Clinical evaluation.

<p><b>A: Pedigree of the five-generation Chinese family with autosomal dominant congenital cataract (ADCC).</b> Squares and circles indicate males and females, respectively. Filled symbols indicate affected members and empty symbols indicate unaffected individuals. The diagonal line indicates a deceased family member and the arrow indicates the proband. Family members whose DNA was analyzed by sequencing and restriction enzyme digestion are indicated by asterisks. <b>B: Photograph of the right eye of the proband.</b> The photograph (diffuse illumination) of the proband (V: 1) before surgery shows a posterior polar cataract with cotton-like opacities in the posterior subcapsular cortex. The same phenotype was noted bilaterally.</p

Bioinformatics analysis of the mutant CRYGD Y151*.

<p>(A) The predicted secondary structure showing the reduced extended strand and random coil in the mutant. (B) The C-terminal domain of the truncated CRYGD favors the unfolded state. The residue-specific stability constant (K_f) for each residue of the protein was predicted by the BEST/COREX server.</p

A Novel Insertion Variant of <i>CRYGD</i> Is Associated with Congenital Nuclear Cataract in a Chinese Family

<div><p>Objective</p><p>To investigate a novel insertion variant of <i>CRYGD</i> identified in a Chinese family with nuclear congenital cataract.</p><p>Methods</p><p>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.</p><p>Principal Findings</p><p>A novel insertion variant, c.451_452insGACT, in <i>CRYGD</i> 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.</p><p>Conclusions</p><p>We have identified a novel mutation, c.451_452insGACT, in <i>CRYGD</i>, which is associated with nuclear cataract. This is the first insertion mutation of <i>CRYGD</i> 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.</p></div

Comparison of CRYGD wildtype and mutant.

<p>The physical and chemical parameters of the protein were analyzed by ProtParam.</p><p>Comparison of CRYGD wildtype and mutant.</p

Western blot analysis of CRYGD over-expression in HEK293T cells.

<p>(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).</p

Confirmation of the c.451_452insGACT (p.Tyr151*) insertion mutation of <i>CRYGD</i>.

<p>(A) Sequence chromatogram showing the heterozygous c.451_452insGACT insertion mutation of <i>CRYGD</i> in the proband. The mutation was numbered according to GenBank NM_006891.3. (B) Polyacrylamide gel electrophoresis showing different sizes of the PCR fragments in the pedigree. The 134 bp and 130 bp fragments were amplified from affected and unaffected chromosome, respectively. (C) Protein alignment of mammalian samples showing that the regions around the mutation are highly conserved. Numbers on left and right indicate the position of this fragment. The position of the mutant is marked by a black triangle.</p

Subcellular localization of WT-AQP0 and Y219*-AQP0in HEK 293T cells, 24 h after transient transfection.

<p>Bar 5μm. <b>A: Localization of singly transfected FLAG-tagged wild-type (WT) and mutated (219*) AQP0 proteins.</b> Photomicrographs show the distribution of immunoreactive FLAG-tagged AQP0 (green) and DAPI-stained nuclei (blue) <b>B: Localization of co-transfected FLAG-WT-AQP0 and Myc-219*-AQP0 proteins.</b> Photomicrographs show the distribution of immunoreactiveMyc-tagged 219*-AQP0 (red), FLAG-tagged WT-AQP0 (green) and DAPI-stained nuclei (blue)</p

A novel nonsense mutation (c.657C > G; p.Y219*) in <i>MIP</i>/AQP0 in a Chinese family with posterior polar ADCC.

<p><b>A: DNA sequences of <i>MIP</i> in unaffected and affected individuals.</b> The upper chromatogram of the DNA sequence from an unaffected individual (III: 3) shows only the wild-type AQP0 allele, which encodes tyrosine (TAC) at codon 219. The lower sequence chromatogram from the affected proband (V: 1) shows both C and G (S) at position 657 (arrow); thus, the mutant allele contained a C to G transition, which altered the Tyr to a stop codon (TAG).<b>B: A schematic diagram showing the presumed membrane topology of aquaporin0 (AQP0).</b> The depicted mutated portion (gray circles) illustrates the premature truncation of the protein. Amino acid residue 219 is located within the 6th transmembrane domain (blue arrow). <b>C: RFLP analysis shows the C>G transversion, which co-segregated with disease in a family.</b> The PCR product was 187bp in length and contained two <i>Rsa</i>I sites (GTAC). The unaffected allele yielded three fragments (12bp, 45bp, and 130bp) after <i>Rsa</i>I digestion, whereas the affected allele yielded four (12bp, 45bp, 130bp, and 175bp). Only the affected allele displayed the 175bp band (arrow). M indicates the DNA ladder.</p

Localization of Myc-tagged wildtype or Y151* CRYGD in HEK293T cells.

<p>Immunofluorescence of Myc (green fluorescence) showed the distribution of wildtype CRYGD in both cytomembrane and cytoplasm. However, the mutant CRYGD was mainly localized in the nucleus, in the form of granular deposits. Scale bar: 10 μm.</p

Protein expression levels of WT- and Y219*-AQP0 transfected into HEK 293T cells.

<p>Western blots were performed with the anti-FLAG as indicated.β-actin was used as the loading control. Cells with the mutated (Y219*) AQP0 construct showed an 87% reduction in AQP0 protein level compared to cells with wild-type AQP0. ***P<0.01</p