A new mutation in GJC2 associated with subclinical leukodystrophy

Abstract

Recessive mutations in GJC2, the gene-encoding connexin 47 (Cx47), cause Pelizaeus-Merzbacher-like disease type 1, a severe dysmyelinating disorder. One recessive mutation (p.Ile33Met) has been associated with a much milder phenotype--hereditary spastic paraplegia type 44. Here, we present evidence that a novel Arg98Leu mutation causes an even milder phenotype--a subclinical leukodystrophy. The Arg98Leu mutant forms gap junction plaques in HeLa cells comparable to wild-type Cx47, but electrical coupling was 20-fold lower in cell pairs expressing Arg98Leu than for cell pairs expressing wild-type Cx47. On the other hand, coupling between Cx47Arg98Leu and Cx43WT expressing cells did not show such reductions. Single channel conductance and normalized steady-state junctional conductance-junctional voltage (G(j)-V(j)) relations differed only slightly from those for wild-type Cx47. Our data suggest that the minimal phenotype in this patient results from a reduced efficiency of opening of Cx47 channels between oligodendrocyte and oligodendrocyte with preserved coupling between oligodendrocyte and astrocyte, and support a partial loss of function model for the mild Cx47 associated disease phenotypes.

Figure 1. Brain MRI

a, b, c, d. Diffuse hyperintensity in the subcortical, lobar, and periventricular white matter including posterior limb of the internal capsules (a & d) and cerebral peduncles (d) is seen. The signal abnormalities were slightly more marked in the precentral and postcentral gyri (c). e. Normal white matter signal intensity is noted on T1-weighted image. f. Thinning of the corpus callosum, more marked posteriorly, is present. In all the images enlarged ventricles and sulci are noted. a, b, and c are axial T2-weighted images, d is a coronal FLAIR image, and e and f are sagittal T1-weighted images.

Figure 2. The Arg98Leu mutant forms GJ plaques

These are images of HeLa cells that were transiently transfected to express Arg98Leu or wild-type Cx47 (WT), immunostained with a rabbit antiserum against human Cx47 and counterstained with DAPI. Both Arg98Leu and Cx47WT form GJ plaques (arrowheads) at apposed cell membranes. Scale bar: 10 µm

Figure 3. Junctional coupling for Cx47WT, and Cx47Arg98Leu (R98L) mutant and controls

Neuro2a cells were transiently transfected using pIRES-EGFP (iG) or pIRESdsRed (iR) vectors to express wild-type Cx47WT (47WT), Cx47Arg98Leu (R98L), or Cx43WT (43WT). Homotypic R98L/R98L and heterotypic R98L/47WT pairs show substantially lower levels of functional coupling than seen for 47WT/47WT homotypic pairs. The coupling for the R98L/43WT heterotypic pairings is not significantly different than for the 47WT/43WT pairings; R98L/43WT is statistically significantly different than the vector only controls.* p<0.05, ** p<0.01, *** p<0.001.

Figure 4. Single channel recordings of Cx47Arg98Leu/Cx47Arg98Leu homotypic and Cx47Arg98Leu/Cx43WT heterotypic channels

The unitary conductance transitions for homotypic and Cx43WT/Cx47Arg98Leu heterotypic channels were determined by applying voltage ramps (V_j bottom panels) from +100 to −100 mV to cell 1 (expressing Cx47Arg98Leu or Cx43WT) of poorly coupled cell pairs, and measuring the junctional currents (I_j, upper panels) in cell 2 (expressing Cx47Arg98Leu). a. The conductance the of Cx47Arg98Leu/Cx47Arg98Leu channel shows predominant unitary transitions for the homotypic channel of about 48 pS, similar to what we and others have found for the Cx47WT homotypic channels [29, 43]. The residual state is difficult to estimate with certainty because the number of active channels is not known with certainty but appears to be about 5 pS. b. The single channel conductance of the Cx47Arg98Leu/Cx43WT channel shows minimal rectification, with a conductance of about 85 pS at −100 mV with respect to Cx43WT and about 75 to 80 pS at +100 mV with respect to Cx43WT. Gating of the Cx43 hemichannel likely accounts for closures with negative V_j (downgoing arrows) whereas gating of the Cx47 hemichannel likely accounts for closures when the polarity of V_j is reversed (upgoing arrows), because both of these hemichannels have negative gating polarities. Traces were filtered at 150 Hz (a) and 200 Hz (b).

Figure 5. Representative current traces (a, c) and G_j-V_j relations (b, d), for Arg98Leu expressed in Neuro2a cells and paired homotypically or heterotypically with Cx43 WT

a. Macroscopic junctional currents for homotypically paired cells expressing the Cx47Arg98Leu mutant are similar to those seen for Cx47WT. b. Steady state G_j-V_j relations for homotypically paired cells expressing the Arg98Leu mutant are similar to those seen for Cx47WT, though the Arg98Leu mutant shows a slightly decreased G_min. c. Macroscopic junctional currents between heterotypic cells pairs in the Cx43WT/Cx47Arg98Leu configuration are similar to those seen for Cx43WT/Cx47WT. d. Steady state G_j-V_j relations for heterotypic Cx43WT/Cx47Arg98Leu junctions are, for the most part, similar to those seen for Cx43WT/Cx47WT junctions though the negative limb of the Cx43WT/ Arg98Leu G_j-V_j relation (corresponding to the closure of the Cx47 hemichannel) shows a modest inward shift (indicating closure at smaller V_j) and a slightly decreased G_min compared to that for Cx43WT/Cx47WT. Filled squares are average normalized conductances. Error bars represent SEM. Solid lines are Boltzmann fits to the steady state Cx47Arg98Leu/Cx47Arg98Leu and Cx43WT/Cx47Arg98Leu data. Dashed lines are Boltzmann fits to steady state Cx47WT/Cx47WT and Cx43WT/Cx47WT G_j-V_j relations.