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Charcot-Marie-Tooth Disease (CMT) Genomics

Charcot-Marie-Tooth disease (CMT) is a clinically distinct autosomal dominant disease caused by a 1.5 Mb tandem duplication on chromosome 17. Only recently with the help of molecular genetic studies has the gene PMP22 appeared to be associated with hereditary neuropathies. CMT is the first autosomal dominant disease in which the disease phenotype results from three copies of the normal PMP22 gene. Epidemiological studies show that 1 in every 2,500 people have a form of CMT resultant of duplications, deletions or point mutations of the PMP22 gene. The most common mutation is the PMP22 duplication in which 20-64% of CMT patients carry the duplication. Other forms of CMT are associated with mutations in the MPZ and Cx32 genes. The PMP22 protein is estimated to make up 2-5% of all total myelin proteins in the peripheral nervous system as an integral protein of myelin. The intricate mechanism of PMP22 is still unknown although it is though to function as a homeostasis mechanism of myelin, and axonal integrity. PMP22-related diseases disrupt the organisation of myelin, which is responsible for the disabilities in patients with PMP22 mutations. These findings suggest that therapeutic intervention of CMT duplication patients may be possible by normalizing the amount of PMP22 mRNA levels. Furthermore, increasing evidence suggests that PMP22 mRNA levels are highly variable among patients, suggesting that dysregulation of PMP22 expression of excessive fluctuation of PMP22 is the cause behind the disease. Further studies are required to relate the mechanical intracellular defects caused by PMP22 mutations to the resulting demyelintating nerve phenotypes in order to determine appropriate treatment techniques. Currently methods are being developed to stimulate damaged nerve growth through gene therapy, an approach that can be considered for all hereditary neuropathies distinct from PMP22 mutation.

Charcot-Marie-Tooth disease is a heterogeneous group of autosomal dominant disorders associated with chronic progressive neuropathy, and is one of the most common inherited neurological disorders (Timmerman, Strickland et al. 2014). Three physicians, Jean-Martin Charcot, Pierre Marie, and Howard Henry Tooth initially described Charcot-Marie-Tooth disease over 100 years ago in 1886 (Murakami, Garica et al. 1996).  Only recently however have molecular genetic studies of CMT uncovered the underlying causes of most forms of the disease. Majority of cases of CMT are associated with a 1.5-Mb heterogeneous duplication of chromosome 17, a DNA segment that contains the gene coding for peripheral myelin protein 22 (PMP22) among others (Visigalli, Catagnola et al. 2016). Although many genes exist in this DNA segment almost 60% of diagnosed hereditary neuropathies and 90% of de novo mutations are due to PMP22 duplication (Visigalli, Catagnola et al. 2016). To date, increased expression of PMP22 represents the most likely molecular mechanism underlying CMT1 (Visigalli, Catagnola et al. 2016). The disease phenotype results from over-expression, deletion, or point mutation of the PMP22 gene. 50% of all patients with inherited peripheral neuropathies result from mutations of the PMP22 gene (Paassen, Kooi et al. 2014). Over-expression and point mutations result in a gain-of-function phenotype, whereas deletion results in a loss-of-function phenotype (Paassen, Kooi et al. 2014). These findings suggest that therapeutic intervention in CMT1 duplication patients may be possible by normalizing the amount of PMP22 mRNA levels (Murakami, Garica et al. 1996). Alternatively, other forms of CMT are associated with mutations in the MPZ (CMT1B) and Cx32 (CMTX) genes, thus causing similar CMT phenotypes (Murakami, Garica et al. 1996). All three genes thus far identified appear to play an important role in the myelin formation of maintenance of peripheral nerves, studies suggest that PMP22 is particularly important in protecting the nerves from physical pressure to allow restoration following compression (Visigalli, Catagnola et al. 2016). Over the past decade, understanding of the molecular basis of CMT has increased enormously, and the neurophysiologic deficits and clinical problems associated with CMT have been more clearly delineated (Ho, Tai et al. 2017). Advances in molecular biology and genetic manipulation techniques have allowed the development of animal models of some CMT types, introducing more productive scientific exploration of possible treatments (Verkhratsky and Butt 2007). In addition a CMT gene panel comprising of 27 genes now has the ability to assess multiple co-existing variants of PMP22 gene mutation, resulting in a faster and highly specific diagnosis as opposed to traditional molecular diagnostic pathways, allowing the genetic epidemiology to be studied further (Ho, Tai et al. 2017). The understanding of the molecular basis of CMT and related disorders has allowed accurate DNA diagnosis and genetic counselling of inherited peripheral neuropathies and will make it possible to develop rational strategies for therapy (Majer, Berger et al. 2002, Watila and Balarabe 2015, Ho, Tai et al. 2017)

