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The GJA8 allele encoding CX50I247M is a rare polymorphism, not a cataract-causing mutation.
Graw J
,
Schmidt W
,
Minogue PJ
,
Rodriguez J
,
Tong JJ
,
Klopp N
,
Illig T
,
Ebihara L
,
Berthoud VM
,
Beyer EC
.
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The aim of this study was the genetic, cellular, and physiological characterization of a connexin50 (CX50) variant identified in a child with congenital cataracts. Lens material from surgery was collected and used for cDNA production. Genomic DNA was prepared from blood obtained from the proband and her parents. PCR amplified DNA fragments were sequenced and characterized by restriction digestion. Connexin protein distribution was studied by immunofluorescence in transiently transfected HeLa cells. Formation of functional channels was assessed by two-microelectrode voltage-clamp in cRNA-injected Xenopus oocytes. Ophthalmologic examination showed that the proband suffered from bilateral white, diffuse cataracts, but the parents were free of lens opacities. Direct sequencing of the PCR product produced from lens cDNA showed that the proband was heterozygous for a G>T transition at position 741 of the GJA8 gene, encoding the exchange of methionine for isoleucine at position 247 of CX50 (CX50I247M). The mutation was confirmed in the genomic DNA, but it was also present in the unaffected mother. When expressed in HeLa cells, both wild type CX50 and CX50I247M formed gap junction plaques. Both CX50 and CX50I247M induced gap junctional currents in pairs of Xenopus oocytes. Although the CX50I247M substitution has previously been suggested to cause cataracts, our genetic, cellular, and electrophysiological data suggest that this allele more likely represents a rare silent, polymorphic variant.
Figure 2. Molecular analysis of proband LB and her parents. A: Sequence analysis of GJA8 cDNA indicates a T/G heterozygosity at position 741 (red arrow) for the proband (II.1). B: The T→G exchange at position 741 leads to an amino acid exchange from Ile to Met at position 247 (I247M) and creates an SfaN/LweI restriction site in the mutated sequence. C: Restriction digest using LweI in the members of the family demonstrates its presence also in the unaffected mother (red arrows).
Figure 3. Immunodetection of wild type and mutant CX50 in transfected HeLa cells. A, B: HeLa cells transfected with CX50 or CX50I247M were fixed 48 h after transfection and subjected to immunofluorescence using anti-CX50 antibodies. The distribution of CX50 immunoreactivity appeared similar in both groups of cells; cells expressing either CX50 or CX50I247M showed a significant number of gap junctional plaques (arrows). The scale bar represents 13 μm in A and 17 μm in B.
Figure 4. Gap junctional conductances induced by wild type or mutant CX50. Graph shows a summary plot of steady-state gap junctional conductances in pairs of Xenopus oocytes injected with cRNAs encoding wild type CX50 or CX50I247M or injected with no connexin cRNA (AS). Results are presented as mean ± .E.M. Numbers in parentheses indicate the number of oocyte pairs studied in each case.
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