Four mutations identified in Chinese families with autosomal dominant congenital cataracts by next-generation sequencing.

BACKGROUND: Congenital cataracts, which can arise due to a combination of factors like environmental influences and genetic predisposition, significantly impact children's visual health globally. The occurrence rate of congenital cataracts varies from 0. 63 to 9.74 per 10,000 births. There are 7.4 instances per 10,000 children, with the highest occurrence seen in Asia. Symptoms of the disease include clouding of the lens and visual impairment. Timely identification of the condition plays a crucial role in the management and outlook of pediatric patients. OBJECTIVE: This investigation aimed to discover causative mutations in four separate Chinese family lineages. METHODS: The detailed clinical data and family history of four Chinese families with autosomal dominant congenital cataracts were carefully documented. Examination of the Whole Exome Sequencing was utilized to identify the genetic anomalies present in the familial cases. Subsequent validation of the identified mutations was carried out using PCR and Sanger sequencing. Following this, various computational predictive programs were utilized to evaluate how the mutations impact the structure and function of the protein. RESULTS: The sequencing results reveal four potential disease-causing mutations: c.436G > A (p.V146M) of CRYBB2 Family 1, c.26G > T (p.R9I) of GJA3 in family 2, c.227G > A (p.R76H) of GJA8 in family 3, c.-168G > T of FTL in family 4. Among them, the causative mutation in Family GJA3 is novel, and Family FTL is a rare cataract syndrome. These familial mutations showed complete co-segregation with the affected individuals, with no presence in unaffected family members or the 100 controls. Several bioinformatic prediction tools also support the likely pathogenicity of these mutations. CONCLUSION: Our findings expand the mutational and phenotypic spectrum of genes associated with congenital cataracts and provide clues to the pathogenesis of congenital cataracts. These data also demonstrate the importance of NGS technology for the molecular diagnosis of congenital cataract patients.

The Functional Significance of High Cysteine Content in Eye Lens γ-Crystallins.

Cataract disease is strongly associated with progressively accumulating oxidative damage to the extremely long-lived crystallin proteins of the lens. Cysteine oxidation affects crystallin folding, interactions, and light-scattering aggregation especially strongly due to the formation of disulfide bridges. Minimizing crystallin aggregation is crucial for lifelong lens transparency, so one might expect the ubiquitous lens crystallin superfamilies (α and ßγ) to contain little cysteine. Yet, the Cys content of γ-crystallins is well above the average for human proteins. We review literature relevant to this longstanding puzzle and take advantage of expanding genomic databases and improved machine learning tools for protein structure prediction to investigate it further. We observe remarkably low Cys conservation in the ßγ-crystallin superfamily; however, in γ-crystallin, the spatial positioning of Cys residues is clearly fine-tuned by evolution. We propose that the requirements of long-term lens transparency and high lens optical power impose competing evolutionary pressures on lens ßγ-crystallins, leading to distinct adaptations: high Cys content in γ-crystallins but low in ßB-crystallins. Aquatic species need more powerful lenses than terrestrial ones, which explains the high methionine content of many fish γ- (and even ß-) crystallins. Finally, we discuss synergies between sulfur-containing and aromatic residues in crystallins and suggest future experimental directions.

Oxidation of active cysteines mediates protein aggregation of S10R, the cataract-associated mutant of mouse GammaB-crystallin.

