Identification of connexin43 (alpha 1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE)

Gap junction channels formed by the connexin43 protein are considered to pl ay crucial roles in development and function because they allow the direct cell-to-cell exchange of molecules that mediate multiple signaling events. Previous results have shown that connexin43 channels are intricately gated by phosphorylation and that disruption of this regulation gives rise to sev ere heart malformations and defects of laterality in human, chick and frog. Here we report the identification of connexin43 gene mutations that re res ent a minor population of connexin43 alleles, which could be reliably detec ted by using denaturing gradient gel electrophoresis (DGGE) to visualize no rmal and mutant DNAs that were separately sequenced. In contrast, sequencin g of total PCR products without DGGE-pre-selection failed to consistently i dentify these mutations. Forty-six controls and 20 heart transplant recipie nts were examined in this study. In the latter group, 14 children had hypop lastic left heart syndrome (HLHS) in which connexin43 gene defects were det ected in eight. The remaining six transplant patients with HLHS and all con trols showed no defects. All eight HLHS children with gene defects had the same four substitutions: two that were silent polymorphisms, and two that w ere missense, replacing arginine codons at positions 362 and 376 with codon s for glutamines. All four of these substitutions are identical to the nucl eotide sequence of the connexin43 pseudogene, suggesting the possibility of an illicit recombination. A breakpoint region was identified 5 ' to the mu tation site in a 63 bp domain that is 100% identical in the gene and pseudo gene. Results from in vitro phosphorylation indicate that the absence of ar ginines 362 and 376 completely, abolishes phosphorylation in the connexin43 channel regulation domain suggesting a possible mechanism for the patholog ies associated with HLHS. (C) 2001 Elsevier Science B.V. Ali rights reserve d.