Charcot-Marie-tooth disease causing mutation (p.R158H) in pyruvate dehydrogenase kinase 3 (PDK3) affects synaptic transmission, ATP production and causes neurodegeneration in a CMTX6 C. elegans model.

Charcot-Marie-Tooth (CMT) is a commonly inherited, non-fatal neurodegenerative disorder that affects sensory and motor neurons in patients. More than 90 genes are known to cause axonal and demyelinating forms of CMT. The p.R158H mutation in the pyruvate dehydrogenase kinase 3 (PDK3) gene is the genetic cause for an X linked form of axonal CMT (CMTX6). In vitro studies using patient fibroblasts and iPSC-derived motor neurons have shown that this mutation causes deficits in energy metabolism and mitochondrial function. Animal models that recapitulate pathogenic in vivo events in patients are crucial for investigating mechanisms of axonal degeneration and developing therapies for CMT. We have developed a C. elegans model of CMTX6 by knocking-in the p.R158H mutation in pdhk-2, the ortholog of PDK3. In addition, we have developed animal models overexpressing the wild type and mutant form of human PDK3 specifically in the GABAergic motor neurons of C. elegans. CMTX6 mutants generated in this study exhibit synaptic transmission deficits, locomotion defects and show signs of progressive neurodegeneration. Furthermore, the CMTX6 in vivo models display energy deficits that recapitulate the phenotype observed in patient fibroblasts and iPSC-derived motor neurons. Our CMTX6 animals represent the first in vivo model for this form of CMT and have provided novel insights into the cellular function and metabolic pathways perturbed by the p.R158H mutation, all the while closely replicating the clinical presentation observed in CMTX6 patients.

Whole Genome Sequencing Identifies a 78 kb Insertion from Chromosome 8 as the Cause of Charcot-Marie-Tooth Neuropathy CMTX3.

With the advent of whole exome sequencing, cases where no pathogenic coding mutations can be found are increasingly being observed in many diseases. In two large, distantly-related families that mapped to the Charcot-Marie-Tooth neuropathy CMTX3 locus at chromosome Xq26.3-q27.3, all coding mutations were excluded. Using whole genome sequencing we found a large DNA interchromosomal insertion within the CMTX3 locus. The 78 kb insertion originates from chromosome 8q24.3, segregates fully with the disease in the two families, and is absent from the general population as well as 627 neurologically normal chromosomes from in-house controls. Large insertions into chromosome Xq27.1 are known to cause a range of diseases and this is the first neuropathy phenotype caused by an interchromosomal insertion at this locus. The CMTX3 insertion represents an understudied pathogenic structural variation mechanism for inherited peripheral neuropathies. Our finding highlights the importance of considering all structural variation types when studying unsolved inherited peripheral neuropathy cases with no pathogenic coding mutations.

SOX10 regulates an alternative promoter at the Charcot-Marie-Tooth disease locus MTMR2.

Schwann cells are the myelinating glia of the peripheral nervous system and dysfunction of these cells causes motor and sensory peripheral neuropathy. The transcription factor SOX10 is critical for Schwann cell development and maintenance, and many SOX10 target genes encode proteins required for Schwann cell function. Loss-of-function mutations in the gene encoding myotubularin-related protein 2 (MTMR2) cause Charcot-Marie-Tooth disease type 4B1 (CMT4B1), a severe demyelinating peripheral neuropathy characterized by myelin outfoldings along peripheral nerves. Previous reports indicate that MTMR2 is ubiquitously expressed making it unclear how loss of this gene causes a Schwann cell-specific phenotype. To address this, we performed computational and functional analyses at MTMR2 to identify transcriptional regulatory elements important for Schwann cell expression. Through these efforts, we identified an alternative, SOX10-responsive promoter at MTMR2 that displays strong regulatory activity in immortalized rat Schwann (S16) cells. This promoter directs transcription of a previously unidentified MTMR2 transcript that is enriched in mouse Schwann cells compared to immortalized mouse motor neurons (MN-1), and is predicted to encode an N-terminally truncated protein isoform. The expression of the endogenous transcript is induced in a heterologous cell line by ectopically expressing SOX10, and is nearly ablated in Schwann cells by impairing SOX10 function. Intriguingly, overexpressing the two MTMR2 protein isoforms in HeLa cells revealed that both localize to nuclear puncta and the shorter isoform displays higher nuclear localization compared to the longer isoform. Combined, our data warrant further investigation of the truncated MTMR2 protein isoform in Schwann cells and in CMT4B1 pathogenesis.

A 1.35 Mb DNA fragment is inserted into the DHMN1 locus on chromosome 7q34-q36.2.

Distal hereditary motor neuropathies predominantly affect the motor neurons of the peripheral nervous system leading to chronic disability. Using whole genome sequencing (WGS) we have identified a novel structural variation (SV) within the distal hereditary motor neuropathy locus on chromosome 7q34-q36.2 (DHMN1). The SV involves the insertion of a 1.35 Mb DNA fragment into the DHMN1 disease locus. The source of the inserted sequence is 2.3 Mb distal to the disease locus at chromosome 7q36.3. The insertion involves the duplication of five genes (LOC389602, RNF32, LMBR1, NOM1, MNX1) and partial duplication of UBE3C. The genomic structure of genes within the DHMN1 locus are not disrupted by the insertion and no disease causing point mutations within the locus were identified. This suggests the novel SV is the most likely DNA mutation disrupting the DHMN1 locus. Due to the size and position of the DNA insertion, the gene(s) directly affected by the genomic re-arrangement remains elusive. Our finding represents a new genetic cause for hereditary motor neuropathies and highlights the growing importance of interrogating the non-coding genome for SV mutations in families which have been excluded for genome wide coding mutations.