The Ser10 to Arg mutation in mouse γB-crystallin (MGB) has been associated with protein aggregation, dense nuclear opacity, and the degeneration of fiber cells in the lens core. Overexpression of the gap junction protein, connexin 46 (Cx46), was found to suppress the nuclear opacity and restore normal cell-cell contact. However, the molecular basis for the protein aggregation and related downstream effects were not evident from these studies. Here, we provide a comparison of the structures and solution properties of wild type MGB and the S10R mutant in vitro and show that, even though the mutation does not directly involve cysteine residues, some cysteines in the mutant protein are activated, leading to the enhanced formation of intermolecular disulfide-crosslinked protein aggregates relative to the wild-type. This occurs even as the protein structure is essentially unaltered. Thus, the primary event is enhanced protein aggregation due to the disulfide crosslinking of the mutant protein. We suggest that these aggregates eventually get deposited on fiber cell membranes. Since the gap junction protein, Cx46 is involved in the transport of reduced glutathione, we posit that these deposits interfere in Cx46-mediated glutathione transport and facilitate the oxidative stress-mediated downstream changes. Overexpression of Cx46 suppresses such oxidative aggregation. These studies provide a plausible explanation for the protein aggregation and other changes that accompany this mutation. If indeed cysteine oxidation is the primary event for protein aggregation also in vivo, then the S10R mutant mouse, which is currently available, could serve as a viable animal model for human age-onset cataract.

Cataract-causing mutation R48C increases γA-crystallin susceptibility to oxidative stress and ultraviolet radiation.

Among all congenital cataracts caused by genetic mutations, approximately half are caused by a mutation in crystallin genes, and accounts the leading cause of blindness in children globally. In this study, we investigated the underlying molecular mechanism of R48C mutation (c.142C > T; p.[Arg48Cys]) of γA-crystallin in a Mexican-Mestizo descent family causing congenital cataracts. We purified γA-crystallin wild-type (WT) and R48C mutant and compared their structural characteristics and biophysical properties by Spectroscopic experiments and environmental stress (oxidative stress, ultraviolet irradiation, pH disorders, thermal shock, or chemical denaturation). The R48C mutant did not affect the secondary and tertiary structure of monomer γA-crystallin, nor did it affect its stability to heat shock and chemicals. However, the R48C mutant destroys the oxidative stability of γA-crystallin, which makes the protein more prone to aggregation and precipitation under oxidative conditions. These might be the pathogenesis of γA-crystallin R48C mutant related to congenital cataract and help to develop anti-cataract strategies from the perspective of γA-crystallin.

Surface Exposed Free Cysteine Suppresses Crystallization of Human γD-Crystallin.

Human γD-crystallin (HGD) has remarkable stability against condensation in the human lens, sometimes over a whole lifetime. The native protein has a surface exposed free cysteine that forms dimers (Benedek, 1997; Ramkumar et al., 1864)1,2 without specific biological function and leads to further protein association and/or aggregation, which creates a paradox for understanding its stability. Previous work has demonstrated that chemical modification of the protein at the free cysteine (C110), increases the temperature at which liquid-liquid phase separation occurs (LLPS), lowers protein solubility and suggests an important role for this amino acid in maintaining its long-term resistance to condensation. Here we demonstrate that mutation of the cysteine does not alter the structure or solubility (liquidus) line for the protein, but dramatically increases the protein crystal nucleation rate following LLPS, suggesting that the free cysteine has a vital role in suppressing crystallization in the human lens.

The aging mouse lens transcriptome.

Age is a major risk factor for cataract (ARC). However, the influence of aging on the lens transcriptome is under studied. Lens epithelial (LEC) and fiber cells (LFC) were isolated from young (3 month old) and aged (24 month old) C57BL/6J mice, and the transcriptome elucidated via RNAseq. EdgeR estimated differential gene expression in pairwise contrasts, and Advaita's Ipathway guide and custom R scripts were used to evaluate the potential biological significance of differentially expressed genes (DEGs). This analysis revealed age-dependent decreases in lens differentiation marker expression in both LECs and LFCs, with gamma crystallin transcripts downregulating nearly 50 fold in aged LFCs. The expression of the transcription factors Hsf4 and Maf, which are known to activate lens fiber cell preferred genes, are downregulated, while FoxE3, which represses gamma crystallin expression, is upregulated in aged fibers. Aged LECs upregulate genes controlling the immune response, complement pathways, and cellular stress responses, including glutathione peroxidase 3 (Gpx3). Aged LFCs exhibit broad changes in the expression of genes regulating cell communication, and upregulate genes involved in antigen processing/presentation and cholesterol metabolism, while changes in the expression of mitochondrial respiratory chain genes are consistent with mitochondrial stress, including upregulation of NDufa4l2, which encodes an alternate electron transport chain protein. However, age did not profoundly affect the response of LECs to injury as both young and aged LECs upregulate inflammatory gene signatures at 24 h post injury to similar extents. These RNAseq profiles provide a rich data set that can be mined to understand the genetic regulation of lens aging and how this impinges on the pathophysiology of age related cataract.

