Molecular genetics of charcot-marie-tooth disease: from genes to genomes.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders of the peripheral nervous system, mainly characterized by distal muscle weakness and atrophy leading to motor handicap. With an estimated prevalence of 1 in 2,500, this condition is one of the most commonly inherited neurological disorders. Mutations in more than 30 genes affecting glial and/or neuronal functions have been associated with different forms of CMT leading to a substantial improvement in diagnostics of the disease and in the understanding of implicated pathophysiological mechanisms. However, recent data from systematic genetic screening performed in large cohorts of CMT patients indicated that molecular diagnosis could be established only in ∼50-70% of them, suggesting that additional genes are involved in this disease. In addition to providing an overview of genetic and functional data concerning various CMT forms, this review focuses on recent data generated through the use of highly parallel genetic technologies (SNP chips, sequence capture and next-generation DNA sequencing) in CMT families, and the current and future impact of these technologies on gene discovery and diagnostics of CMTs.

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

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.

The characteristics of gait in Charcot-Marie-Tooth disease types I and II.

Certain typical gait characteristics such as foot-drop and foot supination are well described in Charcot-Marie-Tooth disease. These are directly related to the primary disease and due to the weakness of ankle dorsiflexors and everters characteristic of this hereditary neuropathy. We analysed 16 subjects aged 8-52 years old (11 with type I, 5 with type II Charcot-Marie-Tooth disease) using three-dimensional gait analysis and identified kinematic features previously unreported. These patients showed a combination of tight tendo achillei, foot-drop, failure of plantar flexion and increased foot supination, but also presented with excessive internal rotation of the knee and/or tibia, knee hyperextension in stance, excessive external rotation at the hips and decreased hip adduction in stance (typical of a broad based gait). These proximal features could have been an adaptation to or consequence of the disrupted ankle and foot biomechanics, however a direct relation to the neuropathy is also possible since sub-normal muscle power was observed at the proximal levels in most subjects on both manual testing and kinetic analysis. Gait analysis is a useful tool in defining the characteristic gait of patients with Charcot-Marie-Tooth disease.

Expression of mitofusin 2(R94Q) in a transgenic mouse leads to Charcot-Marie-Tooth neuropathy type 2A.

Charcot-Marie-Tooth disease type 2A is an autosomal dominant axonal form of peripheral neuropathy caused by mutations in the mitofusin 2 gene. Mitofusin 2 encodes a mitochondrial outer membrane protein that participates in mitochondrial fusion in mammalian cells. How mutations in this protein lead to Charcot-Marie-Tooth disease type 2A pathophysiology remains unclear. We have generated a transgenic mouse expressing either a mutated (R94Q) or wild-type form of human mitofusin 2 in neurons to evaluate whether the R94Q mutation was sufficient for inducing a Charcot-Marie-Tooth disease type 2A phenotype. Only mice expressing mitofusin 2(R94Q) developed locomotor impairments and gait defects thus mimicking the Charcot-Marie-Tooth disease type 2A neuropathy. In these animals, the number of mitochondria per axon was significantly increased in the distal part of the sciatic nerve axons with a diameter smaller than 3.5 microm. Importantly, the analysis of R94Q transgenic animals also revealed an age-related shift in the size of myelinated axons leading to an over-representation of axons smaller than 3.5 microm. Together these data suggest a link between an increased number of mitochondria in axons and a shift in axonal size distribution in mitofusin 2(R94Q) transgenic animals that may contribute to their neurological phenotype.

SH3TC2/KIAA1985 protein is required for proper myelination and the integrity of the node of Ranvier in the peripheral nervous system.

Charcot-Marie-Tooth disease type 4C (CMT4C) is an early-onset, autosomal recessive form of demyelinating neuropathy. The clinical manifestations include progressive scoliosis, delayed age of walking, muscular atrophy, distal weakness, and reduced nerve conduction velocity. The gene mutated in CMT4C disease, SH3TC2/KIAA1985, was recently identified; however, the function of the protein it encodes remains unknown. We have generated knockout mice where the first exon of the Sh3tc2 gene is replaced with an enhanced GFP cassette. The Sh3tc2(DeltaEx1/DeltaEx1) knockout animals develop progressive peripheral neuropathy manifested by decreased motor and sensory nerve conduction velocity and hypomyelination. We show that Sh3tc2 is specifically expressed in Schwann cells and localizes to the plasma membrane and to the perinuclear endocytic recycling compartment, concordant with its possible function in myelination and/or in regions of axoglial interactions. Concomitantly, transcriptional profiling performed on the endoneurial compartment of peripheral nerves isolated from control and Sh3tc2(DeltaEx1/DeltaEx1) animals uncovered changes in transcripts encoding genes involved in myelination and cell adhesion. Finally, detailed analyses of the structures composed of compact and noncompact myelin in the peripheral nerve of Sh3tc2(DeltaEx1/DeltaEx1) animals revealed abnormal organization of the node of Ranvier, a phenotype that we confirmed in CMT4C patient nerve biopsies. The generated Sh3tc2 knockout mice thus present a reliable model of CMT4C neuropathy that was instrumental in establishing a role for Sh3tc2 in myelination and in the integrity of the node of Ranvier, a morphological phenotype that can be used as an additional CMT4C diagnostic marker.

