Cause Of CMT Research Articles

O.08 Biallelic variants in the mitochondrial form of phosphoenolpyruvate carboxykinase (PCK2) cause a recessive form of Charcot-Marie-Tooth disease

Mutations in over 100 genes have been associated with Charcot-Marie-Tooth disease (CMT). Despite testing availability, many patients remain genetically unresolved. In this study, a new cause of CMT was identified in <i>PKC2</i>. Phosphoenolpyruvate carboxykinase (PCK) plays a critical role in cytosolic gluconeogenesis. PCK is encoded by <i>PCK1</i>, the cytosolic gene associated with hypoglycemia during fasting, and <i>PCK2</i>, the mitochondrial gene not yet associated with human disease. Whole exome sequencing (WES) and nerve conduction studies (NCS) were performed. PCK enzymatic activity and protein production were assessed from patient fibroblasts. To validate the association, a mouse knockout model of <i>PCK2</i> deficiency was generated. Three patients from 2 families were identified with biallelic variants in <i>PCK2</i> via WES. Compound heterozygous variants (p.Ser23Ter/p.Pro170Leu) were identified in one patient and a homozygous variant (p.Arg193Ter) in two siblings. All 3 patients presented with weakness and gait abnormalities. Fibroblast cultures showed a reduction or absence of <i>PCK2</i> protein and profound reduction in <i>PCK2</i> activity with no obvious metabolic phenotype. NCS showed reduction in conduction velocities with temporal dispersion and conduction block, consistent with a demyelinating peripheral neuropathy. Corroborating the human phenotype, the mouse model had abnormal NCS and peripheral nerve pathology. In all, this study suggests a new cause of CMT, biallelic variants in <i>PCK2</i>. This supports the growing evidence for the importance of mitochondrial dysfunction in CMT which may inform future therapeutic targets.

Charcot-Marie-Tooth disease due to MORC2 mutations in Spain.

MORC2 mutations have been described as a rare cause of axonal Charcot-Marie-Tooth disease (CMT2Z). The aim of this work was to determine the frequency and distribution of these mutations throughout Spain, toprovide a comprehensivephenotypical description and, if possible, to establish a genotype-phenotype correlation. Retrospectively, data on patients diagnosed with CMT2Z in Spain werecollected and clinical, electrophysiological and muscle imaging information were analysed. Fifteen patients with CMT2Z were identified throughout Spain, seven of them belonging to a single kindred, whilst the rest were sporadic. The most common mutation was p.R252W, and four new mutations were identified. Eleven patients were categorized as having a scapuloperoneal phenotype, with asymmetric muscle weakness, early proximal upper limb involvement and frequent spontaneous muscular activity with distal sensory impairment and pes cavus, whilst two presented with a more classic length dependent sensory motor phenotype. This distinction was corroborated by the distribution of musclefatty infiltration in muscle imaging. Two other patients were classified as having a neurodevelopmental phenotype consisting in congenital or early onset, delay in motor milestones, and global developmental delay in one of them. Nerve conduction studies revealed an unequivocally axonal neuropathy with frequent spontaneous activity, and serum creatine kinase levels were increased in 50% of the patients. MORC2 mutations are a rare cause of CMT in Spain, but in-depth phenotyping reveals a recognizable phenotypic spectrum that will be clinically relevant for future identification of this disease.

Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2

Although mutations in more than 90 genes are known to cause CMT, the underlying genetic cause of CMT remains unknown in morethan 50% of affected individuals. The discovery of additional genes that harbor CMT2-causing mutations increasingly depends on sharing sequence data on a global level. In this way-by combining data from seven countries on four continents-we were able to define mutations in ATP1A1, which encodes the alpha1 subunit of the Na+,K+-ATPase, as a cause of autosomal-dominant CMT2. Seven missense changes were identified that segregated within individual pedigrees: c.143T>G (p.Leu48Arg), c.1775T>C (p.Ile592Thr), c.1789G>A (p.Ala597Thr), c.1801_1802delinsTT (p.Asp601Phe), c.1798C>G (p.Pro600Ala), c.1798C>A (p.Pro600Thr), and c.2432A>C (p.Asp811Ala). Immunostaining peripheral nerve axons localized ATP1A1 to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures of myelin sheaths. Two-electrode voltage clamp measurements on Xenopus oocytes demonstrated significant reduction in Na+ current activity in some, but not all, ouabain-insensitive ATP1A1 mutants, suggesting a loss-of-function defect of the Na+,K+ pump. Five mutants fall into a remarkably narrow motif within the helical linker region that couples the nucleotide-binding and phosphorylation domains. These findings identify a CMT pathway and a potential target for therapy development in degenerative diseases of peripheral nerve axons.