Single cell transcriptomics of the developing zebrafish lens and identification of putative controllers of lens development.

The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos and larvae at single cell resolution through five days of development, identifying a number of novel putative regulators of lens development. Our data address open questions about the temperospatial expression of α-crystallins during lens development that will support future studies of their function and provide the first detailed view of ß- and γ-crystallin expression in and outside the lens. We describe divergent expression in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, membrane associated, RNA-binding, and transcription factor genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact.

The long noncoding RNA CRYBG3 induces aneuploidy by interfering with spindle assembly checkpoint via direct binding with Bub3.

Aneuploidy is a hallmark of genomic instability that leads to tumor initiation, progression, and metastasis. CDC20, Bub1, and Bub3 form the mitosis checkpoint complex (MCC) that binds the anaphase-promoting complex or cyclosome (APC/C), a crucial factor of the spindle assembly checkpoint (SAC), to ensure the bi-directional attachment and proper segregation of all sister chromosomes. However, just how MCC is regulated to ensure normal mitosis during cellular division remains unclear. In the present study, we demonstrated that LNC CRYBG3, an ionizing radiation-inducible long noncoding RNA, directly binds with Bub3 and interrupts its interaction with CDC20 to result in aneuploidy. The 261-317 (S3) residual of the LNC CRYBG3 sequence is critical for its interaction with Bub3 protein. Overexpression of LNC CRYBG3 leads to aneuploidy and promotes tumorigenesis and metastasis of lung cancer cells, implying that LNC CRYBG3 is a novel oncogene. These findings provide a novel mechanistic basis for the pathogenesis of NSCLC after exposure to ionizing radiation as well as a potential target for the diagnosis, treatment, and prognosis of an often fatal disease.

ATP differentially antagonizes the crowding-induced destabilization of human γS-crystallin and its four cataract-causing mutants.

αßγ-crystallins account for ∼90% of ocular proteins in lens with concentrations ≥400 mg/ml, which has to be soluble for the whole life-span and their aggregation results in cataract. So far, four cataract-causing mutants G18V, D26G, S39C and V42 M have been identified for human γS-crystallin. Mysteriously, lens maintains ATP concentrations of 3-7 mM despite being a metabolically-quiescent organ. Here by DSF and NMR, we characterized the binding of ATP to three cataract-causing mutants of human γS-crystallin as well as its effect on the solution conformations and thermal stability. The results together decode several novel findings: 1) ATP shows no detectable binding to WT and mutants, as well as no significant alternation of their conformations even at molar ratio of 1:200.2) Cataract-causing mutants show distinctive patterns of the crowding-induced destabilization. 3) ATP differentially antagonizes their crowding-induced destabilization. Our studies suggest that the crowding-induced destabilization of human γS-crystallin is also critically dependent of the hydration shell which could be differentially altered by four mutations. Most unexpectedly, ATP acts as an effective mediator for the protein hydration shell to antagonize the crowding-induced destabilization.

Cataract-Associated New Mutants S175G/H181Q of ßΒ2-Crystallin and P24S/S31G of γD-Crystallin Are Involved in Protein Aggregation by Structural Changes.

