium concentration Ctrl 201±37 vs primidone 137±8 nM, n=5, p<.001). In cultures lens epithelium, agents that increase cellular cAMP, including IBMX, forskolin and 8p-CPT-cAMP, all caused a marked reduction of Rb uptake. Conclusions: TRPM3 activation causes a significant reduction in the rate of active Na-K transport and the response is associated with calcium entry. Because most lens Rb uptake is ouabain-sensitive, the response points to Na,K-ATPase inhibition. Studies are under way to test whether the Na-K transport response to TRPM3 activation involves cAMP signaling. This work was supported by NIH grants R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel single-base deletional mutation of MIP impairs protein distribution and cell-to-cell adhesion in autosomal dominant cataracts in a Chinese family.

Congenital cataracts are the leading cause of irreversible visual disability in children, and genetic factors play an important role in their development. In this study, targeted exome sequencing revealed a novel single-base deletional mutation of MIP (c.301delG; p.Ala101Profs*16) segregated with congenital punctate cataract in a Chinese family. The hydrophobic properties, and secondary and tertiary structures for truncated MIP were predicted to affect the function of protein by bioinformatics analysis. When MIP-WT and MIP-Ala101fs expression constructs were singly transfected into HeLa cells, it was found that the mRNA level showed no significant difference, while the protein level of the mutant was remarkably reduced compared to that of the wild-type MIP. Immunofluorescence images showed that the MIP-WT was principally localized to the plasma membrane, whereas the MIP-Ala101fs protein was aberrantly trapped in the cytoplasm. Furthermore, the cell-to-cell adhesion capability and the cell-to-cell communication property were both significantly reduced for MIP-Ala101fs compared to the MIP-WT (all *p < 0.05). This is the first report of the c.301delG mutation in the MIP gene associated with autosomal dominant congenital cataracts. We propose that the cataract is caused by the decreased protein expression and reduced cell-to-cell adhesion by the mutant MIP. The impaired trafficking or instability of the mutant protein, as well as compromised intercellular communication is probably a concurrent result of the mutation. The results expand the genetic and phenotypic spectra of MIP and help to better understand the molecular basis of congenital cataracts.

Human γS-Crystallin Mutation F10_Y11delinsLN in the First Greek Key Pair Destabilizes and Impairs Tight Packing Causing Cortical Lamellar Cataract.

Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of βγ-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human γS-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 °C (wild type 73.1 °C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with Cm = 2.72 M GuHCl). The mutant self-aggregates upon heating at 60 °C, which is inhibited by α-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human γS-crystallin impairs the protein's tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.

A novel missense mutation in the CRYBA2 caused autosomal dominant presenile cataract in a Chinese family.

Presenile cataract is a relatively rare type of cataract, but its genetic mechanisms are currently not well understood. The precise identification of these causative genes is crucial for effective genetic counseling for patients and their families. The aim of our study was to identify the causative gene associated with presenile cataract in a Chinese family. In February 2020, a four-generation pedigree of presenile cataract patients was recruited at the 2nd Affiliated Hospital of Kunming Medical University. One patient and her healthy husbandfrom the family underwent whole exome sequencing. The variant was validated through sanger sequencing, and co-segregation analysis was conducted in all family members to assess its pathogenicity. Molecular dynamics simulation (MDS) was used to analyze the conformation of both the wild type and pathogenic mutant loci p.Y153H of CRYBA2. We identified presenile cataract in the pedigree, which follows an autosomal-dominant pattern of inheritance. The family includes five clinically affected patients who all developed presenile cataract between the ages from 24 to 30. We confirmed the pathogenicity of a heterozygous missense variant (NM_057093:c.457T >C) in CRYBA2 within this family. The affected amino acid demonstrates high conservation across species. Subsequent sanger sequencing confirmed co-segregation of the disease in all family members. MDS analysis revealed that the p.Y153H mutant disrupted hydrogen bond formation between Y153 and R193 within the two β-strands of the fourth Greek key domain, leading to destabilization of the βA2-crystallin. In conclusion, a novel causative mutation (NM_057093:c.457T>C) in CRYBA2 might contribute to autosomal dominant presenile cataract.

A novel mutation of LIM2 causes autosomal dominant membranous cataract in a Chinese family.

