Abstract We previously reported familial cases charac- terized by Charcot-Marie-Tooth disease (CMT) pheno- type with abnormal myelin foldings and MPZ Ile62Phe mutation. To further clarify the molecular mechanisms in this family, we produced wild-type MPZ, Ile62Phe mutant and other mutations in the neighboring regions producing thin myelin sheaths (Ser63del, Ser63Cys and Ser63Phe) by site-specific mutagenesis and transfected these into rat pheochromocytoma cells (PC12). We investigated the ex- pression and aggregation properties of the MPZ protein through immunoblotting, immunohistochemical staining and adhesion assay. MPZ protein with Ile62Phe mutation was immunohistochemically detectable mainly in the plasma membrane of the cells, and it induced a cell ag- gregation behavior different from the other mutations or the wild-type MPZ. These studies suggested that MPZ Ile62Phe mutation in CMT1B with abnormal myelin fold- ing induced dysregulation of adhesion function of the MPZ protein in a manner unlike those seen in cells with other mutations. The present study provides evidence that the site and nature of amino acid substitutions in the MPZ protein are closely related to the abnormal myelination in CMT1B. Keywords CMT1B · MPZ Ile62Phe · Myelin folding · Cellular expression · Aggregation assay Introduction Abnormal myelin foldings have been reported in heredi- tary motor and sensory neuropathy (HMSN) with a broad spectrum of clinical severity and genetic heterogeneity [7, 8, 9, 18, 19, 20, 21, 23, 24, 25]. Most of the reported cases of HMSN with abnormal myelin foldings described as fo- cally folded myelin sheaths were of an autosomal reces- sive form and were classified as Charcot-Marie-Tooth (CMT) type 4B (MIM#601382) with heterogeneous re- sponsible genes [1, 4, 14]. Recently, homozygous muta- tions in MTMR2 (MIM*603557) have been reported in some families with CMT4B [4]. A few cases of HMSN with abnormal myelin foldings or tomacula formation show myelin protein zero (MPZ: MIM*159440) gene mu- tations; Asp61Glu [2], Lys96Glu [24]; Lys130Arg [8, 23] and Ile135Leu [23], and have been diagnosed as CMT1B (MIM#118200). We have reported the familial CMT1B with Ile62Phe mutation in MPZ and abnormal myelin foldings [18]. To clarify the pathomechanism in CMT1B patients with Ile62Phe mutation, we studied the aggrega- tion effects and cellular localization of mutant MPZ pro- tein in culture cells. We report here the altered aggrega- tion behavior and expression of the mutant MPZ in rat pheochromocytoma cells (PC12). Materials and methods Patients reports Details of the clinical and genetic findings in this family have been reported previously [18]. Briefly, patient 1, a 40-year-old woman, showed delay of motor development, and did not begin to walk un- til the age of 2 years. Muscle atrophy and weakness in distal parts of the extremities appeared at the age of 12. On examination at age 40, she showed severe predominantly distal muscle weakness and atrophy, hypotonus, areflexia in all extremities and bilateral pes cavus. Her sensory system was moderately affected in the distal parts of the extremities. She could walk with a waddling and step- page gait. Sural nerve biopsy showed many myelinated fibers with an irregularly folded, or extremely thick myelin sheath and small onion bulbs [26]. Direct sequencing of MPZ in this family showed a heterozygous point mutation A184T. This alternation produced a phenylalanine substitution for isoleucine 62 in exon 2 of MPZ. Her three children also had Ile62Phe mutation and peripheral neuropa- thy with varying clinical severity. Wataru Matsuyama · Masanori Nakagawa · Hiroshi Takashima · Mitsuhiro Osame Altered trafficking and adhesion function of MPZ mutations and phenotypes of Charcot-Marie-Tooth disease 1B Acta Neuropathol (2002) 103 : 501–508 DOI 10.1007/s00401-001-0497-1 Received: 3 September 2001 / Revised, accepted: 5 November 2001 / Published online: 31 January 2002 REGULAR PAPER W. Matsuyama · M. Nakagawa (✉) · M. Osame Third Department of Internal Medicine, Kagoshima University Faculty of Medicine, Sakuragaoka 8-35-1, Kagoshima City 890-8520, Japan e-mail: nakagawa@m2.kufm.kagoshima-u.ac.jp, Tel.: +81-992-755332, Fax: +81-992-657164 H. Takashima Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA © Springer-Verlag 2002