The family pedigree is shown in . There were three affected members—the proband (III-8, arrowhead), his brother (III-11) and their female cousin (III-5). At age 39, the proband presented with an 8-year history of slowly progressive walking difficulties, leg stiffness and slurred speech. He felt ‘awkward’ since his school years, had minimal writing difficulties, but was fit for military service at age 18. At age 21, he underwent surgery for concomitant strabismus. Currently, he is able to walk without assistance, and works as a priest. His legs and gait were spastic, and bilateral Babinski signs were found. The jaw jerk and deep tendon reflexes (DTRs) were brisk throughout, with non-sustained ankle clonus. Mild pes cavus was noted. He had no nystagmus, but pursuit eye movements were slightly saccadic. The following findings were mild: pseudobulbar dysarthria, loss of finger dexterity, dysmetria and intention tremor on finger-to-nose and heel-to-knee testing.
Fig. 1 Family pedigree. The family pedigree is shown, and the genotypes were determined for all individuals indicated by asterisks. The proband (III-8; arrowhead), III-11 and III-5 are homozygous for the I33M mutation; II-2, II-9, III-1, III-2, III-3, III-7, (more ...)
The proband's 36-year-old brother (III-11) had minimal motor difficulties since infancy and mild learning impairment at school. He reported mild intention tremor during late-childhood, and a few tonic-clonic seizures, mainly febrile, between childhood and his early teens. These problems had long been attributed to a dystocic delivery with cyanosis at birth. After age 20, he began to show a slow worsening of walking difficulties and slight dysarthria, followed by decreased dexterity with his hands. Presently, he walks with a cane, but he can still walk a few steps without aid. On neurological examination he had a shuffling, scissor-like gait, marked spasticity with mild weakness of the lower limbs, an increased jaw jerk and increased DTRs in all limbs, with sustained clonus at the ankles and bilateral Babinski signs. He had slight dysarthria, mild loss of finger dexterity, dysmetria, intention tremor on finger-to-nose testing and moderate hypodiadochokinesia. He had pes cavus with hammer toes, lumbar hyperlordosis and concomitant strabismus but no nystagmus.
The proband's 53-year-old cousin (III-5) had always been considered somewhat dull, but completed primary school without help. She walked at 20 months, and developed progressive spastic paraplegia and dysarthric speech beginning in her teens, becoming wheelchair bound at age 30. She later developed urinary incontinence followed by retention, and required a permanent catheter at age 46. More recently, she has developed episodic painful spasms in lower limbs, and marked constipation with rare fecal incontinence. On exam, she was unable to stand or walk without bilateral support. Her arms were mildly spastic, atrophied and weak; her legs were markedly spastic, wasted and completely paralysed. Her jaw jerk and all DTRs were exaggerated, with sustained right ankle clonus and bilateral Babinski signs. She had slightly saccadic pursuits without nystagmus, and moderate dysarthria with nasal, scanning and spastic qualities. She had moderate dysmetria and intention tremor on finger-to-nose testing, with marked hand hypodiadochokinesia. Light touch and pain sensations were reduced in the left upper limb and both legs. Position and vibration sense were severely impaired in the legs. She also had severe dorsal scoliosis, bilateral pes cavus and ankle contractures ().
Summary of clinical and instrumental data for patients with the I33M GJA12/GJC2 mutation
The following blood tests were normal for all affected patients: creatine kinase, uric acid, ammonia, lactate, pyruvate, alanine and other amino acids, very-long-chain fatty acids and phytanic acid. Urinary levels of organic acids, amino acids and sulfatides were also normal. Lysosomal enzyme assessment in blood leukocytes ruled out metachromatic leukodystrophy, Krabbe disease and GM1 and GM2 gangliosidoses. Neuro-ophthalmologic examination revealed only left eye amblyopia in patients III-8 and III-11, attributed to congenital strabismus. Audiometry revealed mild sensorineural hearing loss in Patient III-5 and left ear conduction hypoacusia due to previous trauma in Patient III-8.