To identify mutations in the genes of a four-generation Chinese family with congenital membranous cataracts and investigate the morphologic changes and possible functional damage underlying the role of the mutant gene. Whole exome analysis of thirteen members of a four-generation pedigree affected with congenital membranous cataracts was performed; co-segregation analysis of identified variants was validated by Sanger sequencing. All members underwent detailed physical and complete eye examinations. The physical changes caused by the mutation were analyzed in silico through homology modeling. The lens fiber block from a patient was observed under a scanning electron microscope (SEM). Cell membrane proteins and cytoplasmic proteins from the human lenses donated by one patient with cataract in this family and from the dislocated lens resulted from the penetrating ocular trauma of a patient unrelated with this family were extracted, and the expression and localization of MP20 and Cx46 were detected by Western blot (WB) assay in these proteins. A novel LIM2 heterozygous mutation (c.388C>T, p.R130C) was identified with congenital membranous cataracts inherited by an autosomal dominant (AD) pattern. Nystagmus and amblyopia were observed in all patients of this family, and exotropia and long axial length were observed in most patients. A/B ultrasound scan and ultrasound biomicroscopy revealed obvious thin crystalline lenses from 1.7 to 2.7 mm in central thickness in all cataract eyes. The bioinformatic analysis showed that the mutation was deleterious to the physiological function of LIM2-encoded MP20. Furthermore, by SEM, ultrastructure of the cataract nucleus showed that lens fiber cells (LFCs) remained morphologic characteristics of immature fiber cells, including flap cell surface with straight edges and lacking normal ball-and-socket joint boundaries, which implied that the differentiation of LFCs might be inhibited. Accumulation of MP20 and Cx46 in the cytoplasm was observed in the cytoplasm of the LFCs in human cataract lens. We identify a novel heterozygous LIM2 (c.388C>T, p.R130C) mutation inherited by an AD pattern. This LIM2 mutation causes the abnormal sub-localization of MP20 and Cx46 in LFCs resulting in membranous cataracts.

Alpha-crystallin mutations alter lens metabolites in mouse models of human cataracts.

Cataracts are a major cause of blindness worldwide and commonly occur in individuals over 70 years old. Cataracts can also appear earlier in life due to genetic mutations. The lens proteins, αA- and αB-crystallins, are chaperone proteins that have important roles maintaining protein solubility to prevent cataract formation. Mutations in the CRYAA and CRYAB crystallin genes are associated with autosomal dominant early onset human cataracts. Although studies about the proteomic and genomic changes that occur in cataracts have been reported, metabolomics studies are very limited. Here, we directly investigated cataract metabolism using gas-chromatography-mass spectrometry (GC-MS) to analyze the metabolites in adult Cryaa-R49C and Cryab-R120G knock-in mouse lenses. The most abundant metabolites were myo-inositol, L-(+)-lactic acid, cholesterol, phosphate, glycerol phosphate, palmitic and 9-octadecenoic acids, α-D-mannopyranose, and β-D-glucopyranose. Cryaa-R49C knock-in mouse lenses had a significant decrease in the number of sugars and minor sterols, which occurred in concert with an increase in lactic acid. Cholesterol composition was unchanged. In contrast, Cryab-R120G knock-in lenses exhibited increased total amino acid content including valine, alanine, serine, leucine, isoleucine, glycine, and aspartic acid. Minor sterols, including cholest-7-en-3-ol and glycerol phosphate were decreased. These studies indicate that lenses from Cryaa-R49C and Cryab-R120G knock-in mice, which are models for human cataracts, have unique amino acid and metabolite profiles.

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.

Autosomal dominant optic atrophy and cataract "plus" phenotype including axonal neuropathy.

