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Am J Hum Genet. Oct 2003; 73(4): 926–932.
Published online Aug 19, 2003. doi:  10.1086/378159
PMCID: PMC1180612

Slowed Conduction and Thin Myelination of Peripheral Nerves Associated with Mutant Rho Guanine-Nucleotide Exchange Factor 10


Slowed nerve-conduction velocities (NCVs) are a biological endophenotype in the majority of the hereditary motor and sensory neuropathies (HMSN). Here, we identified a family with autosomal dominant segregation of slowed NCVs without the clinical phenotype of HMSN. Peripheral-nerve biopsy showed predominantly thinly myelinated axons. We identified a locus at 8p23 and a Thr109Ile mutation in ARHGEF10, encoding a guanine-nucleotide exchange factor (GEF) for the Rho family of GTPase proteins (RhoGTPases). Rho GEFs are implicated in neural morphogenesis and connectivity and regulate the activity of small RhoGTPases by catalyzing the exchange of bound GDP by GTP. Expression analysis of ARHGEF10, by use of its mouse orthologue Gef10, showed that it is highly expressed in the peripheral nervous system. Our data support a role for ARHGEF10 in developmental myelination of peripheral nerves.

The phenotype of slowed motor and sensory nerve-conduction velocities (NCVs) in a 4-generation family (De Jonghe et al. 1999) was accidentally discovered on clinical and electrophysiological examination of proband III-16 for vascular problems of the leg. Subsequent examination identified slowed NCVs in 12 of 39 healthy relatives (5 males and 7 females), indicating an autosomal dominant inheritance of the phenotype (fig. 1AA).). NCVs were uniformly slowed in all nerves examined; 34–42 m/s for motor median nerve (normal [gt-or-equal, slanted]49 m/s), 27–36 m/s for motor peroneal nerve (normal [gt-or-equal, slanted]41 m/s), 32–46 m/s for sensory median nerve (normal [gt-or-equal, slanted]46 m/s), 33–45 m/s for sensory ulnar nerve (normal [gt-or-equal, slanted]46 m/s), and 28–35 m/s for sensory sural nerve (normal [gt-or-equal, slanted]44 m/s). Compound muscle-action potentials were normal, and sensory nerve–action potentials were sometimes slightly reduced. None of the affected family members showed any clinical signs of peripheral or CNS dysfunction. The eldest individuals, II-4 and II-7 (aged 87 and 78 years, respectively, at time of neurological examination), had NCVs that were not significantly different from those measured in younger affected individuals. Histological studies of a peripheral-nerve biopsy of proband III-16 at age 54 years showed numerous relatively thin myelin sheaths (mean g-ratio: 0.75 for myelinated fibers, with a range of 2–7 μm) (fig. 1B), slight onion-bulb formation, and few axonal-regeneration clusters (De Jonghe et al. 1999).

Figure  1
Haplotype analysis of chromosome 8p23 STR markers. a, Pedigree of the family with slowed NCVs. Squares = male; circles = female; unblackened = unaffected; blackened = reduced nerve conduction phenotype; slashed = deceased; diagonal arrow = proband. Blood ...
Table A
Primer Conditions for New STR Markers on Chromosome 8p23