Electromyography and motor and sensory nerve conduction studies were normal in all three patients. Motor evoked potential (MEP) studies showed prolongation of central motor conduction time (CMCT) in upper limbs, and no response in lower limbs in Patients III-8 and III-11; in Patient III-5, MEPs could be recorded only from the left upper limb, with central motor conduction time prolongation, whereas no response was obtained from the other limbs. Their electroretinogram was normal, whereas visual evoked potentials showed delayed P100 wave latencies. Brainstem auditory evoked potentials disclosed absence or severe distortion and delay of waves III and V; wave I was normal in Patients III-11 and III-5, but almost unrecognizable in Patient III-8, particularly on the left side. Upper and lower limb somatosensory evoked potential studies demonstrated delayed latencies of central components in all patients; no response could be evoked in lower limbs in Patient III-5. In all patients, EEG showed a posterior background activity of about 8 Hz with a slightly irregular morphology. The cardiovascular reflexes were normal in Patient III-8. Neuropsychological assessment revealed IQ scores of 94 in Patient III-8, 83 in Patient III-11 and 77 in Patient III-5 (normal value > 70).
Brain MR imaging and spectroscopic findings were homogeneous in the three patients and highly suggestive of a hypomyelinating leukoencephalopathy. White matter regions showed diffuse high signal intensity on T2
-weighted and low signal on T1
-weighted images (Fig. 2, Supplementary Fig. 1
-weighted signal hyperintensity was evident at level of the pons, in the region of corticospinal and spinothalamic tracts (B and F). The corpus callosum was thin in all patients. The cervical spine of Patient III-8 appeared normal (data not shown). Ventricular and posterior fossa dilation, likely secondary to white matter volume loss, was mild in Patients III-8 and III-11, but advanced in Patient III-5. Choline (Cho), N
-acetyl-aspartate (NAA) and creatine (Cr) were measured in the white matter of the centrum semiovale by 1
H MR spectroscopic imaging. The NAA/Cr ratios were within normal values, consistent with what has been reported for PMD and PMLD (Lee et al.
; Bizzi et al.
). Average Cho/NAA and Cho/Cr ratios, however, were reduced—0.49 and 0.84 in Patient III-8; 0.47 and 0.80 in Patient III-11; 0.44 and 0.75 in Patient III-5—compared with 0.60 and 1.0, respectively, in normal adults.
Fig. 2 Imaging studies show white matter abnormalities. These are MRI images from patients III-8 (A–D) and III-5 (E–H). Sagittal T1-weighted (T1W) images [spin-echo (SE): repetition time (TR)/echo time (TE) 556/13 ms; 6-mm thickness] shows thinning (more ...)
Affected patients have an I33M mutation in GJA12/GJC2
The above data indicated that the three patients from this family had a recessively inherited hypomyelinating leukoencephalopathy with an unusual clinical phenotype of a late onset complicated HSP. Although the parents of our patients were reported to be unrelated, they all originated from a small village in northern Italy, and Subjects I-2, I-3 and II-1 () had the same family name. Hence, we sequenced amplified genomic DNA of the GJA12/GJC2 gene, and found a novel missense mutation (99C > G), predicted to cause an Ile > Met amino acid substitution (I33M) in Cx47. This mutation was homozygous in the three affected individuals, heterozygous in obligate healthy carriers and some clinically normal relatives (asterisks in denote DNA analysis), and absent in 210 control alleles. Heterozygous relatives () were asymptomatic and had a normal neurological exam (II-9, III-7 and III-12); one of these relatives (III-7) had a normal brain MRI and 1H MRSI.
As shown in , I33 is located in the first transmembrane domain, which is a highly conserved region of connexins (Yeager and Nicholson, 1996
). This amino acid residue is identical in Cx47 orthologues of other vertebrates (cow, mouse, frog and zebrafish; Supplementary Fig. 2
), but Val (Cx30, Cx30.3, Cx31, Cx31.1), Leu (Cx30.2) or even Met (Cx40, Cx59, Cx62) are found at the corresponding position in other connexins (Supplementary Fig. 2
). Although mutations in the genes encoding Cx26, Cx30, Cx30.3, Cx31, Cx32, Cx40, Cx43, Cx46 and Cx50 have been described, mutations in the residues corresponding to I33 have not been identified previously.
Fig. 3 GJA12/GJC2 mutations associated with CNS diseases. This is a schematic drawing of human Cx47, illustrating the position and nature of mutations associated with CNS diseases. I33M (grey circle) is located in the first transmembrane domain. All of the other (more ...)
I33M forms gap junction plaques in HeLa cells
To investigate the underlying molecular defects of the I33M mutant, we expressed it in communication-incompetent HeLa and N2A cells. For comparison, we also expressed WT Cx47 and P87S, a missense mutant that causes PMLD (Uhlenberg et al.
), and results in loss-of-function (Orthmann-Murphy et al.
