Characterization of a variant of gap junction protein α8 identified in a family with hereditary cataract

Purpose: Congenital cataracts occur in isolation in about 70% of cases or are associated with other abnormalities such as anterior segment dysgenesis and microphthalmia. We identified a three-generation family in the University of California San Francisco glaucoma clinic comprising three individuals with congenital cataracts and aphakic glaucoma, one of whom also had microphthalmia. The purpose of this study was to identify a possible causative mutation in this family and to investigate its pathogenesis. Methods: We performed exome sequencing and identified a putative mutation in gap junction protein α8 (GJA8). We used PCR and DNA sequencing of GJA8 in affected and unaffected members of the pedigree to test segregation of the variant with the phenotype. We tested cellular distribution and function of the variant protein by immunofluorescence and intercellular transfer of Neurobiotin in transiently transfected HeLa cells. Results: Exome sequencing revealed a variant in GJA8 (c.658A>G) encoding connexin50 (Cx50) that resulted in a missense change (p.N220D) in transmembrane domain 4. The variant was present in all three affected family members, but was also present in the proband's grandfather who was reported to be unaffected. The mutant protein localized to the plasma membrane and supported intercellular Neurobiotin transfer in HeLa cells. Conclusions: We identified a variant in transmembrane domain 4 of Cx50 in a family with autosomal dominant congenital cataracts. This variant has been previously identified in other cataract cohorts, but it is also present in unaffected individuals. Our study demonstrates that the mutant protein localized to the plasma membrane and formed functional intercellular channels. These data suggest that GJA8 c.658A>G is most likely a benign rare variant.</p

Characterization of a variant of gap junction protein α8 identified in a family with hereditary cataract.

Congenital cataracts occur in isolation in about 70% of cases or are associated with other abnormalities such as anterior segment dysgenesis and microphthalmia. We identified a three-generation family in the University of California San Francisco glaucoma clinic comprising three individuals with congenital cataracts and aphakic glaucoma, one of whom also had microphthalmia. The purpose of this study was to identify a possible causative mutation in this family and to investigate its pathogenesis.We performed exome sequencing and identified a putative mutation in gap junction protein α8 (GJA8). We used PCR and DNA sequencing of GJA8 in affected and unaffected members of the pedigree to test segregation of the variant with the phenotype. We tested cellular distribution and function of the variant protein by immunofluorescence and intercellular transfer of Neurobiotin in transiently transfected HeLa cells.Exome sequencing revealed a variant in GJA8 (c.658A>G) encoding connexin50 (Cx50) that resulted in a missense change (p.N220D) in transmembrane domain 4. The variant was present in all three affected family members, but was also present in the proband's grandfather who was reported to be unaffected. The mutant protein localized to the plasma membrane and supported intercellular Neurobiotin transfer in HeLa cells.We identified a variant in transmembrane domain 4 of Cx50 in a family with autosomal dominant congenital cataracts. This variant has been previously identified in other cataract cohorts, but it is also present in unaffected individuals. Our study demonstrates that the mutant protein localized to the plasma membrane and formed functional intercellular channels. These data suggest that GJA8 c.658A>G is most likely a benign rare variant

List of candidate genes for hereditary cataract.

<p>List of candidate genes for hereditary cataract.</p

Protein sequence alignment.

<p>(A) Orthologous sequences of Cx50 from human, mouse, rat, chick and sheep show that amino acid N220 is highly conserved across species. (B) Alignment of other human gap junction protein sequences also demonstrated conservation of this residue among isoforms.</p

Pedigree of the family with autosomal dominant congenital cataract.

<p>The proband (III-1) is denoted with an arrow. Subject II-3 was also affected by microphthalmia. Asterisks indicate subjects from whom DNA was available.</p

<i>GJA8</i> DNA sequencing.

<p>The c.658A>G variant results in missense mutation N220D within the fourth transmembrane domain of Cx50. PCR of <i>GJA8</i> and DNA sequencing revealed that the 658A>G variant was present in all affected subjects, III-1, II-2, and II-3. The variation was also present in the proband’s maternal grandfather (I-1).</p

Cx50^N220D production, localization and function in transfected cells.

<p>(A) Proteins from homogenates of HeLa cells transiently transfected with wild type Cx50 or Cx50^N220D were resolved by SDS-PAGE and subjected to immunoblotting using rabbit polyclonal anti-human Cx50 antibodies. Samples from cells expressing either Cx50 or Cx50^N220D contain a single immunoreactive band of similar mobility. Molecular mass standards are indicated on the left. (B) Photomicrographs show the distribution of immunoreactivity to anti-Cx50 antibodies in HeLa cells transfected with wild type Cx50 (WT) or Cx50^N220D (N220D). Cells expressing WT and N220D both show substantial labeling at appositional interfaces between cells consistent with gap junction plaques. (C) Photomicrograph showing intercellular neurobiotin transfer from a microinjected cell (*) to neighboring cells in HeLa cells transiently with CX50^N220D. Bar, 16 μm for B and 10 μm for C.</p

Characterization of a variant of gap junction protein α8 identified in a family with hereditary cataract

Congenital cataracts occur in isolation in about 70% of cases or are associated with other abnormalities such as anterior segment dysgenesis and microphthalmia. We identified a three-generation family in the University of California San Francisco glaucoma clinic comprising three individuals with congenital cataracts and aphakic glaucoma, one of whom also had microphthalmia. The purpose of this study was to identify a possible causative mutation in this family and to investigate its pathogenesis.We performed exome sequencing and identified a putative mutation in gap junction protein α8 (GJA8). We used PCR and DNA sequencing of GJA8 in affected and unaffected members of the pedigree to test segregation of the variant with the phenotype. We tested cellular distribution and function of the variant protein by immunofluorescence and intercellular transfer of Neurobiotin in transiently transfected HeLa cells.Exome sequencing revealed a variant in GJA8 (c.658A>G) encoding connexin50 (Cx50) that resulted in a missense change (p.N220D) in transmembrane domain 4. The variant was present in all three affected family members, but was also present in the proband's grandfather who was reported to be unaffected. The mutant protein localized to the plasma membrane and supported intercellular Neurobiotin transfer in HeLa cells.We identified a variant in transmembrane domain 4 of Cx50 in a family with autosomal dominant congenital cataracts. This variant has been previously identified in other cataract cohorts, but it is also present in unaffected individuals. Our study demonstrates that the mutant protein localized to the plasma membrane and formed functional intercellular channels. These data suggest that GJA8 c.658A>G is most likely a benign rare variant