ObjectiveTo characterize the phenotype in individuals with OPA3-related autosomal dominant optic atrophy and cataract (ADOAC) and peripheral neuropathy (PN).MethodsTwo probands with multiple affected relatives and one sporadic case were referred for evaluation of a PN. Their phenotype was determined by clinical ± neurophysiological assessment. Neuropathologic examination of sural nerve and skeletal muscle, and ultrastructural analysis of mitochondria in fibroblasts were performed in one case. Exome sequencing was performed in the probands.ResultsThe main clinical features in one family (n = 7 affected individuals) and one sporadic case were early-onset cataracts (n = 7), symptoms of gastrointestinal dysmotility (n = 8), and possible/confirmed PN (n = 7). Impaired vision was an early-onset feature in another family (n = 4 affected individuals), in which 3 members had symptoms of gastrointestinal dysmotility and 2 developed PN and cataracts. The less common features among all individuals included symptoms/signs of autonomic dysfunction (n = 3), hearing loss (n = 3), and recurrent pancreatitis (n = 1). In 5 individuals, the neuropathy was axonal and clinically asymptomatic (n = 1), sensory-predominant (n = 2), or motor and sensory (n = 2). In one patient, nerve biopsy revealed a loss of large and small myelinated fibers. In fibroblasts, mitochondria were frequently enlarged with slightly fragmented cristae. The exome sequencing identified OPA3 variants in all probands: a novel variant (c.23T>C) and the known mutation (c.313C>G) in OPA3.ConclusionsA syndromic form of ADOAC (ADOAC+), in which axonal neuropathy may be a major feature, is described. OPA3 mutations should be included in the differential diagnosis of complex inherited PN, even in the absence of clinically apparent optic atrophy.

Analysis of the dominant mutation N188T of human connexin46 (hCx46) using concatenation and molecular dynamics simulation.

Connexins (Cx) are proteins that form cell‐to‐cell gap junction channels. A mutation at position 188 in the second extracellular loop (E2) domain of hCx46 has been linked to an autosomal dominant zonular pulverulent cataract. As it is dominantly inherited, it is possible that the mutant variant affects the co‐expressed wild‐type Cx and/or its interaction with other cellular components. Here, we proposed to use concatenated hCx46wt‐hCx46N188T and hCx46N188T‐hCx46wt to analyze how hCx46N188T affected co‐expressed hCx46wt to achieve a dominant inheritance. Heterodimer hCx46wt‐hCx46N188T formed fewer gap junction plaques compared to homodimer hCx46wt‐hCx46wt, while the hCx46N188T‐hCx46N188T homodimer formed almost no gap junction plaques. Dye uptake experiments showed that hemichannels of concatenated variants were similar to hemichannels of monomers. Molecular dynamics simulations revealed that for docking, the N188 of a protomer was engaged in hydrogen bonds (HBs) with R180, N189, and D191 of the counterpart protomer of the adjacent hemichannel. T188 suppressed the formation of HBs between protomers. Molecular dynamics simulations of an equimolar hCx46wt/hCx46N188T gap junction channel revealed a reduced number of HBs between protomers, suggesting reduction of gap junction channels between lens fibers co‐expressing the variants.

A novel mutation of PANK4 causes autosomal dominant congenital posterior cataract.

Though many mutations have been identified to be associated with the occurrence of congenital cataract, pathogenic loci in some affected families are still unknown. Clinical data and genomic DNA were collected from a four-generation Chinese family. Candidate mutations were independently verified for cosegregation in the whole pedigree. Linkage analysis showed that the disease-causing mutation was located between 1p36.21 and 1p36.33. Analysis of the whole-exome sequencing data combined with linkage analysis identified a novel pathogenic variant (g.2451906C>T) at intron 4 of Pantothenate kinase 4 (PANK4 protein, PANK4 gene) in 1p36.32|606162. This variant showed complete cosegregation with the phenotype in the pedigree. The mutation was not detected in 106 normal controls nor in 40 sporadic congenital cataract patients. The mutation was demonstrated to significantly reduce the expression of the PANK4 protein level in the blood of cataract patients than that in normal individuals by ELISA. Pank4-/- mice showed a cataract phenotype with increased numbers of apoptotic lens epithelial cells, fiber cell aggregation, and significant mRNA variation of crystallin family members. Thus, the association of a new entity of an autosomal dominant cataract with mutations in PANK4, which influences cell proliferation, apoptosis of lens epithelial cells, crystallin abnormalities, and fiber cell derangement, subsequently induces cataract.