SH3TC2, a protein mutant in Charcot-Marie-Tooth neuropathy, links peripheral nerve myelination to endosomal recycling

Patients with Charcot-Marie-Tooth neuropathy and gene targeting in mice revealed an essential role for the SH3TC2 gene in peripheral nerve myelination. SH3TC2 expression is restricted to Schwann cells in the peripheral nervous system, and the gene product, SH3TC2, localizes to the perinuclear recycling compartment. Here, we show that SH3TC2 interacts with the small guanosine triphosphatase Rab11, which is known to regulate the recycling of internalized membranes and receptors back to the cell surface. Results of protein binding studies and transferrin receptor trafficking are in line with a role of SH3TC2 as a Rab11 effector molecule. Consistent with a function of Rab11 in Schwann cell myelination, SH3TC2 mutations that cause neuropathy disrupt the SH3TC2/Rab11 interaction, and forced expression of dominant negative Rab11 strongly impairs myelin formation in vitro. Our data indicate that the SH3TC2/Rab11 interaction is relevant for peripheral nerve pathophysiology and place endosomal recycling on the list of cellular mechanisms involved in Schwann cell myelination.

Predictors of endocarditis in isolates from cultures of blood following dental extractions in rats with periodontal disease.

Rats with periodontitis and catheter-induced aortic valve vegetations underwent dental extractions. Cultures of blood obtained 1 min later showed polymicrobial bacteremia in 19 of 19 rats, mostly due to viridans streptococci (18 of 19), Morganella (15 of 19), group G streptococci (13 of 19), and Staphylococcus aureus (10 of 19). Viridans streptococci circulated in higher numbers than did group G streptococci and S. aureus (P less than .01). Three days after dental extractions, 18 of 20 rats had endocarditis. Fifteen (83%) of 18 infections were due to group G streptococci, 9 (50%) of 18 were due to S. aureus, and 2 (11%) of 18 were due to viridans streptococci (P less than .05). In vitro, adherence to platelet-fibrin matrices of endocarditis strain 8 of group G streptococcus was two times greater than that of endocarditis strain S. aureus 23 and three to four times greater than that of Streptococcus sanguis 44 and Morganella morganii 93 (P less than 10(-5)). The inoculum size that produced endocarditis in 90% of rats after iv challenge was 10(5) cfu for group G streptococcus strain 8 and 10(7) for S. sanguis 44.

KIAA1985, a Protein Mutant in Charcot-Marie-Tooth Neuropathy, Links Peripheral Nerve Myelination to Endosomal Recycling Pathways

Charcot-Marie-Tooth neuropathy (CMT) represents a heterogenous group of inherited disorders of the peripheral nervous system. One form of autosomal recessive demyelinating CMT (CMT4C, 5q32) is caused by mutations in the gene encoding KIAA1985, a protein of so far unknown function. Here we show that KIAA1985 is exclusively expressed in Schwann cells. KIAA1985 is tethered to cellular membranes through an N-terminal myristic acid anchor and localizes to the perinuclear recycling compartment. A search for proteins that interact with KIAA1985 identified the small GTPase Rab11, a key regulator of recycling endosome functions. CMT4C-related missense mutations disrupt the KIAA1985/Rab11 interaction. Protein binding studies indicate that KIAA1985 functions as a Rab11 effector, as it interacts only with active forms of Rab11 (WT and Q70L) and does not interact with the GDP locked mutant (S25N). Consistent with a function of Rab11 in Schwann cell myelination, myelin formation was strongly impaired when dorsal root ganglion neurons were co-cultured with Schwann cells infected with Rab11 S25N. Our data indicate that the KIAA1985/Rab11 interaction is relevant for peripheral nerve pathophysiology and place endosomal recycling on the list of cellular mechanisms involved in Schwann cell myelination.