A novel gammaD-crystallin mutation causes mild changes in protein properties but leads to congenital coralliform cataract

Abstract

Purpose: To identify the genetic lesions for congenital coralliform cataract.

Methods: Two Chinese families with autosomal dominant coralliform cataract, 12 affected and 14 unaffected individuals, were recruited. Fifteen known genes associated with autosomal dominant congenital cataract were screened by two-point linkage analysis with gene based single nucleotide polymorphisms and microsatellite markers. Sequence variations were identified. Recombinant FLAG-tagged wild type or mutant gammaD-crystallin was expressed in human lens epithelial cells and COS-7 cells. Protein solubility and intracellular distribution were analyzed by western blotting and immunofluorescence, respectively.

Results: A novel heterozygous change, c.43C>A (R15S) of gammaD-crystallin (CRYGD) co-segregated with coralliform cataract in one family and a known substitution, c.70C>A (P24T), in the other family. Unaffected family members and 103 unrelated control subjects did not carry these mutations. Similar to the wild type protein, R15S gammaD-crystallin was detergent soluble and was located in the cytoplasm. ProtScale and ScanProsite analyses revealed raised local hydrophobicity and the creation of a hypothetical casein kinase II phosphorylation site.

Conclusions: A novel R15S mutation caused congenital coralliform cataract in a Chinese family. R15S possessed similar properties to the wild type gammaD-crystallin, but its predicted increase of hydrophobicity and putative phosphorylation site could lead to protein aggregation, subsequently causing opacification in lens.

Figure 1

Pedigree of coralliform cataract families. The asterisk indicates family members who attended this study. The lens photograph from II-13 in Family A showed a line-shape opacity with a larger white dot end and tubular opacity radiating from the center of the lens. The lens photograph from II-2 in Family B showed a tubular and irregular opacity extending from center of the lens. Large white dots accumulated at the end of tubular protrusions.

Figure 2

R15S mutation in Family A. A: The upper panel showed the forward (on the left) and the reverse (on the right) DNA segments of wildtype CRYGD. The lower panel displayed the forward and the reverse sequences of mutant CRYGD. The mutation of c.43C>A was indicated by arrow both in forward and reverse sequences. B: The DNA sequences in the upper lines and amino acid sequences in the lower lines were compared between wildtype and mutant CRYGD. The arrow denotes the nucleotide change.

Figure 3

Hydrophobicity change of R15S CRYGD. The prediction by ProtScale analysis at Expasy indicated an increase of local hydrophobicity around the site of R15S mutation (Circle in panel B). A: The curve showed the hydrophobicity score of each amino acid of wildtype CRYGD. B: The curve was the hydrophobicity of R15S CRYGD. At the position of the 15th amino acid and its neighboring locations, the hydrophobicity scores increased which was indicated by the circle.

Figure 4

Detergent solubility assay of FLAG-tagged wild type and mutant CRYGD. Western blotting analysis showed cell type specific solubility changes of CRYGD mutants. When expressed in human lens B3 epithelial cells (upper set of blots), wild type (WT) and all known mutant CRYGD (including R15S), except G165fsX8, were completely soluble in 0.5% Triton X-100 extraction. G165fsX8 was mainly Tx-insoluble. When expressed in COS-7 cells (lower set of blots), R15C and R15S CRYGD became moderately Tx-insoluble, unlike the wild type and P24T and G61C mutants. G165fsX8 remained Tx-insoluble.