Transgenic zebrafish models reveal distinct molecular mechanisms for cataract-linked αA-crystallin mutants

Mutations in the small heat shock proteins α-crystallins have been linked to autosomal dominant cataracts in humans. Extensive studies in vitro have revealed a spectrum of alterations to the structure and function of these proteins including shifts in the size of the oligomer, modulation of subunit exchange and modification of their affinity to client proteins. Although mouse models of these mutants were instrumental in identifying changes in cellular proliferation and lens development, a direct comparative analysis of their effects on lens proteostasis has not been performed. Here, we have transgenically expressed cataract-linked mutants of αA- and αB-crystallin in the zebrafish lens to dissect the underlying molecular changes that contribute to the loss of lens optical properties. Zebrafish lines expressing these mutants displayed a range of morphological lens defects. Phenotype penetrance and severity were dependent on the mutation even in fish lines lacking endogenous α-crystallin. The mechanistic origins of these differences were investigated by the transgenic co-expression of a destabilized human γD-crystallin mutant. We found that the R49C but not the R116C mutant of αA-crystallin drove aggregation of γD-crystallin, although both mutants have similar affinity to client proteins in vitro. Our working model attributes these differences to the propensity of R49C, located in the buried N-terminal domain of αA-crystallin, to disulfide crosslinking as previously demonstrated in vitro. Our findings complement and extend previous work in mouse models and emphasize the need of investigating chaperone/client protein interactions in appropriate cellular context.

Detection of c.139G&gt;A (D47N) mutation in GJA8 gene in an extended family with inheritance of autosomal dominant zonular cataract without pulverulent opacities by exome sequencing

The aim of this studywas to identify the gene causing bilateral autosomal dominant zonular congenital cataract (ADZCC) without pulverulent opacities in an extended Muslim family by exome sequencing and subsequent analysis. An extended family of 37 members (14 affected and 23 unaffected) who belong to different nuclear families was screened for causative gene. Proband and her unaffected son were screened for causative variant by exome sequencing followed by Sanger sequencing of the proband's entire nuclear family. The rest of the members were further screened for variants detected, by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractorymutation system-polymerase chain reaction (T-ARMS PCR). Review of exome sequencing data of the proband and her unaffected son for 40 known genes causing congenital nonsyndromic cataracts revealed two variants, namely c.139G>A (p.Asp47Asn; D47N) in the GJA8 gene and c.2036C>T in the FYCO1 gene to be potentially pathogenic. Further, validation of these two variants in the entire family showed cosegregation of c.139G>A variant in GJA8 with ADZCC without pulverulent opacities. Variation of c.2036C>T in FYCO1 was not associated with disease in the family. The mutation c.139G>A in the GJA8 gene detected in the present study was also previously reported in Caucasian and Chinese families but with different phenotypes, i.e. nuclear and nuclear pulverulent cataracts. Thus, the mutation c.139G>A in GJA8 appears to exhibit marked interfamilial phenotypic variability.

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 Analysis of Families with Autosomal Dominant Congenital Cataract: A Recurrent Mutation in the CRYBA1/A3 Gene Causing Congenital Nuclear Cataract

ABSTRACTPurpose: To identify the CRYBA1/A3 mutation spectrum and analyze the genotype-phenotype correlations in Chinese families with congenital cataract.Methods: Family history and clinical data of 47 unrelated families with autosomal dominant congenital cataract (ADCC) were recorded. CRYBA1/A3 gene sequencing was applied to identify the causative mutation. Haplotypes were constructed using closely linked microsatellite markers and intragenic single-nucleotide polymorphisms (SNPs) to compare the affected haplotype in three families.Results: Nuclear cataract was the most common type of ADCC in Chinese families, accounting for 42.6% (20/47). A recurrent CRYBA1/A3 deletion mutation (ΔG91) was identified in three families (6.4%) with nonprogressive nuclear congenital cataract. Different haplotypes segregated with the mutation in each family.Conclusions: A recurrent ΔG91CRYBA1/A3 mutation occurs independently in 6.4% of the Chinese families with autosomal dominant nuclear cataracts and most likely represents a mutational hot spot, which underscores the relations between nonprogressive nuclear cataract and CRYBA1/A3.

Probing the changes in gene expression due to α-crystallin mutations in mouse models of hereditary human cataract.

