Clinical and molecular evaluation of Italian patients affected by Pelizaeus-Merzbacher disease

Pelizaeus-Merzbacher disease (PMD) is a rare form of dysmyelinating leukodystrophy. Recent studies in dysmyelinating mutants led to investigation of the involvement of the myelin PLP (proteolipid protein) gene in this disease. In fact, various single-base mutations in the human PLP gene have been identified in patients with PMD. In addition, a complete deletion and a duplication of the PLP gene have been documented in a kindred with typical PMD and in two boys with PMD. PLP exon mutations have been found only in 10-25% of families carrying the disease analysed (Boespflug-Tanguy et al 1994). In this work 5 patients and two obligate carrier mothers have been studied at the PLP locus by denaturing gradient gel electrophoresis (DGGE) analysis (Myers et al 1987). This method allows the separation of DNA fragments differing by as little as a single base change. It is based on the melting properties of DNA in solution as a result of differences in nucleotide composition. Two modifications that greatly improve the resolution of the method have been applied : (1) a thermostable GC clamp of 40bp has been attached to the 5' end of one of the two primers used for PCR amplification ; (2) analysis of heterduplex molecules (hybrids formed between mutant and wild-type strand).