Human Genetics

, Volume 127, Issue 5, pp 545–551

Novel mutations in the connexin43 (GJA1) and GJA1 pseudogene may contribute to nonsyndromic hearing loss

  • Hui-Mei Hong
  • Jiann-Jou Yang
  • Jia-Ching Shieh
  • Mei-Ling Li
  • Shuan-Yow Li
Original Investigation

DOI: 10.1007/s00439-010-0791-x

Cite this article as:
Hong, HM., Yang, JJ., Shieh, JC. et al. Hum Genet (2010) 127: 545. doi:10.1007/s00439-010-0791-x

Abstract

Connexins (CXs), a large family of membrane proteins, are key components of gap junction channels. Among a cohort of patients with nonsyndromic hearing loss, we have recently identified three novel missense mutations in the GJA1 gene and GJA1 pseudogene (ρGJA1) as likely being causally related to hearing loss. However, the functional alteration of CX43 caused by the mutations of GJA1 and ρGJA1 gene remains unclear. This study compares the intracellular distribution and assembly of three CX43 mutants expressed in HeLa cells with their wild-type (WT) counterparts and the effects of the mutant proteins on those cells. Localization assay of WT CX43 reveals a typical punctuate fluorescence pattern of a gap junction channel between neighboring expression cells. Additionally, immunoblotting analysis of the transfectants confirms the production of mutant proteins, in which their distributions along appositional membranes are determined using immunofluorescent staining procedures. Furthermore, dye transfer assay results demonstrate that gap junctional intercellular communication is less in HeLa cells carrying mutant GJA1 or ρGJA1 gene than in WT-expressing cells. The results of this study suggest that the three mutations in GJA1 or ρGJA1 that we previously reported result in at least partial loss of normal functions carried out by CX43, which may form a basis for the mechanism contributing to hearing loss in patients.

Supplementary material

439_2010_791_MOESM1_ESM.jpg (57 kb)
Supplemental Fig. 1. Topology diagram of the human Cx43 protein. Approximate locations of Cx43 mutations are indicated by black stars, showing that the S69P mutant is located in the E1, 932delC and T326I mutants in the tail of CL. M1-4: transmembrane domains; E1-2: extracellular domains; CL: cytoplasmic linking domain; N: N-terminal domain; C: C-terminal domain. (JPG 310 kb)
439_2010_791_MOESM2_ESM.jpg (310 kb)
Supplemental Fig. 2. ConSeq predictions demonstrated on human Cx43 (NP_000156.1; SWISS-PROT: P17302 (CXA1_Human)), using 50 homologs obtained from the Pfam database. The sequence of the Cx43 protein is displayed with the evolutionary rates at each site color-coded onto it (see legend). The residues of the Cx43 sequence are numbered starting from 1. The first row below the sequence lists the predicted burial status of the site (i.e. “b”—buried versus “e”—exposed). The second row indicates residues predicted to be structurally and functionally important: “s” and “f”, respectively. Vertical arrows indicate amino acid codons (p.S69 and p.T326). (JPG 56 kb)

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Hui-Mei Hong
    • 1
    • 3
  • Jiann-Jou Yang
    • 1
    • 2
  • Jia-Ching Shieh
    • 1
    • 2
  • Mei-Ling Li
    • 1
  • Shuan-Yow Li
    • 1
    • 2
  1. 1.Department of BioMedical SciencesChung Shan Medical UniversityTaichungTaiwan, ROC
  2. 2.Department of Medical ResearchChung Shan Medical University HospitalTaichungTaiwan, ROC
  3. 3.Institute of MedicineChung Shan Medical UniversityTaichungTaiwan, ROC

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