The E368Q Mutant Allele of GJA8 is Associated with Congenital Cataracts with Intrafamilial Variation in a South Indian Family

Abstract

Purpose: To determine the basis of the autosomal dominant congenital cataracts in a three generation south Indian pedigree.

Methods: The proband and several family members underwent a complete ophthalmic examination. The coding regions of eight candidate genes (CRYAA, CRYBB2, CRYGC, CRYGD, GJA3, GJA8, AQP0, and PITX3) were amplified by PCR and directly sequenced. Wild type and mutant connexin50 (Cx50) were expressed by stable transfection of HeLa cells. Their cellular distributions and function were examined by immunofluorescence microscopy and by microinjection of gap junction permeant tracers, respectively.

Results: Congenital cataracts (with some variations in phenotype) segregated as an autosomal dominant trait within a three generation pedigree. Three affected individuals (proband, sibling and mother) showed a sequence variation in the candidate gene GJA8 encoding Cx50: a c.1102G>C transversion encoding a substitution of glutamate for glutamine at position 368 (E368Q). This substitution was absent from an unaffected family member (paternal aunt) and 100 healthy controls of the same ethnicity. In transfected HeLa cells, both wild type Cx50 and E368Q localized to gap junction plaques, and supported similar levels of intercellular transfer of Neurobiotin.

Conclusions: The E368Q mutant allele of GJA8 is associated with autosomal dominant congenital cataracts with phenotypic variability. E368Q forms gap junction plaques and functional channels in transfected HeLa cells.

Figure 1

Pedigree of the family with autosomal dominant congenital cataracts. Black symbols indicate affected individuals and open symbols indicate unaffected individuals. Diagonal lines indicate deceased family members. The proband is indicated with an arrow. The proband (III-4), a sibling (III-1) and the mother (II-6) were affected.

Figure 2

Chromatograms of DNA sequences show the association of a c.1102G>C transversion with the cataract phenotype in affected family members. The chromatogram from an unaffected individual (II-2) shows only the wild type GJA8 allele with G at position 1102. Sequence chromatograms from two affected family members (II-6 and III-1) and the proband (III-4) show both C and G at position 1102. The mutant allele containing a G to C transversion would result in the substitution of glutamine for glutamate at amino acid residue 368 (E368Q).

Figure 3

Diagram of the topology of Cx50 in the plasma membrane and the location of the E368Q mutation.

Figure 4

The E368Q mutant forms gap junction plaques and shows normal levels of intercellular communication. A, B. Immunofluorescence localization of wild type Cx50 (A) and E368Q (B) in stably transfected HeLa cells. Both wild type and mutant proteins show appositional membrane staining characteristic of gap junctions (arrows). Bar, 13 μm in A and 17 μm in B. C. Bar graphs show the extent of intercellular communication determined by counting Neurobiotin-labeled neighboring cells after microinjection of the tracer in untransfected HeLa cells (None) or cells expressing wild type Cx50 or E368Q. The numbers of microinjected cells are indicated in parentheses.