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A Novel Insertion Variant of CRYGD Is Associated with Congenital Nuclear Cataract in a Chinese Family.

Zhuang X, Wang L, Song Z, Xiao W - PLoS ONE (2015)

Bottom Line: A significantly reduced solubility was observed for this mutant.We have identified a novel mutation, c.451_452insGACT, in CRYGD, which is associated with nuclear cataract.The mutant protein, with loss of solubility and localization to the nucleus, is hypothesized to be the major cause of cataract in these patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Shengjing Hospital, China Medical University, Shenyang, China; Department of Ophthalmology, The Fourth People's Hospital of Shenyang, Shenyang, China.

ABSTRACT

Objective: To investigate a novel insertion variant of CRYGD identified in a Chinese family with nuclear congenital cataract.

Methods: A Chinese family with congenital nuclear cataract was recruited for the mutational screening of candidate genes by direct sequencing. Recombinant N-terminal Myc tagged wildtype or mutant CRYGD was expressed in HEK293T cells. The expression pattern, protein solubility and subcellular distribution were analyzed by western blotting and immunofluorescence.

Principal findings: A novel insertion variant, c.451_452insGACT, in CRYGD was identified in the patients. It causes a frameshift and a premature termination of the polypeptide to become Y151*. A significantly reduced solubility was observed for this mutant. Unlike wildtype CRYGD, which existed mainly in the cytoplasm, Y151* was mis-located in the nucleus.

Conclusions: We have identified a novel mutation, c.451_452insGACT, in CRYGD, which is associated with nuclear cataract. This is the first insertion mutation of CRYGD found to cause autosomal dominant congenital cataract. The mutant protein, with loss of solubility and localization to the nucleus, is hypothesized to be the major cause of cataract in these patients.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of CRYGD over-expression in HEK293T cells.(A) The truncated CRYGD showed decreased solubility. In the supernatant, the mutant protein was truncated and was present in much lower amounts than the wildtype. In the precipitant, there was more mutant protein and the wildtype was not detected. (B) Quantification by band densitometry indicated the prominent reduction of mutant CRYGD in the supernatant (p<0.05).
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pone.0131471.g004: Western blot analysis of CRYGD over-expression in HEK293T cells.(A) The truncated CRYGD showed decreased solubility. In the supernatant, the mutant protein was truncated and was present in much lower amounts than the wildtype. In the precipitant, there was more mutant protein and the wildtype was not detected. (B) Quantification by band densitometry indicated the prominent reduction of mutant CRYGD in the supernatant (p<0.05).

Mentions: Myc-tagged wildtype and Y151* CRYGD constructs were transiently expressed in HEK293T cells and were detected using anti-Myc antibody (9E10). Western blotting showed that the molecular weight of recombinant mutant CRYGD had decreased as a result of the loss of 24 amino acids (Fig 4A, upper). Moreover, the mutant CRYGD had a greatly reduced level of protein level in the supernatant when compared with wildtype (Fig 4A, upper; Fig 4B). The mutant protein aggregated mainly in the precipitant where the wildtype was not detected (Fig 4A, lower). The results indicated that the mutant protein possessed decreased solubility.


A Novel Insertion Variant of CRYGD Is Associated with Congenital Nuclear Cataract in a Chinese Family.

Zhuang X, Wang L, Song Z, Xiao W - PLoS ONE (2015)

Western blot analysis of CRYGD over-expression in HEK293T cells.(A) The truncated CRYGD showed decreased solubility. In the supernatant, the mutant protein was truncated and was present in much lower amounts than the wildtype. In the precipitant, there was more mutant protein and the wildtype was not detected. (B) Quantification by band densitometry indicated the prominent reduction of mutant CRYGD in the supernatant (p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493073&req=5

pone.0131471.g004: Western blot analysis of CRYGD over-expression in HEK293T cells.(A) The truncated CRYGD showed decreased solubility. In the supernatant, the mutant protein was truncated and was present in much lower amounts than the wildtype. In the precipitant, there was more mutant protein and the wildtype was not detected. (B) Quantification by band densitometry indicated the prominent reduction of mutant CRYGD in the supernatant (p<0.05).
Mentions: Myc-tagged wildtype and Y151* CRYGD constructs were transiently expressed in HEK293T cells and were detected using anti-Myc antibody (9E10). Western blotting showed that the molecular weight of recombinant mutant CRYGD had decreased as a result of the loss of 24 amino acids (Fig 4A, upper). Moreover, the mutant CRYGD had a greatly reduced level of protein level in the supernatant when compared with wildtype (Fig 4A, upper; Fig 4B). The mutant protein aggregated mainly in the precipitant where the wildtype was not detected (Fig 4A, lower). The results indicated that the mutant protein possessed decreased solubility.

Bottom Line: A significantly reduced solubility was observed for this mutant.We have identified a novel mutation, c.451_452insGACT, in CRYGD, which is associated with nuclear cataract.The mutant protein, with loss of solubility and localization to the nucleus, is hypothesized to be the major cause of cataract in these patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Shengjing Hospital, China Medical University, Shenyang, China; Department of Ophthalmology, The Fourth People's Hospital of Shenyang, Shenyang, China.

ABSTRACT

Objective: To investigate a novel insertion variant of CRYGD identified in a Chinese family with nuclear congenital cataract.

Methods: A Chinese family with congenital nuclear cataract was recruited for the mutational screening of candidate genes by direct sequencing. Recombinant N-terminal Myc tagged wildtype or mutant CRYGD was expressed in HEK293T cells. The expression pattern, protein solubility and subcellular distribution were analyzed by western blotting and immunofluorescence.

Principal findings: A novel insertion variant, c.451_452insGACT, in CRYGD was identified in the patients. It causes a frameshift and a premature termination of the polypeptide to become Y151*. A significantly reduced solubility was observed for this mutant. Unlike wildtype CRYGD, which existed mainly in the cytoplasm, Y151* was mis-located in the nucleus.

Conclusions: We have identified a novel mutation, c.451_452insGACT, in CRYGD, which is associated with nuclear cataract. This is the first insertion mutation of CRYGD found to cause autosomal dominant congenital cataract. The mutant protein, with loss of solubility and localization to the nucleus, is hypothesized to be the major cause of cataract in these patients.

No MeSH data available.


Related in: MedlinePlus