Two Novel De Novo GARS Mutations Cause Early-Onset Axonal Charcot-Marie-Tooth Disease.

BackgroundMutations in the GARS gene have been identified in a small number of patients with Charcot-Marie-Tooth disease (CMT) type 2D or distal spinal muscular atrophy type V, for whom disease onset typically occurs during adolescence or young adulthood, initially manifesting as weakness and atrophy of the hand muscles. The role of GARS mutations in patients with inherited neuropathies in Taiwan remains elusive.Methodology and Principal FindingsMutational analyses of the coding regions of GARS were performed using targeted sequencing of 54 patients with molecularly unassigned axonal CMT, who were selected from 340 unrelated CMT patients. Two heterozygous mutations in GARS, p.Asp146Tyr and p.Met238Arg, were identified; one in each patient. Both are novel de novo mutations. The p.Asp146Tyr mutation is associated with a severe infantile-onset neuropathy and the p.Met238Arg mutation results in childhood-onset disability.Conclusion GARS mutations are an uncommon cause of CMT in Taiwan. The p.Asp146Tyr and p.Met238Arg mutations are associated with early-onset axonal CMT. These findings broaden the mutational spectrum of GARS and also highlight the importance of considering GARS mutations as a disease cause in patients with early-onset neuropathies.

Absence of Dystrophin Related Protein-2 disrupts Cajal bands in a patient with Charcot–Marie–Tooth disease

Using exome sequencing in an individual with Charcot–Marie–Tooth disease (CMT) we have identified a mutation in the X-linked dystrophin-related protein 2 (DRP2) gene. A 60-year-old gentleman presented to our clinic and underwent clinical, electrophysiological and skin biopsy studies. The patient had clinical features of a length dependent sensorimotor neuropathy with an age of onset of 50 years. Neurophysiology revealed prolonged latencies with intermediate conduction velocities but no conduction block or temporal dispersion. A panel of 23 disease causing genes was sequenced and ultimately was uninformative. Whole exome sequencing revealed a stop mutation in DRP2, c.805C>T (Q269*). DRP2 interacts with periaxin and dystroglycan to form the periaxin–DRP2–dystroglycan complex which plays a role in the maintenance of the well-characterized Cajal bands of myelinating Schwann cells. Skin biopsies from our patient revealed a lack of DRP2 in myelinated dermal nerves by immunofluorescence. Furthermore electron microscopy failed to identify Cajal bands in the patient's dermal myelinated axons in keeping with ultrastructural pathology seen in the Drp2 knockout mouse. Both the electrophysiologic and dermal nerve twig pathology support the interpretation that this patient's DRP2 mutation causes characteristic morphological abnormalities recapitulating the Drp2 knockout model and potentially represents a novel genetic cause of CMT.

Exome Sequencing Identifies GNB4 Mutations as a Cause of Dominant Intermediate Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of inherited neuropathies. Mutations in approximately 45 genes have been identified as being associated with CMT. Nevertheless, the genetic etiologies of at least 30% of CMTs have yet to be elucidated. Using a genome-wide linkage study, we previously mapped a dominant intermediate CMT to chromosomal region 3q28-q29. Subsequent exome sequencing of two affected first cousins revealed heterozygous mutation c.158G>A (p.Gly53Asp) in GNB4, encoding guanine-nucleotide-binding protein subunit beta-4 (Gβ4), to cosegregate with the CMT phenotype in the family. Further analysis of GNB4 in an additional 88 unrelated CMT individuals uncovered another de novo mutation, c.265A>G (p.Lys89Glu), in this gene in one individual. Immunohistochemistry studies revealed that Gβ4 was abundant in the axons and Schwann cells of peripheral nerves and that expression of Gβ4 was significantly reduced in the sural nerve of the two individuals carrying the c.158G>A (p.Gly53Asp) mutation. Invitro studies demonstrated that both the p.Gly53Asp and p.Lys89Glu altered proteins impaired bradykinin-induced G-protein-coupled-receptor (GPCR) signaling, which was facilitated by the wild-type Gβ4. This study identifies GNB4 mutations as a cause of CMT and highlights the importance of Gβ4-related GPCR signaling in peripheral-nerve function in humans.