). We confirmed expression by immunoblotting lysates from bulk-selected and transiently transfected cells (Supplementary Fig. 3
). As expected (Orthmann-Murphy et al.
), P87S was localized the endoplasmic reticulum (ER) in both transiently (Supplementary Figs. 4 and 5
) and permanently transfected () cells, whereas I33M (and WT Cx47) formed gap junction plaques at apposed cell borders. We confirmed the cell surface localization of WT Cx47 and I33M by double labelling with a monoclonal antibody that recognizes cadherins. Neither parental HeLa cells, nor bulk-selected HeLa cells that had been transfected to express vector alone, expressed Cx47 (data not shown). We obtained similar results in four separate transient transfection experiments and two different bulk-selected cell lines expressing I33M.
Fig. 4 The I33M mutant forms gap junction plaques. These are confocal images of bulk-selected HeLa cells that express WT Cx47 or the indicated mutants, immunostained with a rabbit antiserum against human Cx47 (red) and a mouse monoclonal antibody against pan-cadherin (more ...)
I33M does not form functional homotypic channels
Because patients expressing I33M have a milder phenotype than do patients with PMLD, and I33M can form gap junction plaques, we hypothesized that I33M might form gap junction channels with altered functional properties. To determine whether I33M could transfer small molecules, we scrape loaded cells (el-Fouly et al.
; Trosko et al.
), using gap junction tracers of different sizes, shapes and charge. A confluent monolayer of cells was injured with a scalpel blade in media that contained 2% neurobiotin (NB; MW 287, + 1) or 0.1% Lucifer Yellow (LY; MW 443, −2). As in parental cells or cells expressing vector alone (data not shown), no transfer was seen past the scrape line in bulk-selected cells expressing I33M or P87S (B–C and E–F), whereas NB and LY transferred to cells beyond the scrape line for cells expressing WT Cx47 (A and D). We also confirmed that the cells along the scrape line expressed the appropriate connexin (Supplementary Fig. 6
). This experiment was repeated at least twice in two different bulk-selected cells lines expressing I33M with similar results.
Fig. 5 I33M is impermeable to low molecular weight tracers. These are representative images of bulk-selected HeLa cells that stably express WT Cx47 or the indicated mutants, scrape-loaded with neurobiotin (A—C) or Lucifer Yellow (D—F). Cells (more ...)
We used a more sensitive assay to determine whether Cx47 mutants can form functional homotypic gap junctions—dual whole-cell patch clamping on transiently transfected N2A cells. In this experiment, each cell was transiently transfected to express a single connexin as well as EGFP. For negative control pairs, cells expressing a single connexin were paired with cells expressing EGFP alone (Cx/EGFP). Voltage ramps or steps were applied to one cell of an EGFP-expressing pair, and current responses were measured in the second cell; the junctional voltage (Vj) corresponds to the difference in voltage between the two cells. As shown in , I33M/I33M homotypic pairings failed to form functional channels (0/6 pairs), whereas cell pairs expressing WT Cx47 were coupled. The difference between I33M/I33M and WT Cx47/Cx47 pairings was statistically significant (I33M/I33M versus Cx47/Cx47, P = 0.0022, Fisher's exact test). Thus, I33M does not appear to form functional homotypic gap junctions by two different assays.
Summary of dual whole-cell patch clamp recordings
I33M forms functional heterotypic channels with Cx43 in a cell model system
To determine whether I33M can form heterotypic channels with Cx43, we applied morphological and functional assays we used previously to show that Cx47 mutants associated with PMLD do not form functional heterotypic gap junctions with WT Cx43 (Orthmann-Murphy et al.
). In the morphological assay, cell lines in which at least 90% of cells stably expressed Cx47 (I33M, P87S or WT Cx47) or Cx43 were transiently transfected to express DsRed (DsRed + cells). The DsRed + cells (expressing Cx47 or Cx43) were mixed with DsRed- cells (expressing Cx43 or Cx47, respectively) at a ratio of 1:20, and, 24 h after plating, immunostained for Cx47 and Cx43. For each combination, we determined whether the connexin puncta at the periphery of the DsRed + cell overlapped with connexin puncta expressed by the surrounding DsRed- cells. As shown in , P87S was localized to the ER, and did not appear to form overlapping puncta with Cx43 (Orthmann-Murphy et al.