ß/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to cataracts. In this study, we identified 10 new mutations of ß/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of ßΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant ß-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of ß/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

Cataract-causing G18V eliminates the antagonization by ATP against the crowding-induced destabilization of human γS-crystallin.

In lens, ∼90% of ocular proteins are αßγ-crystallins with concentrations ≥400 mg/ml, which need to remain soluble for the whole life-span and their aggregation leads to cataract. The G18V mutation of human γS-crystallin causes hereditary childhood-onset cortical cataract. Mysteriously, despite being a metabolically-quiescent organ, lens maintains ATP concentrations of 3-7 mM. Very recently, we found that ATP has no significant binding to γS-crystallin as well as no alternation of its conformation. Nevertheless, ATP antagonizes the crowding-induced destabilization of γS-crystallin even at 1:1, most likely by interacting with the hydration shell. Here by DSF and NMR, we characterized the effect of ATP on binding, conformation, stability of G18V γS-crystallin and its interactions with α-crystallin. The results reveal: 1) G18V significantly accelerates the crowding-induced destabilization with Tm of 67 °C reduced to 50.5 °C at 1 mM. 2) Most unexpectedly, G18V almost completely eliminates the antagonizing effect of ATP against the crowding-induced destabilization. 3) ATP shows no significant effect on the interactions of α-crystallin with both WT and G18V γS-crystallin. Results together decode for the first time that G18V causes cataract not only by accelerating the crowding-induced destabilization, but also by eliminating the antagonizing effect of ATP against the crowding-induced destabilization.

Human γS-Crystallin-Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage.

Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper(II)-induced aggregation. This work was motivated by the observation that zinc(II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper(II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper(II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein-metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γS-crystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-Crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.

Increased hydrophobicity of CRYGD p.(Ala159ProfsTer9): Suspected cause of congenital cataracts in a large Chinese family.

OBJECTIVE: This study aimed to identify the disease-causing mutation of congenital cataract disease in a large northeastern Chinese family. MATERIALS AND METHODS: The subjects' peripheral blood was collected, their genomic DNA was extracted, mutation screening of candidate genes was performed using polymerase chain reaction, and the amplified products were sequenced. Recombinant C-terminal enhanced green fluorescent protein-tagged wild-type or mutant CRYGD was expressed in HEK293T cells, and the expression pattern was observed under a fluorescence microscope. The CRYGD protein mutation was analyzed via bioinformatics analysis. RESULTS: c.475delG, a novel frameshift mutation in CRYGD, was identified in the affected family members. This mutation causes premature termination of the polypeptide, resulting in truncated p.(Ala159ProfsTer9). According to the bioinformatics analysis results, compared with wild-type CRYGD, p.(Ala159ProfsTer9) exhibits significantly decreased hydrophilicity. Fluorescence microscopy revealed that p.(Ala159ProfsTer9) aggregates in the cell in the form of granular deposits. CONCLUSION: In this study, the novel frameshift mutation c.475delG, p.(Ala159ProfsTer9) in CRYGD was identified to cause congenital cataracts in a large Chinese family; increased hydrophobicity of p.(Ala159ProfsTer9) protein may be the underlying mechanism.

Polyphenol-enriched fraction of Vaccinium uliginosum L. protects selenite-induced cataract formation in the lens of Sprague-Dawley rat pups.