4) Results


PMP22 gene, also known as Peripheral Myelin Protein 22 or Growth Arrest Specific Protein 3 (GAS-3), is located on chromosome 17p22 and orientated on the minus strand (Li, Parker et al. 2013). The gene is 35.55 kB in length and consists of 6 exons, with the coding region spanning from exon-2 to exon-5. The first exon is comprised of two alternately transcribed promoters, 1a and 1b, which give rise to two differing transcripts (Li, Parker et al. 2013). The two transcripts are identical in coding sequence but differ in the 5’ untranslated region, which suggests that there are two different PMP22 transcripts produced, exon-1a and exon-1b (Li, Parker et al. 2013).


Majority of the expression of PMP22 transcripts has consistently shown to be in myelinating Schwann cells of the peripheral nervous system (PNS) (Notterpek, Roux et al. 2001). Expression of high levels of PMP22 mRNA outside of the peripheral nervous system has also been indicated, especially epithelial cells of the lungs and intestines, although the role of the protein in these tissues has not been determined (Notterpek, Roux et al. 2001). In general, PMP22 proteins are localised to similar regions of PMP22 mRNA expression (Notterpek, Roux et al. 2001). The constitutive expression of the PMP22 protein is tightly regulated at transcription level by two distinct promoters that give rise to two different transcripts, which is activated by early growth response 2 (EGR2) (Li, Parker et al. 2013). The two alternate PMP22 mRNAs encode the same protein, in which contains four transmembrane regions. Although it has been discovered that myelinating Schwann cells significantly upregulate the expression of PMP22 during axonal contact (Notterpek, Roux et al. 2001, Giambonini-Brugnoli, Buchstaller et al. 2005). The involvement of axonal contact and expression of PMP22 is unknown, although it indicates the involvement of the PMP22 protein in neuronal death (Notterpek, Roux et al. 2001).

Macintosh HD:Users:DaynaCant:Desktop:PMP22 GENE STRUCTURE.jpg

Figure 1. A schematic representation of PMP22 gene structure and predicted protein structure.


PMP22 is an integral membrane glycoprotein of intermodal myelin that consists of 160 amino acids in length with a molecular mass of 17891 Da (Zoidl, D’Urso et al. 1996). PMP22 protein comprises an estimated 2-5% of total myelin proteins in the PNS (Paassen, Kooi et al. 2014). Details of the exact structure of the PMP22 protein is still unknown but it has been suggested that the protein possesses four transmembrane domains, two extracellular domains and one intracellular domain as seen in figure 1 (Zoidl, D’Urso et al. 1996). A prediction regarding the amino acid sequence of the PMP22 protein structure suggests the existence of post-translational modification, including the existence of N-glycolysation site at Asn41 and modification sites at PhosphoSite Plus (Zoidl, D’Urso et al. 1996). Further post-transcriptional regulation of the protein has been found to be by MicroRNAs that target 3’ UTR in a complementary reverse manner (Majer, Berger et al. 2002). Levels of PMP22 need to be tightly regulated since alterations in the levels by mutations are responsible for greater than 50% of patients with inherited peripheral neuropathies (Li, Parker et al. 2013).


