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

Abstract

PurposeCongenital 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.MethodsWe 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.ResultsExome 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.ConclusionsWe 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.

New GJA8 variants and phenotypes highlight its critical role in a broad spectrum of eye anomalies

“…However, in contrast with these previously reported variants, the frequency of the p.(Leu7Met) variant in unaffected individuals, in particular of African/Afro-American ethnicity, suggests that the substitution with a methionine might be tolerated. This substitution of asparagine 220, located in the TM4 domain and predicted deleterious by SIFT and Polyphen-2, has been reported before in a proband with congenital cataract and microcornea (Ma et al, 2016) and in a three generation family with congenital cataracts and aphakic glaucoma (Kuo et al, 2017). However, in those families it did not co-segregate with the phenotype and therefore was classified as benign.…”

“…These were identified in individuals with AMC, but without cataracts, in unaffected parents either in this or previous studies (Ma et al, 2016;Kuo et al, 2017) and in controls (gnomAD).…”

“…However, in those families it did not co-segregate with the phenotype and therefore was classified as benign. This was also supported by functional experiments showing that this rare polymorphism did not abolish intercellular channel function (Kuo et al, 2017).…”

Characterization of a Novel Gja8 (Cx50) Mutation in a New Cataract Rat Model

“…However, only one reported mutation is located in the human TM4 region (p.N220D) while no disorders of TM4 region have been found in rats or mice yet. 6 , 21 We identified a spontaneously new point mutation of TM4 domain in Cx50 which is novel in murine models (See in Supplementary Fig. S1 ).…”

Evaluating gap junction variants for a role in pediatric cataract: an overview of the genetic landscape and clinical classification of variants in the GJA3 and GJA8 genes

“…The LB classification was predominantly due to the population minor allele frequency being greater than that expected for the disorder (rule BS1, Table 3). In GJA3, this was the only synonymous variant included in this review, c.84G>A p.(Val28=) [107] while in GJA8, there were three LB variants p.(Asn220Asp) [134,151], p.(Ile247Met) [152][153][154], and p.(Glu368Gln) [155,156]. In the GJA8 gene, there were four variants classified as VUS due to the application of both pathogenic and benign rules, leading to an aggregate score of 0.…”