G.P.88 Peripheral neuropathies caused by mutations in the myelin protein zero

Abstract Charcot–Marie–Tooth disease (CMT), is a group of clinically and genetically heterogeneous neuropathies divided into demyelinating (CMT1) and axonal forms (CMT2). Mutations in the myelin protein zero (MPZ/P0) gene are the third most frequent cause of CMT. This gene is a member of the immunoglobulin gene superfamily that encodes the most abundant protein of the peripheral myelin, necessary for myelin compaction. There are many mutations in MPZ, most of them cause the CMT1B phenotype with normal early development but disability appears during the first two decades of life; and others, are responsible for severe neuropathies of infancy designated as Dejerine–Sottas syndrome. MPZ mutations have also been shown also in axonal type of CMT2. Hereby, we report the clinical and electrophysiological features of eight patients belonging to eight families affected by CMT 1B. The mean age of the patients is 13 years (range: 4–29). In five patients, the symptoms appeared between 2 and 3 years of age, showing motor clumsiness, difficulty to rise from the floor and for climbing stairs. In two cases, debuted before age of two, with a delay to develop an autonomous gait. The other one, presented an atypical phenotype at the age of seven, with hand tremor and episodes of muscular pain after exercising, with an intermittent increase of the CPK. Most of the patients had a stable evolution; five of them developed scoliosis and had affected their upper extremities. Only one of the patients developed a proximal weakness, and required scoliosis surgery at the age of 13. The electrophysiological study, showed a demyelinating neuropathy in all of the cases. Two patients with the same mutation, C241A, had different clinical evolution. There is a wide clinical spectrum in patients with MPZ gene mutation, which makes difficult to establish a genotype-phenotype correlation. We recommend the study on MPZ gene in patients with demyelinating neuropathy (once mutations in the PMP22 gene have been ruled out).

Exome Sequencing Identifies a New Locus for DI-CMT (P05.143)

Objective: To utilize exome sequencing to identify the genetic cause of CMT in a family with no mutation in previously identified CMT genes. Background Family WSU-75549 has nine living family members with an unidentified form of CMT with intermediate nerve conduction velocities (NCV). There are multiple cases of male-to-male transmission. Previous genetic testing for multiple genes including those known to cause Dominant Intermediate CMT (DI-CMT) have been negative as has been chromosome analysis. Whole exome sequencing is capable of re-sequencing the near complete set of coding exons of an individual. Design/Methods: DNA from all nine affected individuals and one unaffected sibling control was collected. Affected members were evaluated clinically and by electrophysiology. Exome sequencing was performed on three samples. Other family members were then tested for identified mutations. Results: The family phenotype was variable with CMTNS ranging from 8 (mild) in one patient aged 28 years to 32 (severe) in a 50 year old. Progression appears to quicken in this family during the fourth and fifth decades. Median NCV in upper extremities ranged between 29.2 m/s and 39.8 m/s. Exome sequencing did not identify mutations for any known CMT gene. However, utilizing rare variants identified by exome sequencing a novel locus was found for the family at 1p12. The two-point LOD score was 3.27 at a SNV marker in the gene WARS2. Genome-wide comparative genomic hybridization analysis did not reveal any copy number variations in the vicinity of 1p12. Further studies are ongoing to determine the nature of the familial CMT mutation. Conclusions: This is a large family with a previously unidentified form of DI-CMT. Data derived from exome sequencing enabled us to identify a novel chromosomal CMT locus at 1p12. A detailed genetic follow-up studies are under way to identify the causative mutation. Supported by: NINDS, ORD, MDA. Disclosure: Dr. Siskind has nothing to disclose. Dr. Speziani has nothing to disclose. Dr. Gonzalez has nothing to disclose. Dr. Blanton has nothing to disclose. Dr. Shy has nothing to disclose. Dr. Zuchner has received license fee payments from Athena Diagnostics.

MPZ mutation in an early-onset Charcot-Marie-Tooth disease type 1B family by genome-wide linkage analysis

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous peripheral neuropathy. The objective of this study was to find the causative mutation(s) in a demyelinating autosomal dominant CMT family. A high density SNP-based genome-wide linkage scan was performed, and causative mutations were determined by sequencing of candidate genes in the linkage disequilibrium region. Linkage analysis mapped the underlying gene to a region on chromosome 1q22-q23 with a maximum two-point LOD score of 2.036. Sequencing analysis revealed a novel c.243C>G (His81Gln) mutation in the MPZ gene, which encodes the major integral membrane protein of the peripheral nerve system. MPZ is well known as a CMT-causative gene with wide phenotypic spectrum. The clinical symptoms were more similar to those of patients with the His81Arg than patients with the His81Tyr mutation. The novel mutation completely co-segregated with affected members, and was not found in controls. Therefore, we suggest that the identified mutation in MPZ is the underlying cause of CMT in the family. In addition, this study demonstrated that the clinical phenotypes may be variable with different mutations at the same site in the MPZ gene.