The mammalian eye lens expresses a high concentration of crystallins (α, β and γ-crystallins) to maintain the refractive index essential for lens transparency. Crystallins are long-lived proteins that do not turnover throughout life. The structural destabilization of crystallins by UV exposure, glycation, oxidative stress and mutations in crystallin genes leads to protein aggregation and development of cataracts. Several destabilizing mutations in crystallin genes are linked with human autosomal dominant hereditary cataracts. To investigate the mechanism by which the α-crystallin mutations Cryaa-R49C and Cryab-R120G lead to cataract formation, we determined whether these mutations cause an altered expression of specific transcripts in the lens at an early postnatal age by RNA-seq analysis. Using knock-in mouse models previously generated in our laboratory, in the present work, we identified genes that exhibited altered abundance in the mutant lenses, including decreased transcripts for Clic5, an intracellular water channel in Cryaa-R49C heterozygous mutant lenses, and increased transcripts for Eno1b in Cryab-R120G heterozygous mutant lenses. In addition, RNA-seq analysis revealed increased histones H2B, H2A, and H4 gene expression in Cryaa-R49C mutant lenses, suggesting that the αA-crystallin mutation regulates histone expression via a transcriptional mechanism. Additionally, these studies confirmed the increased expression of histones H2B, H2A, and H4 by proteomic analysis of Cryaa-R49C knock-in and Cryaa;Cryab gene knockout lenses reported previously. Taken together, these findings offer additional insight into the early transcriptional changes caused by Cryaa and Cryab mutations associated with autosomal dominant human cataracts, and indicate that the transcript levels of certain genes are affected by the expression of mutant α-crystallin in vivo.

A novel frameshift mutation in CX46 associated with hereditary dominant cataracts in a Chinese family.

To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family. A Chinese family consisting of 20 cataract patients (including 9 male and 11 female) and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46) mutant. Immunoblotting was conduceted for testing the expression of CX46. To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3) were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C) was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400) compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression. The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.

Mutation analysis of connexin 50 gene among Iranian families with autosomal dominant cataracts.

Objective(s):Childhood cataract is a genetically heterogeneous eye disorder that results in visual impairment. The aim of this study was to identify the genetic mutations of connexin 50 gene among Iranian families suffered from autosomal dominant congenital cataracts (ADCC).Materials and Methods:Families, having at least two members with bilateral familial congenital cataract, were selected for the study. Probands were evaluated by detailed ophthalmologist’s examination, and the pedigree analysis was performed. PCR amplifications were performed corresponding to coding region and intron-exon boundaries of GJA8, a candidate gene responsible for ADCC. PCR products were subjected to bidirectional sequencing, and the co-segregation of identified mutations was examined and finally, the impact of identified mutations on biological functions of GJA8 was predicted by in silico examination.Results:Three different genetic alterations, including c.130G>A (p.V44M), c.301G>T (p.R101L) and c.134G>T (p.W45L) in GJA8 gene were detected among three probands. Two identified mutations, W45L and V44M have been already reported, while the R101L is a novel mutation and its co-segregation was examined. This mutation was exclusively detected in the ADCC and could not be found among the healthy control group. The result of bioinformatic studies of R101L mutation predicted that this amino acid substitution within GJA8 could be a disease-afflicting mutation due to its potential effect on the protein structure and biological function.Conclusion:Our results suggest that mutations of lens connexin genes such as GJA8 gene could be one of the major mechanisms of cataract development, at least in a significant proportion of Iranian patients with ADCC.

Mutations of RagA GTPase in mTORC1 Pathway Are Associated with Autosomal Dominant Cataracts.

Cataracts are a significant public health problem with no proven methods for prevention. Discovery of novel disease mechanisms to delineate new therapeutic targets is of importance in cataract prevention and therapy. Herein, we report that mutations in the RagA GTPase (RRAGA), a key regulator of the mechanistic rapamycin complex 1 (mTORC1), are associated with autosomal dominant cataracts. We performed whole exome sequencing in a family with autosomal dominant juvenile-onset cataracts, and identified a novel p.Leu60Arg mutation in RRAGA that co-segregated with the disease, after filtering against the dbSNP database, and at least 123,000 control chromosomes from public and in-house exome databases. In a follow-up direct screening of RRAGA in another 22 families and 142 unrelated patients with congenital or juvenile-onset cataracts, RRAGA was found to be mutated in two unrelated patients (p.Leu60Arg and c.-16G>A respectively). Functional studies in human lens epithelial cells revealed that the RRAGA mutations exerted deleterious effects on mTORC1 signaling, including increased relocation of RRAGA to the lysosomes, up-regulated mTORC1 phosphorylation, down-regulated autophagy, altered cell growth or compromised promoter activity. These data indicate that the RRAGA mutations, associated with autosomal dominant cataracts, play a role in the disease by acting through disruption of mTORC1 signaling.