). In contrast, cells expressing I33M or WT Cx47 formed overlapping puncta with Cx43, suggesting that I33M/Cx43 may form functional channels. We quantified these results by counting the number of puncta at the cell membrane of each central DsRed + cell, and determining whether these puncta overlapped with the connexin signal in the surrounding DsRed- cells (D). In this analysis, the mixtures are designated as ‘CxA*/CxB,’ where the asterisk denotes the connexin expressed by the DsRed + cell. Mixtures containing I33M/Cx43 (I33M*/Cx43 and Cx43*/I33M) produced a significantly larger proportion of DsRed + cells with at least one overlapping punctum than did P87S/Cx43 mixtures (I33M*/Cx43 versus P87S*/Cx43 or Cx43*/P87S, Cx43*/I33M versus P87S*/Cx43, P
< 0.0001; Cx43*/I33M versus Cx43*/P87S, P
= 0.0013, Fisher's test), but were not significantly different than mixtures containing Cx47/Cx43 (Cx47*/Cx43 and Cx43*/Cx47).
Fig. 6 I33M/Cx43 pairings form gap junction plaques. (A–C) HeLa cells stably expressing Cx47 WT or one of the mutants (I33M or P87S) or Cx43 were transiently transfected to express DsRed (DsRed + ) and mixed with cells expressing one of the connexins, (more ...)
In the electrophysiogical assay, we used dual whole-cell patch clamping on pairs of N2A cells. In these experiments, each cell was transiently transfected to express a single connexin as well as EGFP or monomeric DsRed, so that each member of a cell pair could be unambiguously identified. To confirm that the cells expressing EGFP or DsRed also expressed the expected connexin, we immunostained transiently transfected cells (Supplementary Fig. 5
). In this way, we previously showed that three loss-of-function Cx47 mutants that cause PMLD (P87S, Y269D and M283T) do not form functional heterotypic channels with Cx43 (Orthmann-Murphy et al.
). In contrast, we detected Ij
activation for six of 13 (46%) I33M/Cx43 pairs tested, somewhat less than we previously found for all of the WT Cx47/Cx43 heterotypic pairs we have tested (40 of 55; 73%). As shown in , the mean Gj
measured at Vj
= 0 for I33M/Cx43 channels is significantly smaller than that of WT Cx47/Cx43 channels (I33M/Cx43 versus WT Cx47/Cx43, P
< 0.05, Mann–Whitney test). Negative control pairs, in which cells expressing a single connexin were paired with cells expressing EGFP or DsRed alone, showed low levels of coupling probably due to formation of heterotypic channels with an endogenous connexin expressed by parental N2A cells (; Orthmann-Murphy et al.
To better define the functional differences between I33M/Cx43 and Cx47/Cx43 channels, we examined macroscopic current responses and Gj
relations. By convention, pairing designation is ‘connexin expressed by cell 2/connexin expressed by cell 1’. Both cells in the pair were voltage clamped to 0 mV; cell 1 was stepped between −100 and 100 mV in 20 mV increments, and current was recorded from cell 2. For I33M/Cx43 heterotypic pairings, negative pulses (≤−40 mV) applied to the cell expressing Cx43 activate junctional currents (Ij
) (A). Assuming that Gj
is a linear function of Vj
between −20 mV and +20 mV, the normalized Gj
versus junctional voltage (Vj
) relation reveals that I33M/Cx43 channels are about fifty times more likely to be open after a 12.5-s pulse to Vj
= −100 mV than at Vj
= 0 mV (solid line, B). This is strikingly different than WT Cx47/Cx43 channels (dashed line), which are more likely to be open at Vj
= 0 mV than at either −100 mV or + 100 mV (B; Orthmann-Murphy et al.
). The left shift of the Gj
relation for I33M/Cx43 channels is likely explained by an extremely low open probability of the I33M hemichannel when Vj
< −40 mV are applied to the cell expressing Cx43; this can account for the significantly reduced Gj
values detected at Vj
= 0 mV for both I33M/Cx43 and I33M/I33M channels (). Although the WT Cx43 hemichannel has negative Vj
gating polarity (Bukauskas et al.
) and should be closed under the conditions that open I33M hemichannels, Cx43 rarely closes fully (Bukauskas et al.
). It is possible, then, that I33M is forming a channel with a substate of Cx43. Properties of I33M/WT Cx47 channels (in 5/15 pairs) are similar to those described for I33M/Cx43 (data not shown).
Fig. 7 Functional properties of I33M/Cx43 channels. N2A cells were transiently transfected with a pIRES2-EGFP or a pIRES2-DsRed bicistronic expression vector that also contained WT Cx43 or I33M. After 24 h, red and green cell pairs were generated by mixing the (more ...)