Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia type 2 (SPG2) are X-linked developmental defects of myelin formation affecting the central nervous system (CNS). They differ clinically in the onset and severity of the motor disability but both are allelic to the proteolipid protein gene (PLP), which encodes the principal protein components of CNS myelin, PLP and its spliced isoform, DM20. We investigated 52 PMD and 28 SPG families without large PLP duplications or deletions by genomic PCR amplification and sequencing of the PLP gene. We identified 29 and 4 abnormalities respectively. Patients with PLP mutations presented a large range of disease severity, with a continuum between severe forms of PMD, without motor development, to pure forms of SPG. Clinical severity was found to be correlated with the nature of the mutation, suggesting a distinct strategy for detection of PLP point mutations between severe PMD, mild PMD and SPG. Single amino-acid changes in highly conserved regions of the DM20 protein caused the most severe forms of PMD. Substitutions of less conserved amino acids, truncations, absence of the protein and PLP-specific mutations caused the milder forms of PMD and SPG. Therefore, the interactions and stability of the mutated proteins has a major effect on the severity of PLP-related diseases.

Pelizaeus-Merzbacher Disease (PMD) is an X-linked developmental defect of myelination affecting the central nervous system and segregating with the proteolipoprotein (PLP) locus. Investigating 82 strictly selected sporadic cases of PMD, we found PLP mutations in 77%; complete PLP-gene duplications were the most frequent abnormality (62%), whereas point mutations in coding or splice-site regions of the gene were involved less frequently (38%). We analyzed the maternal status of 56 cases to determine the origin of both types of PLP mutation, since this is relevant to genetic counseling. In the 22 point mutations, 68% of mothers were heterozygous for the mutation, a value identical to the two-thirds of carrier mothers that would be expected if there were an equal mutation rate in male and female germ cells. In sharp contrast, among the 34 duplicated cases, 91% of mothers were carriers, a value significantly (chi2=9. 20, P<.01) in favor of a male bias, with an estimation of the male/female mutation frequency (k) of 9.3. Moreover, we observed the occurrence of de novo mutations between parental and grandparental generations in 17 three-generation families, which allowed a direct estimation of the k value (k=11). Again, a significant male mutation imbalance was observed only for the duplications. The mechanism responsible for this strong male bias in the duplications may involve an unequal sister chromatid exchange, since two deletion events, responsible for mild clinical manifestations, have been reported in PLP-related diseases.

The proteolipid protein 1 (PLP1) gene encodes the two major proteins of the central nervous system (CNS) myelin: PLP and DM20. PLP1 gene mutations are associated with a large spectrum of X-linked dysmyelinating disorders ranging from hypomyelinating leukodystrophy, Pelizaeus-Merzbacher disease (PMD), to spastic paraplegia (SPG2) according to the nature of the mutation. Genetic heterogeneity exists and mutations in the gap-junction alpha 12 (GJA12) gene have been related to PMD. About 20% of patients with the PMD phenotype remain without mutation in these two genes and are classified as affected by Pelizaeus-Merzbacher-like disease (PMLD). To study PLP1 splicing abnormalities, we analyzed PLP/DM20 transcripts from nerves and/or skin cultured fibroblasts of 14 PMD/SPG2 patients carrying different PLP1 mutations and 20 PMLD patients. We found that various types of PLP1 mutations result in missplicing, including one considered as a missense in exon 2 and a nucleotide substitution in intron 3 outside the classical donor and acceptor splicing sites. Moreover, we demonstrated for two patients that the fibroblast transcript pattern was in accordance with the one observed in the corresponding CNS/peripheral nervous system (PNS) tissues. Finally, we observed no abnormal splicing in fibroblasts of 20 PMLD patients tested; suggesting that PLP1 gene splicing abnormalities, potentially caused by undetected intronic mutations, are either not involved or are very rarely implicated in the PMLD phenotype. These results confirm that fibroblasts are reliable, accessible cells useful in detecting PLP1 transcript abnormalities, better characterizing the functional consequences of PLP1 mutations for genotype-phenotype correlation, characterizing new PLP1 splicing regulatory elements, and identifying PLP1 mutations undetected by conventional PLP1 screening.

We report a dinucleotide polymorphism in the first intron of the proteolipid protein (PLP) gene with a heterozygosity frequency of 0.69 useful for molecular analysis of families with X-linked neurologic disorders characterized by dysmyelination of the central nervous system, Pelizaeus-Merzbacher Disease (PMD) and X-linked Spastic Paraplegia (SPG2).