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Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of inherited peripheral neuropathies characterized by progressive weakness and atrophy of distal limb muscles. Recently, SIMPLE/LITAF was shown to be responsible for an autosomal dominant demyelinating form of CMT linked to 16p (CMT1C). Although two transcripts encoding different proteins (SIMPLE and LITAF) have been reported from the same gene, we could not confirm the existence of LITAF. Here we show that the LITAF transcript appears to result from a DNA sequencing error. We screened the SIMPLE gene for mutations in a cohort of 192 patients with CMT or related neuropathies, each of whom tested negative for other known genetic causes of CMT. In 16 unrelated CMT families we identified nine different nucleotide variations in SIMPLE that were not detected in control chromosomes. SIMPLE mutations can occur de novo, associated with sporadic CMT1 and may convey both demyelinating and axonal forms. Bioinformatics analyses and other observations of SIMPLE suggest that 1) it could be a member of the RING finger motif-containing subfamily of E3 ubiquitin ligases that are associated with the ubiquitin-mediated proteasome processing pathway, 2) it could interact through its PPXY motifs with a WW domain containing protein, for instance with NEDD4, an E3 ubiquitin ligase, and 3) it could interact through the PSAP motif with TSG10, a protein associated with endosomal multivesicular protein sorting. Since both SIMPLE and Hrs are endosomal proteins and have both PPXY and P(S/T)AP motifs, we hypothesize that SIMPLE, like Hrs, is potentially a clathrin adaptor aiding in the retention of ubiquitinated proteins on to the endosomes. Thus the potential E3 ubiquitin ligase activity of SIMPLE, alteration in its interactions with NEDD4 or TSG101, or changes in its properties as a clathrin coat adaptor may underlie the pathogenesis of Charcot-Marie-Tooth disease.

Proteolipid protein (PLP1) and its alternatively spliced isoform, DM20, are the major myelin proteins in the CNS, but are also expressed in the PNS. The proteins have an identical sequence except for 35 amino acids in PLP1 (the PLP1-specific domain) not present in DM20. Mutations of PLP1/DM20 cause Pelizaeus-Merzbacher Disease (PMD), a leukodystrophy, and in some instances, a peripheral neuropathy. To identify which mutations cause neuropathy, we have evaluated a cohort of patients with PMD and PLP1 mutations for the presence of neuropathy. As shown previously, all patients with PLP1 null mutations had peripheral neuropathy. We also identified 4 new PLP1 point mutations that cause both PMD and peripheral neuropathy, three of which truncate PLP1 expression within the PLP1-specific domain, but do not alter DM20. The fourth, a splicing mutation, alters both PLP1 and DM20, and is probably a null mutation. Six PLP1 point mutations predicted to produce proteins with an intact PLP1-specific domain do not cause peripheral neuropathy. Sixty-one individuals with PLP1 duplications also had normal peripheral nerve function. These data demonstrate that expression of PLP1 but not DMSO is necessary to prevent neuropathy, and suggest that the 35 amino acid PLP1-specific domain plays an important role in normal peripheral nerve function.

Mutations of the ganglioside-induced differentiation-associated protein 1 gene (GDAP1) cause autosomal recessive Charcot-Marie-Tooth disease type 4A. We report four additional families with recessive mutations (487C-->T, Q163X; 359G-->A, R120Q) of GDAP1; Q163X occurred in three unrelated Hispanic families that had the same haplotype suggesting a Spanish founder mutation. Both the Q163X and the R120Q mutation cause demyelination and axonal loss. The patients had symptoms within the first two years of life and involvement of cranial, sensory, and enteric nerves. Neuropathology showed loss of large myelinated fibers, onion bulb formations and focal folding of the outer myelin lamina.

Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disorder that has been associated with alterations of several proteins: peripheral myelin protein 22, myelin protein zero, connexin 32, early growth response factor 2, periaxin, myotubularin related protein 2, N-myc downstream regulated gene 1 product, neurofilament light chain, and kinesin 1B. To determine the frequency of mutations in these genes among patients with CMT or a related peripheral neuropathy, we identified 153 unrelated patients who enrolled prior to the availability of clinical testing, 79 had a 17p12 duplication (CMT1A duplication), 11 a connexin 32 mutation, 5 a myelin protein zero mutation, 5 a peripheral myelin protein 22 mutation, 1 an early growth response factor 2 mutation, 1 a periaxin mutation, 0 a myotubularin related protein 2 mutation, 1 a neurofilament light chain mutation, and 50 had no identifiable mutation; the N-myc downstream regulated gene 1 and the kinesin 1B gene were not screened for mutations. In the process of screening the above cohort of patients as well as other patients for CMT-causative mutations, we identified several previously unreported mutant alleles: two for connexin 32, three for myelin protein zero, and two for peripheral myelin protein 22. The peripheral myelin protein 22 mutation W28R was associated with CMT1 and profound deafness. One patient with a CMT2 clinical phenotype had three myelin protein zero mutations (I89N+V92M+I162M). Because one-third of the mutations we report arose de novo and thereby caused chronic sporadic neuropathy, we conclude that molecular diagnosis is a necessary adjunct for clinical diagnosis and management of inherited and sporadic neuropathy.

Ten laminin alpha2-deficient patients were identified by both immunofluorescence and immunoblotting (30% of congenital muscular dystrophy patients tested). Three of the laminin alpha2-deficient patients were carrying a diagnosis of infantile polymyositis prior to immunostaining studies. The clinical features in the 10 merosin-deficient patients were homogeneous, with severe floppiness at birth, delay in achievement of motor milestones, and magnetic resonance imaging findings of white matter changes with normal intelligence. The 10-kb laminin alpha2-coding sequence was screened for causative mutations by reverse transcriptase-polymerase chain reaction/single-stranded conformational polymorphism analysis in muscle biopsy specimens from 5 patients, followed by automatic sequencing of aberrant conformers. Clear loss-of-function deletion mutations were identified in both alleles of 1 patient. Muscle histopathology in this patient showed a striking inflammatory infiltrate of T cells and B cells. Reexamination of biopsy specimens from other laminin alpha2-deficient patients showed minor signs of inflammation in each. Based on these findings and the histological and clinical picture suggesting failure of muscle regeneration, a pathogenesis model for this major subset of congenital muscular dystrophy is proposed. Our data show that muscle histopathology showing a neonatal inflammatory process should be considered consistent with congenital muscular dystrophy.