PMP22 acts as a component of compact myelin, and is thought to be involved in the formation and maintenance of compacting myelin (Li, Parker et al. 2013). PMP22 protein is also involved in transcriptional activity, as well as normal axonal cytoskeletal organisation in cell-to-cell interactions (Paassen, Kooi et al. 2014). PMP22 knockout mice showed that the total disruption of the PMP22 gene results in hypermyelination and demyelination, indicating that PMP22 regulates the initiation of myelination, myelin sheath thickness, and the stability of myelin (Verkhratsky and Butt 2007). In addition, studies with knockout mice indicates that PMP22 is a binding partner in the integrin/laminin complex and is involved in mediating the interaction of Schwann cells with the basal lamina (Verkhratsky and Butt 2007).

Role in Disease

Variation in PMP22 expression results in inherited peripheral neuropathies (Paassen, Kooi et al. 2014). PMP22 related diseases disrupt the organisation of myelin, and subsequently axonal integrity, which is responsible for disabilities in patients with PMP22 mutations (Paassen, Kooi et al. 2014). PMP22 mutations, including duplication, deletions, and point mutations result in increased apoptosis of myelinating schwann cells in humans (Paassen, Kooi et al. 2014). A 1.5 MB duplication on chromosome 17 that includes the PMP22 gene predominantly causes the disease Charcot-Marie-Tooth disease (Paassen, Kooi et al. 2014). Deletion and recessive point mutation of a similar 1.5-Mb region is associated with hereditary neuropathy with liability to pressure palsies (HNPP), a clinically distinct neuropathy (Roa, Garcia et al. 1993). A point mutation in the PMP22 can account for 5% of patients with CMT (Paassen, Kooi et al. 2014). Increasing copies of PMP22 have proven detrimental in transgenic models, producing pathological phenotypes resembling those with neuropathy (Sereda and Nave 2006). Additionally, transgenic models with extra copies of PMP22 gene provides proof that over-expression is sufficient to cause peripheral demyelination, onion bulb formation, secondary axonal loss, progressive muscle atrophy, all pathological hallmarks of CMT1A (Sereda, Griffiths et al. 1996). In mutant mice that completely lack PMP22 expression, peripheral nervous system myelination is delayed and followed by focal hypermyelination and myelin degeneration (Sereda and Nave 2006). The clinical phenotype of most Charcot-Marie-Tooth disease patients become symptomatic in the first 2 decades of life and consist of foot deformities (high arches and hammer toes), variable degree of hand and foot weakness, absent deep tendon reflexes (Colomban, Micallef et al. 2014).

DNA mutations in the 1.5 Mb region of the PMP22 gene is predominantly associated with the hereditary neuropathy disorders Charcot-Marie-Tooth disease (CMT), and related pressure palsies (HNPP) (Watila and Balarabe 2015). CMT is a disorder caused by heterozygous duplication of the PMP22 gene, resulting in the presence of three copies of the PMP22 gene (Watila and Balarabe 2015). CMT is the most common inherited neuropathy of the peripheral nervous system, with PMP22 duplication accounting for more than half of all CMT cases (Timmerman, Strickland et al. 2014). CMT presents with weakness of the distal leg muscles, calf hypertrophy, foot deformity (in particular the presence of high arches and hammer toes), clawing of the hands and skeletal deformity of the spine in the form of scoliosis (Colomban, Micallef et al. 2014). The PMP22 gene has been found to be expressed predominantly from Schwann cells of the peripheral nervous system, and is thought the play roles in Schwann cell biology and myelin homeostasis (Notterpek, Roux et al. 2001). The functions of PMP22 protein still remains unclear although it is certain that is a cause behind the severe demyelination of peripheral nerves associated with CMT and associated neuropathies (Notterpek, Roux et al. 2001).


