Charcot-Marie-Tooth Disease (CMT) is the most common group of `peripheral nerve monogenic genetic disorders with high clinical and genetic heterogeneity, and 28 disease genes have been cloned. The main clinical symptoms include progressive symmetric distal limb muscle weakness and atrophy, sensory deficits and decreased or absent tendon reflexes. Based on electrophysiological and pathological features, CMT can be classified into CMT1 (demyelinating type) and CMT2 (axonal type). A series of logical diagnostic processes such as clinical and genetic typing through clinical manifestations, electrophysiological pathological features, and selection of possible disease genes for mutation analysis clarify the molecular diagnosis and provide guidance on disease prognosis and genetic counseling. Charcot-Marie-Tooth Disease (CMT), also known as hereditary motor and sensory neuropathy (HMSN), is the most common group of peripheral neurological monogenic disorders with a high degree of clinical and genetic heterogeneity, with a prevalence of approximately 1 in 2500. Currently, 39 CMT loci have been localized and 28 disease genes have been cloned. Based on clinical and genetic typing based on clinical manifestations, electrophysiological and pathological characteristics of peripheral nerves, and mutation analysis of possible disease genes, about 60-70% of CMT patients can be diagnosed genetically, providing guidance for disease prognosis and genetic counseling. The article reviews the clinical typing, mode of inheritance and genotyping of CMT, as well as the genetic diagnosis process based on the above typing. 1. Clinical manifestations and clinical staging of CMT CMT mostly starts in childhood and adolescence with progressive symmetric distal limb muscle weakness and myasthenia gravis, starting from the lower limbs and gradually progressing to the upper limbs. The muscles below the lower third of the thigh are weak and atrophied, resulting in a “crane leg” or inverted bottle-like deformity, difficulty walking and running, and a cross-threshold gait. Weakness and atrophy of the interosseous muscles and interosseous muscles of the hand, claw-like hand or ape hand deformity, muscle atrophy usually does not exceed above the elbow joint, and fine hand movements are not possible. The peripheral sensory deficits, usually pain, warmth and vibration, are diminished. Tendon reflexes are diminished or absent and may be accompanied by autonomic dysfunction and signs of dystrophy. Skeletal deformities such as high arched feet and scoliosis are often present. Other common signs and symptoms include painful muscle spasms (mostly in the feet and legs), coldness, cyanosis and hyperkeratosis of the feet. Very early onset cases can result in hypotonia (soft baby syndrome), delayed motor development, and tiptoe walking. In contrast, the age of onset can be as late as late adulthood, and the presence of similar cases in the family line usually provides a clue to the diagnosis. Based on the neurophysiological and pathological features, CMT can be initially clinically typed into two types: demyelinating type (CMT1): characterized by reduced nerve conduction velocity (median nerve motor conduction velocity below 38 m/s) and significant myelin abnormalities on nerve biopsy (segmental demyelination, hyperplasia of Schwann cells, “onion head”-like changes); axonal type (CMT3): characterized by a decrease in nerve conduction velocity (median nerve motor conduction velocity below 38 m/s); and axonal type (CMT4). Axonal type (CMT2): characterized by normal or mildly slowed nerve conduction velocity (median nerve motor conduction velocity >38m/s) and chronic axonal degeneration and regeneration (axonal degeneration and reduction of myelinated fibers, nerve regeneration cluster formation) on nerve biopsy. In contrast to the classical clinical typing of CMT, intermediate CMT is gradually recognized as a group of CMT variants with median nerve conduction velocity between 25 and 45 m/s and neuropathological features of both demyelination and axonal degeneration. 2, the mode of inheritance and genotyping of CMT The mode of inheritance of CMT is autosomal dominant (AD) inheritance is the most common, seen in most patients of CMT1 and CMT2 families; with no male to male transmission in the family, male heterozygous (hemizygous) is usually more than female heterozygous (heterozygous) Clinical symptoms Severe X-linked (X-linked) dominant inheritance is second; autosomal recessive (AR) inheritance is less common; and disseminated cases are not uncommon. Based on the genetic loci and disease genes, CMT can be further classified into different genotypes (see Table 1). 6 types of AD-CMT1 have been cloned, and all disease genes have been cloned. 1.5 Mb positive tandem repeat mutations in the 17p11.2 region, including the PMP22 gene, cause CMT1A, which is the most common genotype of CMT, accounting for about 40-50% of all CMT. CMT1 patients and 90% of disseminated CMT1 cases are of CMT1A type. Approximately 3-5% of CMT1 patients have CMT1B caused by MPZ point mutations. PMP22 point mutations, SIMPLE/LITAF, EGR2 and NEFL mutations can also cause the CMT1 phenotype, but are relatively rare, being less than 1% of all CMTs. AD-CMT2 is highly genetically heterogeneous, with 10 types having been localized and 9 disease genes cloned. The results of several research groups have shown that CMT2A2, caused by mutations in the MFN2 gene, is the most common CMT2 genotype, accounting for approximately 20% of all CMT2, and its clinical manifestations can be divided into two clinical phenotypes: early-onset myasthenia gravis with severe symptoms and late-onset myasthenia gravis with mild symptoms. hotspot mutation R94Q in the MFN2 gene often leads to juvenile-onset, severe myasthenia gravis axonal type of The MPZ mutation causes the second most common CMT2J, accounting for about 5% of all CMT2, and the NEFL mutation causes about 2% of all CMT2. The clinical phenotype of AR-CMT is usually more severe than that of AD-CMT, with an early age of onset. 