FGD4 mutations in the four families with CMT4H. Arrowheads in the electropherograms indicate the disease-causing mutations. The wild-type sequence is shown in the lower electropherogram in each panel. A, Homozygous nonsense mutation c.670C→T, R224X. B, Homozygous missense mutation c.893T→G, M298R. C, Homozygous frameshift mutation c.1626_1627delAG, E543fs. The reverse sequence is shown. An arrow indicates the frameshift. D, Homozygous nonsense mutation c.1756G→T, G586X.

In silico characterization of the consequences of the c.893T→G mutation in the FGD4 gene. A, Multiple sequence alignment, generated with ClustalW, of the protein region surrounding the site of the putative M298R mutation. Conserved residues are shown on a gray background. M298R (in bold) is conserved through evolution in members of the FGD protein family. B, Scores from different splice-site prediction algorithms (i.e., WebGene, NNSplice, FSPLICE, and SPL). The putative donor splice site created by the c.893T→G change reaches scores similar to the genuine intron 7 donor splice site.

Sural nerve biopsy specimens of patients with FGD4 mutations. A, Transverse section of the sural nerve biopsy sample of patient CMT244 with the FGD4 M298R mutation. There is a moderate decrease in density of myelinated fibers, particularly affecting the large fibers. Many of the remaining fibers are thinly myelinated, whereas others are surrounded by thickened, grossly distorted, and excessively folded myelin. Toluidine blue staining; 400× magnification. B, Teased fiber preparation of the sural nerve biopsy sample of the same patient. There are demyelinated segments and myelin thickenings on virtually every fiber. 100× magnification. C and D, Electron micrographs of the sural nerve biopsy specimen of patient NP447 with the FGD4 E543fs mutation. Complex folding of the myelin sheath outside the axon (C and D) and a loop extending toward the axon (C) are shown. Semithin sections stained with uranyl acetate and lead citrate; 6,500× magnification.

Expression analysis and overexpression of Fgd4/frabin in Schwann cells. A, Western-blot analysis of lysates from rat tissues, Schwann cell lines, and cultured primary Schwann cells. Upper panel, Mouse monoclonal anti-frabin antibody (BD Transduction Labs) detecting a band of the expected size in all lysates analyzed. Lower panel, Blot probed with mouse monoclonal anti–β-actin antibody (Sigma-Aldrich) to ensure similar loading. Lane 1, Spinal cord; lane 2, sciatic nerve; lane 3, whole brain; lane 4, primary rat Schwann cells; lane 5, primary rat oligodendrocytes; lane 6, RN22; and lane 7, RT4-D6P2T. B, Quantitative RT-PCR analysis of Fgd4 (GenBank accession number NM_139232) expression in the developing mouse sciatic nerve. Measurements were normalized against Gapdh levels. p = Postnatal days. C, Frabin-induced cell-shape changes of primary rat Schwann cells. Upper panel, overview; lower panel, detail of cell processes. pCMV-Myc full-length frabin was transfected by use of the liposome-mediated gene transfection. Left, Transfected cells identified by staining with a mouse monoclonal anti-myc antibody (Roche Diagnostics) and a Cy3-conjugated goat anti-mouse antibody (red) (Jackson ImmunoResearch). Middle, Actin cytoskeleton, visualized with Alexa Fluor 488 phalloidin (green) (Molecular Probes). Right, Merge. Arrows indicate filopodia; arrowheads indicate lamellipodia. Note that untransfected cells, visible in the middle and right columns, are virtually devoid of filopodia and lamellipodia.