Mutations in the small GTP-ase late endosomal protein RAB7 cause Charcot-Marie-Tooth type 2B neuropathy

Abstract

Charcot-Marie-Tooth type 2B (CMT2B) is clinically characterized by marked distal muscle weakness and wasting and a high frequency of foot ulcers, infections, and amputations of the toes because of recurrent infections. CMT2B maps to chromosome 3q13-q22. We refined the CMT2B locus to a 2.5-cM region and report two missense mutations (Leu129Phe and Val162Met) in the small GTP-ase late endosomal protein RAB7 which causes the CMT2B phenotype in three extended families and in three patients with a positive family history. The alignment of RAB7 orthologs shows that both missense mutations target highly conserved amino acid residues. RAB7 is ubiquitously expressed, and we found expression in sensory and motor neurons.

Figure 1

Haplotype analysis of chromosome 3q13-22 STR markers in families with CMT2B. Pedigrees of four families with an ulcero-mutilating phenotype linked to the CMT2B locus: A, Austrian family CMT-140; B, Austrian family CMT-126; C, Scottish family CMT-90; and D, American family CMT-195. Symbols: open diamond = unaffected; filled diamond = affected; half-filled diamond = probably affected (i.e., CMT-126 II-3); slashed diamond = deceased; arrow = recombination; box = disease-associated haplotype. Pedigree structure and sex are disguised to preserve anonymity. The best genetic and physical order of STR markers is according to NCBI (GenBank/LocusLink). The six newly developed STR markers (D3SCMT126A, D3SCMT126B, D3SCMT126C, D3SCMT126D, D3SCMT126F, and D3SCMT126G) are localized between D3S2324 and D3S1587. Genotypes are represented by allele sizes in base pairs, and 0-0 = failed genotype. The RAB7 gene is located on the same contig (NT_005523) as markers D3SCMT126B and D3SCMT126C. For genotyping, fragment analysis was performed on an ABI Prism3700 DNA Analyzer and processed with the ABI GENESCAN 3.5 and GENOTYPER 3.6 software (Perkin Elmer Applied Biosystems).

Figure 2

DNA and protein sequence analysis of RAB7. A, Electropherogram showing the c.385C→T sequence variation in part of exon 3, resulting in the Leu129Phe missense mutation in families CMT-140 and CMT-126 and patient HSN-15.1. B, Electropherogram of the c.484G→A sequence variation in part of exon 4, resulting in the Val162Met missense mutation in families CMT-90 and CMT-195 and patients HSN-8.1 and PN-626.1. The corresponding genomic sequence of a control person is shown below. DNA sequencing was performed using the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech), and the sequencing reactions were loaded on the ABI Prism3700 DNA Analyzer (Perkin Elmer Applied Biosystems). The data were collected and analyzed via the ABI DATA COLLECTION version 1.1 and DNA SEQUENCING ANALYSIS version 3.6 software, respectively. C, ClustalW multiple protein alignment of the RAB7 orthologs of the region surrounding the Leu129Phe and Val162Met mutations. RAB7 orthologs: Human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), fly (Drosophila melanogaster), slime mold (Dictyostelium discoideum), nematode (Caenorhabditis elegans), mouse-ear cress (Arabidopsis thaliana), and baker’s yeast (Saccharomyces cerevisiae). The highly conserved motif involved in guanine nucleotide binding is boxed. Both amino acid mutations are shaded and indicated by an arrow.

Figure 3

Expression analysis of RAB7. A, Human 12-lane Multiple Tissue Northern blot (Clontech) containing poly A+ RNA from adult tissues: lane 1, brain; lane 2, heart; lane 3, skeletal muscle; lane 4, colon; lane 5, thymus; lane 6, spleen; lane 7, kidney; lane 8, liver; lane 9, small intestine; lane 10, placenta; lane 11, lung; and lane 12, peripheral blood leukocyte. The probe used for hybridization is a cDNA fragment of 800 bp, obtained from plasmid clones containing partial human RAB7 cDNA sequences (IMAGp956B0837, IMAGp956M0263, and IMAGp956M2246 [Resource Center of the German Human Genome Project]). B, Mouse multiple tissue northern blot containing poly A+ RNA from adult tissues (Clontech): lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; and lane 8, testis. The probe used for hybridization is a full-length mouse Rab7 cDNA cloned into the pCRII-TOPO vector (Invitrogen). The mouse Rab7 cDNA was generated via RT-PCR and SMART RACE cDNA Amplification (Clontech) from mouse brain RNA. Both northern blots (A and B) were also hybridized with a β-actin cDNA probe (Clontech) as a control for RNA loading (lower part of figure). C, PCR amplification of a mouse Rab7 cDNA fragment of 854 bp: M, 100-bp size marker; C+, Rab7 full-length cDNA cloned into pCRII-TOPO; VH, ventral horn cDNA; DRG, dorsal root ganglia cDNA; C-, blanco. VH and DRG were isolated from 13-d-old mouse embryos, total RNA was extracted using the Totally RNA Kit (Ambion), and RT-PCR was performed using the SMART PCR cDNA Synthesis Kit (Clontech). Mouse Rab7 cDNA primers (MRAB7-2F = 5′-CTGACCAAGGAGGTGATGGT-3′ and MRAB7-2R = 5′-GAACAGTTCTCTCACTCTCC-3′) were used to PCR amplify the Rab7 cDNA fragment of 854 bp in both neuron populations.