Purpose: As the aging population is increasing, the incidence of age-related cataract is expected to increase globally. The surgical intervention, a treatment for cataract, still has complications and is limited to developed countries. In this study, we investigated whether the polyphenol-enriched fraction of Vaccinium uliginosum L. (FH) prevents cataract formation in Sprague-Dawley (SD) rat pups. Methods: Sixty rat pups were randomly divided into six groups: CTL, Se, FH40, FH80, FH120, and Cur80. The cataract was induced with subcutaneous injection of sodium selenite (18 µmol/kg bodyweight) on postnatal (P) day 10. All groups, except CTL, were injected with sodium selenite, and the FH40, FH80, and FH120 groups were given gastric intubation with FH40 mg/kg, 80 mg/kg, and 120 mg/kg on P9, P10, and P11. The Cur80 group was also given gastric intubation with curcumin 80 mg/kg on P9, P10, and P11. All rat pups were euthanized on P30. Results: Lens morphological analysis showed that FH dose-dependently inhibited cataract formation. In the Se group, soluble proteins were insolubilized, and the gene expression of the α-, ß-, and γ-crystallins was downregulated. However, FH treatment statistically significantly inhibited insolubilization of soluble proteins and downregulation of the gene expression of the α-, ß-, and γ-crystallins. In the Se group, the gene and protein levels of m-calpain were downregulated, which were attenuated with FH treatment. In addition, sodium selenite injection caused reduced antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)), glutathione (GSH) depletion, and malondialdehyde (MDA) production in the lens. The administration of FH inhibited sodium selenite-induced oxidative stress in a dose-dependent manner. The mechanism of protection against oxidative stress by FH involves NF-E2-related factor (Nrf-2) and hemoxygenase-1 (HO-1). FH treatment inhibited decrease of Nrf-2 in the nucleus fraction and HO-1 in the cytosol fraction. Finally, the FH treatment protected poly (ADP)-ribose polymerase (PARP) from cleavage, determined with western blotting. Conclusions: FH showed a preventive effect against cataract formation by inhibiting m-calpain-mediated proteolysis and oxidative stress in the lens. These results suggest that FH could be a potential anticataract agent in age-related cataract.

γ-Crystallin redox-detox in the lens.

In the vertebrate eye, limiting oxidation of proteins and lipids is key to maintaining lens function and avoiding cataract formation. A study by Serebryany et al. identifies a surprising contributor to the eye's oxidative defense in their demonstration that γD-crystallin (HγD) functions as an oxidoreductase and uses disulfide exchange to initiate aggregation of mutant crystallins that mimic oxidative damage. These insights suggest a mechanism by which a dynamic pool of closely packed proteins might avoid oxidation-driven protein-folding traps, providing new avenues to understand the basis of a human disease with global impact.

Dynamic disulfide exchange in a crystallin protein in the human eye lens promotes cataract-associated aggregation.

Increased light scattering in the eye lens due to aggregation of the long-lived lens proteins, crystallins, is the cause of cataract disease. Several mutations in the gene encoding human γD-crystallin (HγD) cause misfolding and aggregation. Cataract-associated substitutions at Trp42 cause the protein to aggregate in vitro from a partially unfolded intermediate locked by an internal disulfide bridge, and proteomic evidence suggests a similar aggregation precursor is involved in age-onset cataract. Surprisingly, WT HγD can promote aggregation of the W42Q variant while itself remaining soluble. Here, a search for a biochemical mechanism for this interaction has revealed a previously unknown oxidoreductase activity in HγD. Using in vitro oxidation, mutational analysis, cysteine labeling, and MS, we have assigned this activity to a redox-active internal disulfide bond that is dynamically exchanged among HγD molecules. The W42Q variant acts as a disulfide sink, reducing oxidized WT and forming a distinct internal disulfide that kinetically traps the aggregation-prone intermediate. Our findings suggest a redox "hot potato" competition among WT and mutant or modified polypeptides wherein variants with the lowest kinetic stability are trapped in aggregation-prone intermediate states upon accepting disulfides from more stable variants. Such reactions may occur in other long-lived proteins that function in oxidizing environments. In these cases, aggregation may be forestalled by inhibiting disulfide flow toward mutant or damaged polypeptides.

Reactive cysteine residues in the oxidative dimerization and Cu2+ induced aggregation of human γD-crystallin: Implications for age-related cataract.

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in ß- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu2+-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.

[Analysis of disease-causing gene mutation in three Chinese families with congenital inherited cataract].

