PLEKHG5 deficiency leads to an intermediate form of autosomal-recessive Charcot-Marie-Tooth disease

Abstract

Charcot-Marie-Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of peripheral neuropathies characterized by progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. Following the analysis of two consanguineous families affected by a medium to late-onset recessive form of intermediate CMT, we identified overlapping regions of homozygosity on chromosome 1p36 with a combined maximum LOD score of 5.4. Molecular investigation of the genes from this region allowed identification of two homozygous mutations in PLEKHG5 that produce premature stop codons and are predicted to result in functional null alleles. Analysis of Plekhg5 in the mouse revealed that this gene is expressed in neurons and glial cells of the peripheral nervous system, and that knockout mice display reduced nerve conduction velocities that are comparable with those of affected individuals from both families. Interestingly, a homozygous PLEKHG5 missense mutation was previously reported in a recessive form of severe childhood onset lower motor neuron disease (LMND) leading to loss of the ability to walk and need for respiratory assistance. Together, these observations indicate that different mutations in PLEKHG5 lead to clinically diverse outcomes (intermediate CMT or LMND) affecting the function of neurons and glial cells.

Figure 1.

Characterization of two families affected with an intermediate form of CMT. (A) Haplotype reconstruction for 20 SNPs and 12 microsatellite markers of the chromosome 1p36 region in families 221 and NP72. Microsatellite and SNP markers are ordered from telomere (top) to centromere (bottom) and show overlapping segments of autozygosity. Deduced haplotypes of the mother of the siblings in family 221 are bracketed. The critical region of segregation is defined by ancestral recombination events revealed by haplotype reconstruction in family NP72. +, wild-type allele; -, mutation. (B) Pes cavus deformity is evident in both affected individuals from family 221. The distal amyotrophy is more pronounced in lower than in upper limbs. (C) Transverse section of sural nerve biopsy sample of individual NP72.5 (Thionin-stained semi-thin section of a glutaraldehyde fixed, osmium tetroxidepost-fixed, epon-embedded nerve biopsy specimen; ×400 magnification). Most axons are surrounded by a disproportionally thin myelin sheath. In addition, myelinated nerve fiber density is reduced to about 70% of normal with predominant loss of larger fibers.

Figure 2.

PLEKHG5 mutations identified in families 221 and NP72. The autozygous region on chromosome 1 common to both families (vertical red bar) contains 25 genes (horizontal black bars), including PLEKHG5 (horizontal red bar). Chromatograms of DNA from the index case of each family, detailing the mutations identified, are presented, along with control sequences. Genomic structure of PLEKHG5 and nucleotide numbering is based on reference sequence NM_198681.3. Amino acid numbering is based on reference sequence NP_941374.2.

Figure 3.

Plekhg5 expression in mouse nervous system. (A) The relative level of Plekhg5 expression was evaluated by quantitative PCR (qPCR) in dorsal root ganglia (DRG), brain, optic nerve, spinal cord and sciatic nerve endoneurium. mRNA levels are presented as fold change over the mRNA level in the DRG. The data represent the mean ± SD of triplicate measurements. (B) Relative mRNA levels of Plekhg5 were determined by qPCR and microarrays (E17, P0, P2, P4 and P10: whole mouse sciatic nerve; P14, P28 and P56: sciatic nerve endoneurium). For each time point, the mRNA levels are presented as fold increase over the mRNA level at E17.5 (for qPCR measurements) or at P0 (for microarray experiments). The qPCR data represent the mean ± SD of triplicate measurements. (C) Relative mRNA levels of Plekhg5 were determined by qPCR in purified mouse Schwann cells (mSCs) and in the mouse SC line (MSC80). The data are represented as fold change over the mRNA levels in mSCs. The presented qPCR results were normalized using Ube2l3 as the reference gene.

Figure 4.

Disruption of Plekhg5 function in mice leads to mild neuropathy. (A) Schematic diagram showing the targeted Plekhg5 allele (based on Ensembl transcript ENSMUST00000118648). Homologous recombination between two loxP sites (black arrowheads positioned between exons 10–11 and 17–18) led to the deletion of exons 11–17 which contain the complete Rho-GEF domain and a small portion of the pleckstrin homology (PH) domain. The deletion of exons 11–17 (816 nucleotides) does not disrupt the reading frame of the truncated mRNA. The position of the previously described missense mutation (12) is indicated as well as the positions of mutations identified in this study. RBD: Ras binding domain; PDZ: Psd95-Dlg1-Zo1 domain (B) Electrophysiological characterization of 6- and 12-month-old wild-type (WT, Plekhg5^+/+) and knockout (KO, Plekhg5^{ΔEx11-Ex17/ΔEx11-Ex17}) animals revealed reduced motor nerve conduction velocity (MNCV) in affected mice (n = 4–5). (C) Both proximal CAP and distal CAP were delayed in affected animals. (D) Knockout mice exhibit deficits in rotarod performance test; however, the observed difference does not reach statistical significance. Error bars: SEM, statistically significant differences are indicated by * (P < 0.05).

Figure 5.

Disruption of Plekhg5 function does not lead to detectable defects in motoneurons or NMJ. (A) Characterization of lumbar α-motoneurons in Plekhg5^{ΔEx11-Ex17/ΔEx11-Ex17} mice at the age of one year. No difference in the number (B) or size (C) of α-motoneurons (MN) per ventral horn was detected between control (WT, Plekhg5^+/+) and affected (KO, Plekhg5^{ΔEx11-Ex17/ΔEx11-Ex17}) animals. (D) Confocal images of presynaptic nerve terminals (detected with α-neurofilament antibodies, in green) and postsynaptic endplates (detected with α-bungarotoxin CF594, in red). We did not observe obvious structural abnormalities in the soleus and EDL muscles of Plekhg5 deficient animals.