We performed a 10-cM density genomewide linkage analysis and obtained a conclusive linkage with D8S264 on chromosome 8p23 (table 1). The region around D8S264 was saturated with known and novel microsatellite markers, and a maximum LOD score of 9.33, at recombination fraction (θ) 0, was calculated with marker AF009213 (table 1). Haplotype analysis identified meiotic recombinants, with STR2 and D8S504 restricting the candidate region to 1.5 Mb, on the basis of sequence contigs NT_008060, NT_037694, and NT_023744 (NCBI, LocusLink) (fig. 2a). Within the sequence, a total of five known genes was identified, which we analyzed for mutations by genomic sequencing of exons and flanking splice sites in two affected individuals and two control individuals: KIAA0711 (hypothetical protein KIAA0711 [GenBank accession number NM_014867]), MYOM2 (myomesin [M-protein] 2 [GenBank accession number NM_003970]), CLN8 (ceroid-lipofuscinosis, neuronal 8 [GenBank accession number NM_018941]), DLGAP2 (discs, large [Drosophila] homolog-associated protein 2 [GenBank accession number NM_004745]), and ARHGEF10 (Rho guanine-nucleotide exchange [GEF] factor 10 [GenBank accession number NM_014629]) (intronic primers for ARHGEF10 shown in table B [online only]). A heterozygous mutation, C→T (c.326C→T; g.6110C→T), was observed in exon 3 of ARHGEF10, encoded by 22 exons, as shown by sequence alignment of its 8,467-bp transcript (GenBank accession number NM_014629), with the genomic sequence contained in NT_023744 (fig. 2b). The presence of the mutation was confirmed by BanI digestion of PCR-amplified exon 3, generating one mutant fragment of 329 bp and two wild-type fragments of 172 bp and 157 bp. Complete cosegregation of the mutation was observed in the family (LOD=9.93) (fig. 1; table 1), and the mutation was absent in 600 control chromosomes. The mutation predicts an amino acid substitution of threonine (Thr) to isoleucine (Ile) at codon 109 (Thr109Ile) of the ARHGEF10 protein (GenBank accession number NP_055444). Clustal W protein alignment of the human ARHGEF10 protein—with its orthologues in macaque (GenBank accession number BAB12119), puffer fish (GenBank accession number FuguGenscan_7637), and mouse (Gef10) (GenBank accession number NP_766339)—showed that Thr109 is a conserved amino acid in all four species (table C [online only]). Screening of patients with different clinical subtypes of Charcot-Marie-Tooth disease did not identify mutations in ARHGEF10. This is not unexpected, given that the individuals in our family were clinically unaffected and had minor disturbances in NCVs and myelination.myelination.

Figure  2
Positional cloning and expression analysis of ARHGEF10. a, Linked region on chromosome 8p23. Meiotic recombinants with STR2 and D8S504 defined a critical region of 1.5 Mb. Shaded boxes indicate the five known genes in the region. b, Genomic structure ...
Table 1
Two-Point Linkage Results with Chromosome 8p23 Markers[Note]
Table B
Oligonucleotide Primer Sequences Used for Mutation Analysis and Intron/Exon Boundaries of ARHGEF10
Table C
Clustal W Multiple-Protein Alignment of the Human, Mouse, Fugu, and Macaque GEF10 Orthologues[Note]
Table D
Oligonucleotide Primer Sequences Used for Analysis of Alternative Splice Variants of Gef10 cDNA (GenBank Accession Number NM_172751)

We examined expression of ARHGEF10, using its mouse orthologue, Gef10. Alignment of the Gef10 transcript of 4,481 bp (GenBank accession number NM_172751) with the genomic sequence NT_039455 identified 24 exons; exons 1 and 2 were absent from ARHGEF10 (fig. 2b). Multiple-tissue northern blot analysis of Gef10 indicated ubiquitous expression (data not shown). Overlapping primer sets covering the mouse cDNA sequence were used in PCR analysis on cDNA of E13 mouse brain, dorsal root ganglia (DRG), and ventral horn (VH) and demonstrated Gef10 expression in all three neuronal tissues. Extra PCR fragments that indicated the presence of alternative transcripts were observed (fig. 2c–2e). Sequencing of these fragments identified three splice variants of Gef10: one in all three tissues missing exon 4 (fig. 2c), one specific for DRG missing exon 21 (fig. 2d), and one specific to VH, with an insertion of an additional exon of 165 bp between exons 22 and 23 (fig. 2e). Exon 5, which corresponds to exon 3 in ARHGEF10 and contains the Thr109Ile mutation, is present in all three variant transcripts. Whole-mount in situ hybridization experiments in mouse embryos at E8.5 showed Gef10 expression in the neuroepithelium of the meninges, including the optic sulcus (fig. 3a and 3b). At E9.5, high levels of Gef10 expression were detected in the roof plate of the rhombencephalon (fig. 3c and 3d). In E12.5 embryos, Gef10 is ubiquitously expressed, with a pronounced expression in the neuroepithelium of brain vesicles, the neural tube, the ganglia, DRG, and the neural layer of retina (fig. 3e–3h).

Figure  3
Expression pattern of Gef10 mRNA in mouse embryo. a and b, Frontal view on an E8.5 embryo (a) and blowup of the head region (b) hybridized with the GEF10 probe. GEF10 mRNA is expressed in the neuroepithelium (ne) of brain vesicles and optic stalk (os). ...

