PubMed · 10979110
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G E Green, R J Smith, J P Bent, E S Cohn. 2000-09-13. Genetic testing to identify deaf newborns.. https://doi.org/10.1001/jama.284.10.1245
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High-resolution, fluorescence deconvolution (DV) microscopy was implemented to obtain a detailed view of the organization and structural composition of gap junctions assembled from one or two different connexin isotypes in live and fixed cells. To visualize gap junctions, the structural protein components of gap junction channels, the connexin polypeptides alpha1(Cx43), beta1(Cx32), and beta2(Cx26), were tagged on their C-termini with the autofluorescent tracers green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants. Tagged connexins were expressed in transiently transfected HeLa cells. Comprehensive analysis including dye-transfer analysis demonstrated that the tagged connexins trafficked, assembled, and packed normally into functional gap junction channel plaques. Such gap junction plaques were examined by single, dual, and triple-color DV microscopy. High-resolution images and three-dimensional volume reconstructions of gap junction plaques were obtained by this technique, which revealed several new aspects of gap junction structure. Specifically, the studies demonstrated that the mode of channel distribution strictly depends on the connexin isotypes. Here we present such images, and volume reconstructions in context with images obtained by other light, and electron microscopic techniques, such as laser scanning confocal, conventional wide-field fluorescence, thin section, and freeze-fracture electron microscopy. In addition, we give a simple description of the principal mechanisms of DV microscopy, name advantages and disadvantages, and discuss issues such as dual-color imaging using CFP and YFP, spatial resolution, colocalization, and avoiding imaging artifacts.
Mutations in the gap junction beta2 (GJB2) gene, Connexin 26 (Cx26), cause nonsyndromic sensorineural recessive deafness (NSRD). Two frameshift mutations, 167delT and 35delG, are the most frequent Cx26 lesions causing NSRD. The 35delG mutation is panethnic, while the 167delT lesion occurs almost exclusively in the Ashkenazi Jewish population at a carrier frequency of 2 to 4%. To facilitate carrier detection, a simple nonradioactive allele-specific oligonucleotide (ASO) hybridization assay was developed for the 167delT and 35delG mutations. Screening of 1012 anonymous Ashkenazi Jewish individuals from the New York Metropolitan area revealed carrier frequencies for 167delT and 35delG of 3.96% (95% CI: 2.75-5.15%) and 0.69% (95% CI: 0.18-1.20%), respectively. This sensitive, specific, and relatively inexpensive method can reliably identify affected newborns and patients with NSRD as well as facilitate carrier screening for Connexin 26 deafness in the Ashkenazi Jewish community.
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