Distal enhancers upstream of the Charcot-Marie-Tooth type 1A disease gene PMP22.

Myelin insulates axons in the peripheral nervous system to allow rapid propagation of action potentials, and proper myelination requires the precise regulation of genes encoding myelin proteins, including PMP22. The correct gene dosage of PMP22 is critical; a duplication of PMP22 is the most common cause of the peripheral neuropathy Charcot-Marie-Tooth Disease (CMT) (classified as type 1A), while a deletion of PMP22 leads to another peripheral neuropathy, hereditary neuropathy with liability to pressure palsies. Recently, duplications upstream of PMP22, but not containing the gene itself, were reported in patients with CMT1A like symptoms, suggesting that this region contains regulators of PMP22. Using chromatin immunoprecipitation analysis of two transcription factors known to upregulate PMP22-EGR2 and SOX10-we found several enhancers in this upstream region that contain open chromatin and direct reporter gene expression in tissue culture and in vivo in zebrafish. These studies provide a novel means to identify critical regulatory elements in genes that are required for myelination, and elucidate the functional significance of non-coding genomic rearrangements.

Structural variations causing inherited peripheral neuropathies: A paradigm for understanding genomic organization, chromatin interactions, and gene dysregulation.

Inherited peripheral neuropathies (IPNs) are a clinically and genetically heterogeneous group of diseases affecting the motor and sensory peripheral nerves. IPNs have benefited from gene discovery and genetic diagnosis using next-generation sequencing with over 80 causative genes available for testing. Despite this success, up to 50% of cases remain genetically unsolved. In the absence of protein coding mutations, noncoding DNA or structural variation (SV) mutations are a possible explanation. The most common IPN, Charcot-Marie-Tooth neuropathy type 1A (CMT1A), is caused by a 1.5 Mb duplication causing trisomy of the dosage sensitive gene PMP22. Using genome sequencing, we recently identified two large genomic rearrangements causing IPN subtypes X-linked CMT (CMTX3) and distal hereditary motor neuropathy (DHMN1), thereby expanding the spectrum of SV mutations causing IPN. Understanding how newly discovered SVs can cause IPN may serve as a useful paradigm to examine the role of topologically associated domains (TADs), chromatin interactions, and gene dysregulation in disease. This review will describe the growing role of SV in the pathogenesis of IPN and the importance of considering this type of mutation in Mendelian diseases where protein coding mutations cannot be identified.

Unique clinical and neurophysiologic profile of a cohort of children with CMTX3.

OBJECTIVE: To describe in detail the clinical profile of Charcot-Marie-Tooth disease subtype 3 (CMTX3) to aid appropriate genetic testing and rehabilitative therapy. METHODS: We reviewed the clinical and neurophysiologic profile and CMT Pediatric Scale (CMTPedS) assessments of 11 children with CMTX3. RESULTS: Compared with the more common forms of CMT, CMT1A and CMTX, CMTX3 was characterized by early onset with early and progressive hand weakness. Most affected children were symptomatic within the first 2 years of life. The most common presentation was foot deformity in the first year of life. CMTPedS analysis in these children revealed that CMTX3 progressed more rapidly (4.3 ± 4.1 points over 2 years, n = 7) than CMT1A and CMTX1. Grip strength in affected boys was 2 SDs below age- and sex-matched normative reference values (z score -2.05 ± 1.32) in the second decade of life. The most severely affected individual was wheelchair bound at 14 years of age, and 2 individuals had no movement in the small muscles of the hand in the second decade of life. Nerve conduction studies showed a demyelinating sensorimotor neuropathy with motor conduction velocity ≤23 m/s. CONCLUSIONS: CMTX3 had an earlier onset, severe hand weakness, and more rapidly progressive disability compared to the more common forms of CMT. Understanding the unique phenotype of CMTX3 is essential for directing genetic testing because the CMTX3 insertion will not be seen on a routine microarray or neuromuscular gene panel. Early diagnosis will enable rehabilitation to be started early in this rapidly progressive neuropathy.

Improved inherited peripheral neuropathy genetic diagnosis by whole-exome sequencing.

Inherited peripheral neuropathies (IPNs) are a group of related diseases primarily affecting the peripheral motor and sensory neurons. They include the hereditary sensory neuropathies (HSN), hereditary motor neuropathies (HMN), and Charcot-Marie-Tooth disease (CMT). Using whole-exome sequencing (WES) to achieve a genetic diagnosis is particularly suited to IPNs, where over 80 genes are involved with weak genotype-phenotype correlations beyond the most common genes. We performed WES for 110 index patients with IPN where the genetic cause was undetermined after previous screening for mutations in common genes selected by phenotype and mode of inheritance. We identified 41 missense sequence variants in the known IPN genes in our cohort of 110 index patients. Nine variants (8%), identified in the genes MFN2, GJB1, BSCL2, and SETX, are previously reported mutations and considered to be pathogenic in these families. Twelve novel variants (11%) in the genes NEFL, TRPV4, KIF1B, BICD2, and SETX are implicated in the disease but require further evidence of pathogenicity. The remaining 20 variants were confirmed as polymorphisms (not causing the disease) and are detailed here to help interpret sequence variants identified in other family studies. Validation using segregation, normal controls, and bioinformatics tools was valuable as supporting evidence for sequence variants implicated in disease. In addition, we identified one SETX sequence variant (c.7640T>C), previously reported as a putative mutation, which we have confirmed as a nonpathogenic rare polymorphism. This study highlights the advantage of using WES for genetic diagnosis in highly heterogeneous diseases such as IPNs and has been particularly powerful in this cohort where genetic diagnosis could not be achieved due to phenotype and mode of inheritance not being previously obvious. However, first tier testing for common genes in clinically well-defined cases remains important and will account for most positive results.