ARHGEF10 encodes a guanine-nucleotide exchange factor for the Rho family of GTPase proteins (RhoGEFs) and contains a Dbl homology (DH) domain (codons 177–359), a common feature of all RhoGEFs (Zheng 2001). RhoGEFs activate RhoGTPases by catalyzing the exchange of bound GDP for GTP, which induces a conformational change in the GTP-bound GTPase that allows its interaction with downstream effector proteins (Bourne et al. 1990; Boguski and McCormick 1993; Hart et al. 1994). Within the RhoGEF family, DH domains are invariably followed by a pleckstrin homology (PH) domain, supposed to be involved in subcellular localization of RhoGEFs (Hoffman and Cerione 2002). However, in ARHGEF10, we could not detect a PH-domain consensus motif using several bioinformatic tools (BlastP, ScanProsite, or InterPro). So far, only one other mammalian RhoGEF family member, p164-RhoGEF, lacking the PH domain has been reported (Rumenapp et al. 2002). ARHGEF10 appears to lack an equivalent protein in Caenorhabditis elegans, Drosophila melanogaster, Dictyostelium discoideum, and Saccharomyces cerevisiae, which suggests that the ARHGEF10 signaling pathway is unique to vertebrates. This confirms the overall picture of plasticity, when comparing the RhoGTPases and their interacting proteins between species, with certain species gaining or losing RhoGTPase and RhoGEF family members to give rise to unique sets of signaling proteins (Wherlock and Mellor 2002). RhoGTPases play a pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane-transport pathways, and transcription-factor activity is probably just as significant (Etienne-Manneville and Hall 2002). Recent evidence has implicated RhoGTPases in neuronal morphogenesis, including cell migration, axonal growth and guidance, dendrite elaboration and plasticity, and synapse formation. Several GEFs play a central role in defining the temporal and spatial activation of the corresponding GTPase within neuronal cells (Etienne-Manneville and Hall 2002).

Here, we report a Thr109Ile mutation in ARHGEF10 cosegregating with slowed NCVs of peripheral nerves without clinical phenotype in a 4-generation family. RhoGEFs have been implicated elsewhere in human genetic disorders. A mutation in the DH domain of FGD1 GEF cosegregates with faciogenital dysplasia, a developmental disorder (Pasteris et al. 1994). Mutations in ARHGEF6 (also known as “αPIX”) are associated with X-linked nonsyndromic mental retardation (Kutsche et al. 2000). Loss-of-function mutations in the ALS2 (alsin) gene cause autosomal recessive amyotrophic lateral sclerosis (ALS2), a neurodegenerative disorder affecting the large motor neurons of the CNS (Hadano et al. 2001; Yang et al. 2001). The identification of ARHGEF10 as a gene implicated in peripheral-nerve conduction raises questions about its role during the development of the peripheral nervous system in vertebrates. All affected individuals in the family had slowed NCVs with normal amplitudes at all ages, which indicates that the phenotype is nonprogressive. Together with the numerous thin myelinated axons in the absence of gross signs of demyelination or axonal degeneration and regeneration in the peripheral nerve biopsy of the proband, these observations are indicative of a congenital nonprogressive phenotype, which suggests that ARHGEF10 is most likely involved in normal development of peripheral nerves, though its actual biological function remains to be elucidated. Also, the upstream signaling cascade necessary for ARHGEF10 activation and the RhoGTPase subsequently activated by ARHGEF10 remain to be clarified.


The authors gratefully acknowledge the cooperation and participation of all family members in this study and the contribution of the Flanders Interuniversity Institute for Biotechnology Genetic Service Facility in the genetic analyses. We also thank Christine Van Broeckhoven for critical reading of the manuscript and helpful discussions. This research project was supported by the Fund for Scientific Research (FWO-Flanders), special research funds of the University of Antwerp, the Medical Foundation Queen Elisabeth, the Association Belge contre les Maladies Neuro-Musculaires, and the Interuniversity Attraction Poles programme P5/19 of the Federal Office for Scientific, Technical and Cultural Affairs Belgium. K.V., A.Z., and E.N. are postdoctoral fellows of the FWO-Flanders. N.V. is supported by a Ph.D. fellowship of the Institute for Science and Technology, Belgium.

Electronic-Database Information

Accession numbers and URLs for data presented herein are as follows:

GenBank, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide (for mRNA sequences: MYOM2 [accession number NM_003970], KIAA0711 [accession number NM_014867], GEF10 [ARHGEF10] [accession number NM_014629], CLN8 [accession number NM_018941] DLGAP2 [accession number NM_004745], Homo sapiens RhoGEF10 [accession number NP_055444], and Mus musculus RIKEN cDNA [accession number NM_172751])


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