Genotype-Phenotype correlations of MPZ mutations

Background: Myelin protein zero (MPZ) is the mai structural protein of peripheral myelin, costituiting approximately the 50% of total myelin proteins. MPZ is a single transmembrane glycoprotein, belonging to the immunoglobulin superfamily and is supposed to play an important role in myelin compaction, actins as an adhesion molecule. Missense mutations in MPZ cause inherited neuropathies charachterized by different grades of severity. Recently MPZ mutations were classified in two main groups: mutations causing an early onset phenotype with motor development delay and severe demyelinating neuropathy and the late onset mutations associated with weakness and sensory loss appearing in the third or fouth decade of life and with electrophysiological signs consistent with a mixed demyelinationg and axonal neuropathy. Aim of the project: To study how mutations associated to an early and late onset phenotype interfere with protein trafficking and adhesion. Material and Methods The human cDNA coding for the human MPZwt was cloned in a mammalian expression vector: pDsRed1-N1 (Clontech) in frame with DsRed. Five MPZ mutations associated with early onset (H52R), R69C, Deletion 22-28) and late onset phenotype (T95M, H10P) were cloned in another vector: pEGFP-N1 (Clonetech) in frame with EGFP. Chinese ovary cells (CHO) were transiently transfected with the plasmids coding for the MPZ mutations, both singularly or in double with MPZwt and the cellular localization of the proteins was visualized using a fluorescence microscope. An aggregation assay (M.Filbin 1990) was performed comparing the adhesive proprieties of MPZwt, T95M (late onset phenotype), deletion 22-28 (early onset) and pEGF-N1 control trasfected cells. Results Both the mutations associated to a late onset phenotype reached the cell membrane in a comparable way with the MPZwt. The early onset mutations behaved in a variable way, reaching (H52R), reaching only in a limited amount (deletion 22-28) or not reaching at all (R69C) the plasma membrane. In all cases, when the mutated MPZ was co-trasfected with the wild type protein, the latter was normally expressed on the plasma membrane. The aggregation test demonstrated that MPZwt expressing cells are more adhesive than cells transfected with pEGFP-Nl or Deletion 22-28, while T95M expressing cells perform the aggregation assay in a comparable way to MPZwt cells. Conc!usions These data suggest a more disruptive effect, on the protein structure, of MPZ early onset mutations, compared with MPZ late onset ones. A clear dominant negative effect was not demonstrated for any of the studied mutations, since the co-transfected MPZwt was never prevented from reaching the cellular membrane.