OBJECTIVE: To identify the disease-causing gene mutations in three Chinese pedigrees affected with congenital inherited cataract, in ordre to provide genetic counseling and prenatal diagnosis. METHODS: Using exons combined target region capture sequencing chip to screen the candidate disease-causing mutations, Sanger sequencing was used to confirm the disease-causing mutations. RESULTS: Family 1 was polymorphic cataract, family 2 was cerulean cataract, family 3 was coralliform cataract. The inheritance mode of the three pedigrees consisted with autosomal dominant inheritance. In family 1, a nonsense mutation of CRYßB2 gene c.463C>T in exon 6 result in a p.Q155X amino acid change. In family 2, a missense mutation of of CRYGD gene c.43C>T in exon 2 result in a p.R14C amino acid change. In family 3, a missense mutation of CRYGD gene c.70C>A in exon 2 result in a p.P23T amino aid change. No above-mentioned mutations were found in normal individuals. CONCLUSION: The nonsense mutation c.463C>T (p.Q155X) of CRYßB2 gene, the heterozygous mutations c.43C>T(p.R14C) of CRYGD gene and c.70C>A( p.P23T) of CRYGD gene was the disease-causing gene mutation in family 1, 2 and 3 respectively, our results provid genetic counseling and prenatal diagnosis for these three families.

Two novel mutations identified in ADCC families impair crystallin protein distribution and induce apoptosis in human lens epithelial cells.

Congenital cataract (CC) is a clinical and genetically heterogeneous eye disease that primarily causes lens disorder and even amblyopic blindness in children. As the mechanism underlying CC is genetically inherited, identification of CC-associated gene mutations and their role in protein distribution are topics of both pharmacological and biological research. Through physical and ophthalmic examinations, two Chinese pedigrees with autosomal dominant congenital cataract (ADCC) were recruited for this study. Mutation analyses of CC candidate genes by next-generation sequencing (NGS) and Sanger sequencing revealed a novel missense mutation in CRYBB2 (p.V146L) and a deletion mutation in CRYAA (p.116_118del). Both mutations fully co-segregated were not observed in unaffected family members or in 100 unrelated healthy controls. The CRYBB2 missense mutation disrupts the distribution of CRYBB2 in human lens epithelial cells (HLEpiCs), and the CRYAA deletion mutation causes hyperdispersion of CRYAA. Furthermore, these two crystallin mutations result in aberrant expression of unfolded protein response (UPR) marker genes as well as apoptosis in HLEpiCs. Collectively, these findings broaden the genetic spectrum of ADCC.

Segregation of a novel p.(Ser270Tyr) MAF mutation and p.(Tyr56∗) CRYGD variant in a family with dominantly inherited congenital cataracts.

A bilaterally blind woman, with a three generation family history of autosomal dominant congenital cataracts, variably associated with iris colobomata and microcornea, sought preconception genetic consultation. Whole-exome sequencing was performed in three affected family members, one unaffected first degree relative, and one spouse. The sequence variant c.168C>G; p.(Tyr56∗) in CRYGD, previously reported as pathogenic, and a novel mutation c.809C>A; p.(Ser270Tyr) in MAF, were identified in two affected family members; the grandmother, and half-brother of the proband. The proband inherited only the MAF mutation, whereas her clinically unaffected sister had the CRYGD change. In silico analysis supported a pathogenic role of p.(Ser270Tyr) in MAF, which was absent from publicly available whole-exome datasets, and 1161 Czech individuals. The frequency of CRYGD p.(Tyr56∗) in the ExAC dataset was higher than the estimated incidence of congenital cataract in the general population. Our study highlights that patients with genetically heterogeneous conditions may exhibit rare variants in more than one disease-associated gene, warranting caution with data interpretation, and supporting parallel screening of all genes known to harbour pathogenic mutations for a given phenotype. The pathogenicity of sequence variants previously reported as cataract-causing may require re-assessment in light of recently released datasets of human genomic variation.