Mutations in the gene encoding gap junction protein alpha 12 (connexin 46.6) cause Pelizaeus-Merzbacher-like disease

Abstract

The hypomyelinating leukodystrophies X-linked Pelizaeus-Merzbacher disease (PMD) and Pelizaeus-Merzbacher-like disease (PMLD) are characterized by nystagmus, progressive spasticity, and ataxia. In a consanguineous family with PMLD, we performed a genomewide linkage scan using the GeneChip Mapping EA 10K Array (Affymetrix) and detected a single gene locus on chromosome 1q41-q42. This region harbors the GJA12 gene, which encodes gap junction protein alpha 12 (or connexin 46.6). Gap junction proteins assemble into intercellular channels through which signaling ions and small molecules are exchanged. GJA12 is highly expressed in oligodendrocytes, and, therefore, it serves as an excellent candidate for hypomyelination in PMLD. In three of six families with PMLD, we detected five different GJA12 mutations, including missense, nonsense, and frameshift mutations. We thereby confirm previous assumptions that PMLD is genetically heterogeneous. Although the murine Gja12 ortholog is not expressed in sciatic nerve, we did detect GJA12 transcripts in human sciatic and sural nerve tissue by reverse-transcriptase polymerase chain reaction. These results are in accordance with the electrophysiological finding of reduced motor and sensory nerve conduction velocities in patients with PMLD, which argues for a demyelinating neuropathy. In this study, we demonstrate that GJA12 plays a key role in central myelination and is involved in peripheral myelination in humans.

Figure  A1

Genome scan using the GeneChip Mapping EA 10K Array (Affymetrix). Vertical bars separate the chromosomes. The scanning revealed linkage of PMLD to SNPs on chromosome 1q41-q42. Multipoint linkage analysis yielded a maximum LOD score of 4.83 for SNP markers rs1563353–rs544528.

Figure 1

Axial T2-weighted magnetic resonance images of the brain at the level of the basal ganglia. A, Patient with PMLD at 6 years of age (patient III:3; fig. 3). B, Patient with PMD at 7 years of age. The patients in panels A and B show nearly identical hypomyelination patterns of central white matter, as indicated by diffusely enhanced signal intensity. C, Low signal of normal myelination in an unaffected child.

Figure 2

Domains of the GJA12 gene product. Blackened circles depict the locations of mutations. The missense mutations lie within the second (P90S) and fourth (Y272D) transmembrane and third cytoplasmic (M286T) domains.

Figure 3

Haplotypes in family 1 with PMLD. Haplotype analysis indicated that the cosegregating segment of the PMLD locus is flanked proximally by marker rs3902857 and distally by marker rs1321257 on chromosome 1q41-q42.

Figure 4

Segregation of GJA12 mutations (RFLP analysis). In family 2 (f2), the affected boy was compound heterozygous, carrying the paternal c.268C→T missense mutation and the maternal c.989delC frameshift deletion. In family 3 (f3), the affected boy carried the paternal c.718C→T nonsense mutation and the maternal c.814T→G missense mutation. Family numbers correspond to those in table 1. co = control; f1–f3 = families 1–3; mut = mutated GJA12; wt = wild-type GJA12.

Figure 5

Alignment of selected regions of human GJA12 with orthologs of other species, including the Japanese pufferfish Fugu rubripes. Arrows indicate positions of the missense and nonsense mutations in patients with PMLD. Family numbers correspond to those in table 1. f1–f3 = families 1–3.

Figure 6

PCR products of GJA12 and GJB1 cDNA after various numbers of PCR cycles in human brain, spinal cord, sciatic and sural nerve, and skeletal muscle. GJA12 and GJB1 are more highly expressed in brain and spinal cord than in peripheral nerve tissue. Untranscribed RNA was used as a control (co).