The scientific community essentially worked in reverse regarding the development and understanding of the gene PMP22 and its effect on the human body to cause disease. Inherited neuropathies were known to have a genetic cause but studies on patients with CMT in particular notified scientists that all patients had varying elevated levels of the PMP22 protein (as in CMT1) (Visigalli, Catagnola et al. 2016). In 1991, the genetic causes of CMT were completely unknown. To date more than 60 genes have been implicated in CMT and related syndromes (Rossor, Polke et al. 2013). This accomplishment has led to genetic testing for many types of CMT, which has greatly improved diagnosis (Ho, Tai et al. 2017). The recent advances in molecular genetic techniques, such as next-generation sequencing, have been useful in validating the recognisable clinical phenotypes or pathological signs associated with CMT (Ho, Tai et al. 2017). Knowledge regarding clinical presentation of CMT and the expanding possibilities of genetic testing has become a prerequisite for medical professionals dealing with inherited neuropathy patients. Research so far has largely focused on CMT1 due to its prevalence and the rationale of decreasing the gene dosage of PMP22, potentially reversing the phenotype. The problem is that PMP directed therapy can’t be applied to all cases of CMT due to its differing mutations. This suggests that genetic subtyping of CMT may be essential in the future to select cases that may be eligible for targeted treatment strategies once they become available.

Of equal importance, the ongoing hunt for CMT genes has given insights into treatments that might be used to stop or reverse the disorder. Scientists have begun to investigate how and why specific genetic mutations lead to different types of CMT. This knowledge has essentially enabled physicians to more accurately predict the course of CMT in patients and improve prognosis.  Recent studies have identified small molecule suppressors for the over-expression of PMP22 following the random insertion of PMP22 (Jang, Lopez-Anido et al. 2012). Understanding of how the duplicated DNA segment disrupts the homeostasis of PMP22 expression needs to be investigated to develop an understanding on the possible treatments to normalise PMP22 levels in CMT. Increasing evidence suggests that PMP22 levels are highly variable among patients with CMT1, raising an alternative mechanism causing the disease by dysregulation of PMP22 expression or excessive fluctuation of PMP22 levels, not the absolute increase of PMP22 (Katona, Wu et al. 2009). A clear understanding of these issues could radically change how therapies should be developed against CMT1. Understanding this upstream mechanism may give us better opportunity to normalize PMP22 levels in CMT1.


In addition to genetic advances of the disease, scientists have begun to make progress in understanding the disease process and its affects on the axons and Schwann cells- the cells that make myelin in the peripheral nerves. The formation and maintenance of myelin seems to require a finely tuned interaction between axons and Schwann cells in which the PMP22 proteins seems to disrupt, although the intricate mechanism is still widely unknown (Verkhratsky and Butt 2007). With the development of a partial understanding regarding PMP22 and its supposed role in myelin formation the scientific community have begun to treat CMT by finding ways to improve this axon-Schwann cell interaction or axonal transport. A method is currently being developed regarding gene therapy for CMT, in which damaged nerves are supplied with genes that encode neurotropic factors, which are naturally occurring proteins that stimulate nerve cell growth (Stephan, Marie-Noelle et al. 2011). This approach could perhaps be used to treat all types of CMT, regardless of the underlying defect.


How and to what extent the PMP22 gene defection contributes to defective Schwan cell physiology remains to be seen. The structure and function of the PMP22 protein remains elusive although it is known that PMP22 mutations are found in conjunction with abnormal function of myelin (Sereda and Nave 2006). A conceptually important issue concerns the question of whether PMP22 overexpression is the actual cause of Charcot-Marie-Tooth disease or the 60 other genes found in the same location of the apparent DNA mutation is the causative defect. Since the discovery of PMP22 it has demonstrated its importance in the peripheral nervous system, especially apparent in knockout mice studies in which PMP22 appears to have an important role in myelin integrity (Sereda and Nave 2006). Further studies on the recombination mechanism of CMT and HNPP may reveal the causes of site-specific homologous recombination in the human genome, as well as the need to link PMP22 genetic modifications to its fine-tuning regulation in peripheral myelination. The link between PMP22 overexpression and PMP22 point mutations indicate that they share related disease mechanisms although it remains a challenge for the future to relate mechanistically the intracellular defects caused by PMP22 alterations within the cell to the resulting demyelinating nerve phenotypes.

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