Fig 1.
A four-generation Chinese family affected with autosomal dominant cataract is shown. Squares and circles indicate males and females, respectively. The black symbols represent the affected members and open symbols represent the unaffected individuals. The diagonal line indicates a deceased family member and the black arrow indicates the proband. The family members attending this study are marked with asterisks.
Fig 2.
Clinical features of the proband (A, B) and her son (C, D).
Slit-lamp photographs (diffuse illumination and silt lamp) show the phenotype of the congenital cataract is punctate cortical opacities. In younger affected one, the punctate opacities were only seen in the peripheral cortex of the lens. The punctate opacities became denser and invaded into central cortex of the lens gradually with increasing age.
Fig 3.
Forward sequence analysis of exon 2 of MIP in the normal and affected members of this family. The DNA sequence chromatogram shows a heterozygous G>C nucleotide change (black arrow) in exon 2 of MIP (c.448G>C), which leads to the replacement of aspartic acid (GAC) with histidine (CAC) at codon 150 (p.D150H).
Fig 4.
Haplotype of the cataractous family.
Eight locus around MIP were genotyped. The disease-susceptibility haplotype (indicated by a vertical box) showed cosegregation with affected members in this family from D12S1632 to D12S1691.
Table 1.
Multipoint LOD scores for linkage analysis between cataract locus and markers on 12q13.
Fig 5.
Bioinformatics analysis of the p.D150H mutation and multiple sequence alignment.
(A) The mutation was predicted to be probably damaging with a score of 1.00 by PolyPhen-2, which meant it may have deleterious effect on the structure and function of the protein. (B) the score and media information content from SIFT was 0.00 and 2.63 respectively, predicting the effect of this amino acid substitution on protein function was damaging. (C) The result of a multiple sequence alignment from various species showed that the aspartic acid at position 150 of AQP0 is highly conserved (marked in green, the mutation in red).
Fig 6.
Subcellular location of WT-AQP0 and AQP0-D150H in two expressing cells.
(A) Representative fluorescence microscopy images show the distributions of immunoreactive AQP0 and a Golgi apparatus resident protein (GM130) in HeLa cells which were transiently transfected with wild type AQP0 or AQP0-D150H. The wild type AQP0 was detected mainly at the plasma membrane (white arrow) and in cytoplasm. By contrast, AQP0-D150H was not observed at the plasma membrane other than cytoplasmic sites which extensively overlapped with that of GM130. Scale bar = 10μm. (B) The quantities of wild type and p.D150H mutant AQP0 in membrane-enriched lysates of HEK-293T cells were assessed by western blotting, after WT-AQP0 or AQP0-D150H transfected, GAPDH was used as control.