A novel frameshift mutation in CX46 associated with hereditary dominant cataracts in a Chinese family

Abstract

Aim: To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family.

Methods: A Chinese family consisting of 20 cataract patients (including 9 male and 11 female) and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46) mutant. Immunoblotting was conduceted for testing the expression of CX46.

Results: To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3) were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C) was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400) compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression.

Conclusion: The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.

Keywords: autosome dominant heredity; cataract; congenital; connexin 46; genetic mutation; ocular lens.

Figure 1. A novel cx46fs400 frameshift mutation is associated with this Chinese autosomal dominant cataract pedigree

A: Pedigree of a Chinese family affected with congenital cataracts. The empty symbols represent the unaffected members and filled symbols represent the affected members. Symbol with a question marker indicate members with unknown phenotype. The proband is indicated with an arrow. There is no consanguinity in this pedigree. +/+: Wild-type; +/ins: Insert mutation. B: DNA sequencing profile of a novel cytosine insertion site around position c.1195 from the proband (IV-12) and unaffected individual (III-8).

Figure 2. Characterization of cx46fs400 frameshift mutant

A: A membrane topology diagram illustrating the location of cx46fs400 at the C-terminus of CX46. The substituted 35 animo acids are colored in red, the blue represents the 31 more amino acids extension. N: N-terminus; C: C-terminus. B: Alignment of the amino acid sequences of the wild type CX46 C-terminal tail among different species. Asterisks indi-cate the highly conserved residues. C: Hydrophobicity analysis of the C-terminal tail of CX46 frameshifted mutant vs wild type. The red line represents the hydrophobicity. D: Predicted secondary protein structure of CX46 C-terminal tail of frameshift mutant vs wild type. H: Alpha helix; E: Beta sheet; T: Turn; C: Coil.

Figure 3. Cx46fs400 frameshift mutant impairs CX46 protein expression in the proband's cataract lens tissue

A: The amount of CX46, alpha A-crystallin and alpha B-crystallin protein in lens anterior epithelia from proband (lane 1) and other age-matched cataracts (lanes 2 and 3) were analyzed by immunoblot. B: Immunoblotting the protein expression of CX46, CX50, alpha A-crystallin and alpha B-crystallin in lens fiber tissues of proband (lane 1) and age-matched cataracts (lanes 2 and 3). ctrl1 and ctrl2 are controls of age matched cataract tissues. GAPDH was used as protein loading control.