PMC4422749_01
Family pedigree.. 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.
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The pedigree of the family (Fig 1) revealed an autosomal dominant inheritance pattern for them.
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.
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As shown in Fig 2A and 2B, the phenotype of congenital cataract was punctate cortical opacities. In younger affected one, the punctate opacities were only seen in the peripheral cortex of the lens (Fig 2C and 2D).
Mutation screening.. 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).
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The direct sequencing of candidate genes revealed a heterozygous change (G>C),at position 448 (c.448G>C) of MIP, which leads to the replacement of a conserved aspartic acid with histidine at codon 150 (p.D150H) (Fig 3).
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.
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For chromosome 12q13, around the MIP locus, seven locus genotypes and inferred haplotypes were showed (Fig 4) and multi-point LOD scores were summarized (Table 1).
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).
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The mutation was predicted to be probably damaging with a score of 1.00 by PolyPhen-2 (Fig 5A), which meant it may have deleterious effect on the structure and function of the protein. Similarly, the score and media information content from SIFT was 0.00 and 2.63 respectively (Fig 5B), predicting the effect of this amino acid substitution on protein function was damaging.
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 = 10mum. (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.
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By contrast, AQP0-D150H was not observed at the plasma membrane other than cytoplasmic sites which extensively overlapped with that of GM130, a protein that localizes within Golgi apparatus (Fig 6A).