11 genotypes of AR-CMT1 (also known as CMT4, a demyelinating type) and 10 disease genes were cloned; 4 genotypes of AR-CMT2 (axonal type) and 2 disease genes were cloned. mutations in GDAP1 gene were the most common for both CMT4 and AR-CMT2 X-linked CMT has been localized to 5 genotypes, of which 2 disease genes have been cloned, CMTX1 (Cx32), CMTX2 (Xq24-q26), CMTX3 (Xp22.2). CMTX4 (Xq26-q28) and CMTX5 (PRPS1). The vast majority of X-linked CMT genotypes are CMTX1 due to mutations in the Cx32 gene, which is the second most common CMT genotype, accounting for about 7-12% of all CMTs. Distinct from the classical CMT autosomal dominant intermediate CMT (DI-CMT) three genotypes have been localized and two of the disease genes have been cloned. the development of DI-CMT is rare and has not been reported in China. Notably, mutations in AD-CMT1, AD-CMT2 disease genes (e.g. MPZ, NFL), CMTX1 disease gene GJB1 and CMT4A disease gene GDAP1 are known to cause both demyelinating and axonal degenerative changes in peripheral nerves, with an intermediate clinical presentation. 3. Molecular diagnosis process of CMT Because of the high genetic heterogeneity of CMT, it is inappropriate to detect mutations in all disease genes one by one in the molecular diagnosis of CMT patients, and the corresponding genes should be selected according to the clinical typing and genetic typing of CMT and combined with the frequency of mutations in different genotypes of CMT. AD-CMT1 and disseminated CMT1 patients, the detection of duplicated mutations in large segments of the PMP22 gene should be carried out first. If the test is negative and there is no male-to-male transmission in the family line, CMTX1 should be considered as a possibility and mutation analysis of the GJB1 gene should be performed. If negative, further point mutation analysis for MPZ and PMP22 genes should be performed. If still negative and conditions permit, analysis for mutations in other AD-CMT1 disease genes SIMPLE, EGR2, and NFL should be performed. In AD-CMT2 cases and in patients with disseminated CMT2, mutation testing for the MFN2 gene should be performed first. In cases without male to male transmission in the family line, especially in female CMT2 cases, the possibility of CMTX1 should be considered and mutation analysis of the GJB1 gene should be performed [20]. If the MFN2 and GJB1 gene tests are negative, mutation testing for CMT2 disease genes such as MPZ, NFL, HSPB1, and HSPB8 should be performed sequentially. For patients with intermediate nerve conduction velocity, mutation analysis of GJB1, MPZ, NFL, and GDAP1 genes should be performed first. If negative, further mutation analysis of DNM2 and YARS genes should be performed [3]. In patients with AR-CMT, whether demyelinating (CMT4) and axonal (AR-CMT2), GDAP1 gene mutation testing should be performed first. If negative, mutation analysis of AR-CMT corresponding disease genes such as LMNA, MTMR2 , and NDRG1 should be selected in the context of comprehensive analysis of ethnic genetic background, neuropathological specific features and disease course. In addition, the issues that need special attention in following the molecular diagnostic process of CMT are: (1) disseminated cases: not uncommon and often pose difficulties for accurate molecular diagnosis, de novo mutation (de novo mutation) is mostly seen in CMT1A and MFN2 mutations in the PMP22 gene with repeated mutations leading to CMT2A2. Due to the high clinical heterogeneity of CMT, some (2) Recognition of specific concomitant symptoms: concomitant symptoms such as cerebral nerve involvement, vocal cord palsy, abnormal pupillary changes, optic nerve atrophy, cone bundle symptoms, upper limb involvement dominated by muscle weakness and atrophy, and severe sensory disturbances, can often provide important clues to the molecular diagnosis. A number of studies have found that hearing loss with Adie pupillary changes often suggests CMT2J type due to MPZ hotspot mutation T124M; CMT2A2 patients with MFN2 mutation may have optic nerve atrophy symptoms; AR-CMT patients with GDAP1 mutation have an early age of onset and may have vocal cord palsy symptoms; the occurrence of multiple limb ulcers often suggests RAB7 and CMT2C caused by mutations in TRPV4 gene is characterized by hoarseness and dyspnea due to the involvement of vocal pharyngeal and diaphragm muscles. Therefore, patients with certain concomitant symptoms suggesting a specific gene mutation should first be tested for mutations in the relevant genes. (3) The value of nerve biopsy: it is not essential for most cases because of its invasive nature, but it is still important for some cases (e.g., differential diagnosis of disseminated cases, or the need for pathological testing to provide diagnostic information when mutation analysis is negative for common diseases). For example, peripheral nerve myelin sparing and salami-like structure formation suggest MPZ mutation; abnormal myelin proliferation folding is a common characteristic pathological change of CMT4 due to MTMR2, MTMR13, and FGD4 mutations; giant axon is seen in NEFL mutation; basement membrane proliferation of Schwann cells forming substrate onion bulb-like structure is a characteristic pathological change of CMT4C due to SH3TC2 mutation. Characteristic pathological changes. If the above characteristic pathological changes occur, the corresponding disease gene mutation detection should be carried out. 4. Conclusion and outlook Through the above series of logical diagnostic processes, the selection of possible disease genes for mutation analysis can clarify the molecular diagnosis in about 60-70% of CMT patients. Accurate genetic diagnosis can bring benefits to the prevention and treatment of CMT in many ways: 1) provide guidance for prognostic evaluation and genetic counseling; 2) effectively carry out prenatal diagnosis of CMT pre-documented patients to avoid the birth of affected children and achieve eugenics; 3) provide timely information to the corresponding CMT affected population when specific gene therapy and drugs for different genotypes are developed, and give appropriate treatment guidance. 3) To provide timely information and appropriate treatment guidance to CMT patients when genotype-specific treatments and drugs are developed.