Mutations in the Proteolipid Protein Gene in Japanese Families with Pelizaeus-Merzbacher Disease

Pelizaeus-Merzbacher disease (PMD) is a rare X-linked dysmyelinating disorder ^[1] resulting from a reduced number of mature oligodendrocytes in the CNS. ^[2] Recent studies have demonstrated that abnormalities of proteolipid protein (PLP), the most abundant transmembrane protein in myelin in the CNS, are responsible for the pathogenesis of PMD. PLP is critical not only for myelin formation but for the predifferentiational survival of oligodendrocytes as well. ^[3] There are mutations in the PLP gene, which encodes PLP and its splicing variant DM20, in patients and animal models of PMD (reviewed in Hodes et al. ^[4]). To date, there have been only two mutations reported in Japanese families with PMD. ^[5,6]

We investigated the clinical manifestations of five Japanese families with PMD followed by PLP gene screening using polymerase chain reaction (PCR)-heteroduplex analysis to search for point mutations and small deletions/insertions. We identified two families carrying novel point mutations, both of which were in exon 5; however, we found no mutations in the other three families. On careful investigation of the clinical manifestations, two patients not carrying a mutation revealed milder features than those with PLP mutations. Since Boespflug-Tanguy et al. ^[7] proposed genetic homogeneity of PMD, our results suggested the possibility of genetic abnormalities other than exonic mutations in the PLP gene that may cause a mild PMD phenotype.

Materials and methods.

Five patients and their mothers from five independent Japanese families with PMD (PM1 to 5) were examined. Clinical characteristics of each patient are summarized in Table 1. They all had the classical form of PMD. Of note, patients from PM3 and PM4 could speak several words and hold up their heads by themselves. One could walk with assistance.

Total DNA was extracted from peripheral blood cells. All seven exons and intron sequences neighboring the splice junctions and the 5 prime promoter regions of the PLP gene were amplified by PCR, as described elsewhere. ^[8] Amplified products from the mothers were used directly in PCR-heteroduplex analysis and those from the patients were mixed with equal amounts of products from a normal control subject before analysis. Denatured and reannealed samples were electrophoretically separated on a highresolution polymer gel (Hydrolink-MDE, AT Biochem, Malvern, PA, USA) according to the manufacturer's directions, followed by staining with ethidium bromide. ^[8]

The PCR products from the patients were sequenced using an automatic sequencing system (373A, Applied Biosystems, USA) after subcloning into the vector pCR-II (Invitrogen, USA). At least three clones were analyzed from each exon. Exon 5 PCR products from members of the families and 70 unrelated normal Japanese controls (34 males and 36 females) were digested with Sau3A I or Sty I before separation on 2% agarose gels.

Results.

Of the five families screened by PCR-heteroduplex analysis, PM1 and PM2 showed doublet bands in exon 5 of the PLP gene (Figure 1A) in both the mothers and patient/normal mixtures. Sequencing of these fragments identified two novel point mutations at T623A and C629T, indicating amino acid changes of V208N and P210L, respectively (Figure 1B). We also sequenced the remaining PCR products from the other three patients in which we did not identify doublet bands, and found no mutations.

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Figure 1. (A) PCR-heteroduplex analysis. Both carrier mothers (lane 1, 3, 5, 7, 9) and patient/normal mixtures (lane 2, 4, 6, 8, 10) were examined. Lanes 1 and 2, PM1; lanes 3 and 4, PM2; lanes 5 and 6, PM3; lanes 7 and 8, PM4; lanes 9 and 10, PM5. PM1 and PM2 showed duplet bands along with their mothers. Negative control, C. (B). Sequencing analysis of exon 5 from two PMD patients. PM1 showed a substitution of T623A, indicating amino acid change of V208N (sense strand). PM2 revealed a nucleotide change of C629T, altering amino acid of P210L (antisense strand).

We confirmed these mutations by restricted fragment length polymorphism (RFLP) analysis. The first substitution created a new Sau3A I site (GATC) and the second abolished the Sty I site (CCWWGG). The original exon 5 band (315 bp) was digested by Sau3A I into two fragments (221 bp and 94 bp) due to the substitution at T623A in PM1 but not in normal controls. In PM2, the lack of a Sty I site caused by substitution at C629T resulted in the appearance of a 226-bp band, which was cut into 124-bp and 89-bp bands in the normal controls. Each mother showed a heterozygous pattern for these mutations. We also examined 106 normal X chromosomes obtained from unrelated Japanese controls by RFLP analysis and found that none of them carried these substitutions (data not shown).

Discussion.

We described five families with PMD screened by PCR-heteroduplex analysis for PLP gene mutations and identified two novel nucleotide substitutions in exon 5, resulting in amino acid changes. Since these substitutions were not present in 106 X chromosomes in normal Japanese subjects, we conclude that they are pathologic mutations responsible for PMD and not simply polymorphisms.

The PLP gene spans seven exons and is transcribed as two splicing variants, PLP and DM20. In addition to their role in the formation of the myelin sheath, Hudson et al. ^[9] suggested that they represent membrane-bound signals involved in regulating oligodendrocyte proliferation. According to the topologic model with four hydrophobic alpha-helix domains spanning the membrane with both the N- and C-termini facing the cytosol, ^[10] the only extended region facing the extracellular medium is the loop between the third and fourth transmembrane domains in which the exon 5 mutations of our cases are located. Moreover, 12 mutations in this region account for 52% of PMD mutations, suggesting that this loop plays an important role that has yet to be established. Further studies might explain how the mutations in this loop cause dysfunction of PLP.

All patients analyzed in this study showed typical clinical features, histories, and unique MRI findings that satisfy the criteria for the classical form of PMD. However, two families, PM3 and PM4, with no PLP mutations showed milder clinical manifestations than those carrying the mutations. In previous reports of PMD with exonic mutations in the PLP gene, most cases exhibited more severe manifestations than our cases with mild phenotype. Therefore, we considered that cases with mild PMD phenotype may not carry exonic mutations in PLP gene. Patients with X-linked spastic paraplegia 2 (SPG2), showing much milder manifestations than PMD, also carry PLP mutations. ^[8,11] Individuals show spastic gait starting in their teens, normal intelligence, and lack of nystagmus. Although our two cases showed mild PMD phenotypes, their manifestations began in their infancy and their development was seriously affected. They are clinically distinct from SPG2.

Considering the genetic homogeneity of PMD, ^[7] our cases in which we did not identify mutations may carry other PLP gene abnormalities, which we could not identify in this study but which cause a mild PMD phenotype. There have been recent reports of PLP overdosage due to PLP gene duplication ^[12] or excessive transcription ^[13] causing PMD. Therefore, we must propose the PLP dosage effect as another genetic abnormality in these cases. Further studies of the PLP gene may help in understanding PMD without exonic mutations and in determining the correlation between PLP mutations and PMD phenotype.