Identification and functional analysis of two novel connexin 50 mutations associated with autosome dominant congenital cataracts

Abstract

Autosomal dominant congenital cataracts (ADCC) are clinically and genetically heterogeneous diseases. The present study recruited two Chinese families with bilateral nuclear cataract or zonular pulverulent phenotype. Direct sequencing of candidate genes identified two novel missense mutations of Cx50, Cx50P59A (c.175C > G) and Cx50R76H (c.227G > A), both co-segregated well with all affected individuals. Bioinformatics analysis predicted deleterious for both mutations. Functional and cellular behaviors of wild type and mutant Cx50 examined by stably transfecting recombinant systems revealed similar protein expression levels. Protein distribution pattern by fluorescence microscopy showed that Cx50R76H localized at appositional membranes forming gap junctions with enormous cytoplasmic protein accumulation, whereas the Cx50P59A mutation was found inefficient at forming detectable plaques. Cell growth test by MTT assay showed that induction of Cx50P59A decreased cell viability. Our study constitutes the first report that the Cx50P59A and Cx50R76H mutations are associated with ADCC and expands the mutation spectrum of Cx50 in association with congenital cataracts. The genetic, cellular, and functional data suggest that the altered intercellular communication governed by mutated Cx50 proteins may act as the molecular mechanism underlying ADCC, which further confirms the role of Cx50 in the maintenance of human lens transparency.

Figure 1. Pedigrees and phenotypes.

(A,D) Pedigrees of families. Squares indicate men and circles women; black and white symbols represent affected and unaffected individuals, respectively. The proband is marked with an arrow, and asterisks indicate those members enrolled in this study. (B,E) Photographs of lens of the proband (IV:2 in Family 1 and III:3 in Family 2) presented as a dense congenital nuclear cataract and lamellar cataract involving the embryonal nucleus, respectively. Both were taken during cataract extraction. (C) Slit-lamp photograph of the right eye of the affected individual III:5 in Family 1, showing lens material absorption and pupillary membrane organization. (F,G) Front and oblique view of the same lens of the affected individual II:3 in Family 2, showing a perinuclear cataract with fine punctate opacities involving the central zone of the lens.

Figure 2. Mutation analysis in Cx50.

(A) DNA sequence chromatograms of the affected members and unaffected members in Family 1 show a C/G transition at codon 59 that changed Pro(CCT) to Ala(GCT) (c.175C > G, p.P59A). (B) Sanger sequence of Cx50 showing a heterozygous G > A alteration, resulting in the substitution of an Arg(CGC) by His(CAC) at amino acid residue 76 (c.227G > A, p.R76H). (C) Multiple-sequence alignment of a section of the Cx50 amino acid sequence from different species revealed high conservation of Pro59 and Arg76 (indicated by red arrows). (D) Schematic diagram of the predicted domain structure of human Cx50 showing the positions at which the P59A and R76H mutations occur. As predicted, Cx50P59A lies within the first extracellular domain and Cx50R76H is located in the boundary of the first extracellular loop and the second transmembrane domain (indicated by solid black square). NT, NH2-terminus; CT, COOH-terminus; M1–M4, transmembrane domains; E1 and E2, extracellular loop domains.

Figure 3. Hydrophilicity analysis of the wild type and mutant proteins.

Protscale results shows slight change of hydrophilicity.

Figure 4. Western blot of the wild type and mutant Cx50 in stably transfected cells.

Similar levels of protein with a band of 97 kDa were detected. The blots were probed with anti-Cx50 antibody. β-actin was used as a loading control.

Figure 5. Sub-cellular localization pattern of Cx50 proteins.

Both Cx50WT (A–C) and Cx50R76H (G–I) formed abundant gap junction plaques, as observed at appositional membranes and perinuclear cytoplasmic locations (arrows). Note the enormous aggregation blobs of Cx50R76H in the cytoplasm and plasma membrane (H,I). Images of Cx50P59A transfected cells showed a lack of gap junction plaques; instead, a very low level of expression protein was accumulated in the cytoplasm. Green: GFP; blue: DAPI staining of cell nuclei. The scale bar represents 5 μm.

Figure 6. Quantitative analysis by MTT assay of cell viability.

Induction of CxP59A expression decreased the proliferation and growth capability of cells. *P < 0.05.