A novel mutation in the nerve‐specific 5′UTR of the GJB1 gene causes X‐linked Charcot‐Marie‐Tooth disease

X-linked Charcot-Marie-Tooth disease (CMT1X) is the second most common cause of CMT, and is usually caused by mutations in the gap junction protein beta 1 (GJB1) gene which codes for connexin 32 (CX32). CX32 has three tissue-specific promoters, P1 which is specific for liver and pancreas, P1a specific for liver, oocytes and embryonic stem cells, and P2 which is nerve-specific. Over 300 mutations have been described in GJB1, spread throughout the coding region. We describe two families with X-linked inheritance and a phenotype consistent with CMT1X who did not have mutations in the GJB1 coding region. The non-coding region of GJB1 was sequenced and an upstream exon-splicing variant found at approximately - 373G>A which segregated with the disease in both families and was not present in controls. This substitution is located at the last base of the nerve-specific 5'UTR and thus may disrupt splicing of the nerve-specific transcript. Online consensus splice-site programs predict a reduced score for the mutant sequence vs. the normal sequence. It is likely that other mutations within the GJB1 non-coding regions account for the CMT1X families who do not have coding region mutations.

Charcot‐marie‐tooth disease subtypes and genetic testing strategies

Charcot-Marie-Tooth disease (CMT) affects 1 in 2,500 people and is caused by mutations in more than 30 genes. Identifying the genetic cause of CMT is often necessary for family planning, natural history studies, and for entry into clinical trials. However genetic testing can be both expensive and confusing to patients and physicians. We analyzed data from 1,024 of our patients to determine the percentage and features of each CMT subtype within this clinic population. We identified distinguishing clinical and physiological features of the subtypes that could be used to direct genetic testing for patients with CMT. Of 1,024 patients evaluated, 787 received CMT diagnoses. A total of 527 patients with CMT (67%) received a genetic subtype, while 260 did not have a mutation identified. The most common CMT subtypes were CMT1A, CMT1X, hereditary neuropathy with liability to pressure palsies (HNPP), CMT1B, and CMT2A. All other subtypes accounted for less than 1% each. Eleven patients had >1 genetically identified subtype of CMT. Patients with genetically identified CMT were separable into specific groups based on age of onset and the degree of slowing of motor nerve conduction velocities. Combining features of the phenotypic and physiology groups allowed us to identify patients who were highly likely to have specific subtypes of CMT. Based on these results, we propose a strategy of focused genetic testing for CMT, illustrated in a series of flow diagrams created as testing guides.

Variable phenotypes are associated with PMP22 missense mutations

Charcot-Marie-Tooth disease (CMT) is the commonest hereditary neuropathy encompassing a large group of clinically and genetically heterogeneous disorders. The commonest form of CMT, CMT1A, is usually caused by a 1.4 megabase duplication of chromosome 17 containing the PMP22 gene. Mutations of PMP22 are a less common cause of CMT. We describe clinical, electrophysiological and molecular findings of 10 patients carrying PMP22 missense mutations. The phenotype varied from mild hereditary neuropathy with liability to pressure palsies (HNPP) to severe CMT1. We identified six different point mutations, including two novel mutations. Three families were also found to harbour a Thr118Met mutation. Although PMP22 point mutations are not common, our findings highlight the importance of sequencing the PMP22 gene in patients with variable CMT phenotypes and also confirm that the PMP22 Thr118Met mutation is associated with a neuropathy albeit with reduced penetrance.

July 26 Highlights

Lawson et al. describe the clinical features in three families with CMT2 associated with novel mutations in the mitofusin 2 gene, a common cause of axonal CMT (23% of cases). Affected individuals showed age-independent variability of expression, and clinical examination was more sensitive than electrophysiology in identifying patients. see page 197 The accompanying editorial by Mary M. Reilly places this newly characterized cause of CMT2 into context of CMT 1 and the 15 or more other causes of CMT 2. The GTPase, mitofusin 2 gene ( MFN2 ) (a gene encoding MFN2, a mitochondrial fusion protein), is the primary gene mutated in CMT2A. MFN2 mutations cause a classical CMT2 phenotype: distal predominant lower limb wasting and weakness, sensory loss, and hyporeflexia. An important clinical point identified by Lawson et al. is the greater sensitivity of the clinical examination in determining affected status vs neurophysiology—confirming the impression of clinicians evaluating CMT2. see page 186 Goh et al. found …