A variant in the carboxyl-terminus of connexin 40 alters GAP junctions and increases risk for tetralogy of Fallot

Abstract

GJA5 gene (MIM no. 121013), localized at 1q21.1, encodes for the cardiac gap junction protein connexin 40. In humans, copy number variants of chromosome 1q21.1 have been associated with variable phenotypes comprising congenital heart disease (CHD), including isolated TOF. In mice, the deletion of Gja5 can cause a variety of complex CHDs, in particular of the cardiac outflow tract, corresponding to TOF in many cases. In the present study, we screened for mutations in the GJA5 gene 178 unrelated probands with isolated TOF. A heterozygous nucleotide change (c.793C4T) in exon 2 of the gene leading to the p.Pro265Ser variant at the carboxyl-terminus of the protein was found in two unrelated sporadic patients, one with classic anatomy and one with pulmonary atresia. This GJA5 missense substitution was not observed in 1568 ethnically-matched control chromosomes. Immunofluorescent staining and confocal microscopy revealed that cells expressing the mutant protein form sparse or no visible gap-junction plaques in the region of cell-cell contact. Moreover, analysis of the transfer of the gap junction permanent tracer lucifer yellow showed that cells expressing the mutant protein have a reduced rate of dye transfer compared with wild-type cells. Finally, use of a zebrafish model revealed that microinjection of the GJA5-p.Pro265Ser mutant disrupts overall morphology of the heart tube in the 37% (22/60) of embryos, compared with the 6% (4/66) of the GJA5 wild-type-injected embryos. These findings implicate GJA5 gene as a novel susceptibility gene for TOF.

Phenotypic Variability Associated with a Large Recurrent 1q21.1 Microduplication in a Three-Generation Family

“…This is based on several lines of evidence: (a) Gja5 heterozygous and homozygous knock-out mice show various cardiac malformations [Gu et al, 2003]; (b) germline and somatic mutations in GJA5 predispose to atrial fibrillation [Gollob et al, 2006;Yang et al, 2010;Wirka et al, 2011]; (c) small (100-200 kb) rare duplications encompassing only the GJA5 gene are enriched in patients with non-syndromic TOF [Soemedi et al, 2012]; (d) and lastly, a missense variant (p.Pro265Ser) at the carboxylterminus of GJA5 was detected in 2/178 patients with non-syndromic TOF. This variant was not observed in 1,568 ethnically-matched controls [Guida et al, 2012]. The hypertrabeculated myocardium observed in patient IV-1 has not been reported previously in patients with 1q21.1 microduplications.…”

The diagnostic value of next generation sequencing in familial nonsyndromic congenital heart defects

“…Novel genes have been constantly reported in association with CHD. Since the design of this capture panel, mutations in GATA5, BVES , and GJA5 were shown to cause sporadic or familial TOF [Guida et al, ; Wei et al, ; Wu et al, ], MEF2C mutations were identified in patients with outflow tract defects [Kodo et al, ], NPHP4 and DICER1 variants were described in association with TGA [Foulkes et al, ; French et al, ].…”

Role of connexins in human congenital heart disease: the chicken and egg problem

“…A very interesting study published this year (Guida et al, 2013) characterized over 150 patients with non-syndromic Fallot and found in 1% of their patients a heterozygous nucleotide change in the Cx40 gene leading to altered amino acid sequence. This Pro265Ser variant was not seen in healthy volunteers [amino acid 265 is the binding region for src (sarcoma Rous kinase)].…”