Human βA3/A1-crystallin splicing mutation causes cataracts by activating the unfolded protein response and inducing apoptosis in differentiating lens fiber cells

βγ-Crystallins, having a uniquely stable two domain four Greek key structure, are crucial for transparency of the eye lens,. Mutations in lens crystallins have been proposed to cause cataract formation by a variety of mechanisms most of which involve destabilization of the protein fold. The underlying molecular mechanism for autosomal dominant zonular cataracts with sutural opacities in an Indian family caused by a c.215+1G>A splice mutation in the βA3/A1-crystallin gene CRYBA1 was elucidated using three transgenic mice models. This mutation causes a splice defect in which the mutant mRNA escapes nonsense mediated decay by skipping both exons 3 and 4. Skipping these exons results in an in-frame deletion of the mRNA and synthesis of an unstable p.Ile33_Ala119del mutant βA3/A1-crystallin protein. Transgenic expression of mutant βA3/A1-crystallin but not the wild type protein results in toxicity and abnormalities in the maturation and orientation of differentiating lens fibers in c.97_357del CRYBA1 transgenic mice, leading to a small spherical lens, cataract, and often lens capsule rupture. On a cellular level, the lenses accumulated p.Ile33_Ala119del βA3/A1-crystallin with resultant activation of the stress signaling pathway — unfolded protein response (UPR) and inhibition of normal protein synthesis, culminating in apoptosis. This highlights the mechanistic contrast between mild mutations that destabilize crystallins and other proteins, resulting in their being bound by the α-crystallins that buffer lens cells against damage by denatured proteins, and severely misfolded proteins that are not bound by α-crystallin but accumulate and have a direct toxic effect on lens cells, resulting in early onset cataracts.

A recurrent mutation of CRYGD gene in a northern Chinese family with autosomal dominant congenital nuclear cataract

Background Congenital cataract is a major cause for blindness of childhood. Genetic gene mutation accounts for almost 1/3 of congenital cataract patients. The most common inheritance type is autosomal dominant congenital cataract (ADCC). Over 100 mutations in 26 genes have been found to be associated with ADCC. Objective This study was to identify the disease-causing gene mutation in a family with ADCC. Methods This study was approved by Ethic Committee of Beijing Tongren Hospital and followed Declaration of Helsinki. A northern Chinese family with autosomal dominant congenital nuclear cataract was entrolled in Beijing Tongren Hospital in January 2011. Ocular examinations were performed and periphery blood specimens were collected from each family member under the informed consent. Genomic DNA was extracted. Twenty-one microsatellite markers around 17 ADCC genes were selected for linkage analysis, and two-point LOD score was calculated. CRYGC gene and CRYGD gene were amplified and screened for mutations using direct sequencing. ProtScale software was used to analyze the changes of hydrophobicity of the mutated protein. Co-segregation of the observed change with the disease phenotype was further detected by restriction fragment length polymorphism (RFLP). Results This family included 20 members of 4 generations, and 9 patients were examined in serial 4 passages, which conformed to autosomal dominant inheritance pattern. Clinical examination revealed binocular congenital nuclear cataract in the 9 patients. Maximum two-point LOD score was 4. 68 at marker D2S325 (θ=0). A known T→C change at position 127 of cDNA sequence was found by mutations screening of CRYGD gene. ProtScale programs showed an obvious increase of the local hydrophobicity in the mutant protein. RFLP results indicated that this missense mutation co-segregated with affected members of the family, but was absent in unaffected members and 100 unrelated controls. Conclusions c. T127C mutation of CRYGD gene appears to be the molecular pathogenesis of this ADCC family. Aberrant structure of mutant CRYGD protein caused by hydrophobicity change may lead to opacification of lens. Key words: Cataract/congenital; Cataract/genetics; DNA mutational analysis; Lens nucleus, crystalline/pathology; Pedigree; gamma-Crystallin/genetics; Mutations; Chinese