Review Charcot-Marie-Tooth Disease

Review

Charcot-Marie-Tooth Disease: Lessons in Genetic Mechanisms

James R. Lupski Department of Molecular and Human Genetics, Department of Pediatrics, and Texas Children's Hospital, Baylor College of Medicine, Houston, Texas, U.S.A.

Introduction weakness that now bears their names. Each rec- ognized the hereditary nature of the disease by In recent years, the application of molecular to more occurrence in sib- techniques to the study of human subjects has pointing the frequent lings and observing the disorder in multiple gen- resulted in a virtual explosion of medical genetic erations in one family. Their observations were information. This information has greatly ex- reported decades before Mendel's laws were re- panded our understanding of disease and the discovered. In 1895 Dejerine and Sottas de- mechanisms that cause them. One example is the molecular dissection of the Charcot-Marie- scribed a more severe neuropathy (3), the Dejer- Tooth (CMT) peripheral neuropathy phenotype. ine-Sottas syndrome (DSS), which was thought The study of CMT has (i) revealed large DNA then to be clinically distinct from CMT. In 1939 Allan (3) used Charcot-Marie-Tooth rearrangements as a frequent mutation mecha- disease, also known as peroneal muscular atro- nism, (ii) illuminated the importance of gene phy, to derive two important principles for clin- dosage as a mechanism, (iii) conceptually fused the seemingly disparate categories of Mendelian ical genetic phenotypes. The first was that differ- disorders and chromosomal syndromes, and (iv) ent patterns of inheritance could be observed in illustrated that both allelic variations and locus disorders thought to be caused by a single defective gene if families were examined. This heterogeneity could be responsible for a spec- enough formulated the concept of ge- trum of clinical phenotypes which may include Allan hypothesis netic heterogeneity (or locus heterogeneity) entities thought to be environmental or ac- quired. This review will briefly summarize what that mutations at different loci could be responsible for the same disease in different was known prior to molecular studies, what we phenotype have learned through molecular genetic analysis, families. The second general principle was that age onset and clinical of the dis- and finally, what these lessons mean with re- the of severity spect to other human genetic disorders. ease were somewhat dependent upon the pattern of inheritance. Recessive disorders are caused by two mutant genes and have an earlier onset and increased severity when compared What Was Known? with dominant conditions in which only one In 1886, Charcot and Marie (1) in Paris, France gene in a pair is abnormal. and Tooth (2) in Cambridge, England indepen- Throughout the 1960s and 70s, the clinical dently described the disorder of the peripheral details of CMT subtypes and other related pe- nerves that leads to distal muscle atrophy and ripheral neuropathies were elucidated (reviewed in ref. 4). An important clinical observation was Address correspondence and reprint requests to: Dr. James the recognition that two major CMT types could R. Lupski, Baylor College of Medicine, One Baylor Plaza, Room 609E, Houston, TX 77030, U.S.A. Phone: 713-798- be distinguished on the basis of electrophysi- 6530; Fax: 713-798-5073; E-mail: [email protected] ologic and pathologic studies. CMT type 1 4 Molecular Medicine, Volume 4, Number 1, January 1998

(CMT1), which is the demyelinating form pri- The PMP22 gene encoding peripheral myelin marily affecting the glial cells supporting the protein 22, which is mutated in the mouse mod- neuron, is characterized by reduced or slowed els for human demyelinating neuropathies, motor nerve conduction velocities (NCV) and Trembler and Trembler' (15,16), was shown to "onion bulbs" consisting of defective Schwann map within the 1.5 Mb CMT1A duplication/ cell processes on nerve biopsy. In contrast, CMT HNPP deletion region (17-20). The absence of type 2 (CMT2) is characterized by normal or PMP22 point mutation in CMT1A duplication pa- nearly normal NCV with decreased amplitudes tients further supported a gene dosage model reflecting the axonal involvement in this sub- (21). Rare demyelinating neuropathy patients type. without the CMT1A duplication were found to Although the subtypes of CMT could be dis- have PMP22 point mutations, which are usually tinguished clinically and pathologically, it wasn't associated with a more severe CMT1 phenotype until the 1980s that the application of genetic than observed with duplication (22,23). PMP22 linkage analysis enabled the identification of spe- point mutations were also identified in patients cific genetic loci responsible for CMT1 (5-7). with DSS (24). To underscore a PMP22-specific dosage effect, increased levels of PMP22 mRNA were found in biopsied peripheral nerves of patients with the CMT1A duplication (25). Fur- What Have We Learned? thermore, multiple transgenic animals that over- Chromosomal Duplication as a express wild-type PMP22 (26-28), and a PMP22 Mutational Mechanism knockout mouse heterozygous for a PMP22 null The molecular mechanism responsible for the allele (29,30), recapitulated the phenotypic majority of patients with the CMT phenotype properties of the human demyelinating periph- linked to the proximal short arm of chromosome eral neuropathies. Thus, substantial evidence 17 (17pl 1 .2p12) is a submicroscopic DNA dupli- supports the notion that PMP22 is the dosage- cation. This CMT1A duplication is 3 million base sensitive gene responsible for the demyelinating pairs (3 megabases or 3 Mb) in length! It consists phenotype in patients with CMT1A duplication of a duplicated 1.5 Mb monomeric unit, arranged as well as HNPP deletion. This dosage effect is in tandem, and flanked by a 24,000 base pair (24 manifested by either trisomic overexpression in kilobases or 24 Kb) direct repeat, named CMT1A or monosomic underexpression in HNPP CMT1A-REP (8-10). The molecular mechanism (31,32). responsible for the CMT1A duplication is an unequal crossing-over event mediated by the ho- Bridging the Gap between Chromosomal Syndromes mologous CMTlA-REP repeats. The proposed Disorders mechanism predicted a reciprocal recombination and Mendelian product resulting in a 1.5 Mb deletion (10). This Traditionally, disorders that segregate as Mende- was subsequently shown to be associated with lian traits have been believed to result from mu- the clinically distinct demyelinating peripheral tation in single genes. In contrast, chromosomal neuropathy known as hereditary neuropathy syndromes have been thought to result from ef- with liability to pressure palsies (HNPP) (11,12). fects of many genes within or flanking the region of chromosomal abnormality. Down syndrome associated with trisomy 21 is the most common Gene Dosage as a Mechanism for Disease genetic condition, and yet it has no mutant Several mechanisms were proposed to explain genes; however, a distinct clinical phenotype is how the CMT1A duplication might affect a observed. "CMT1 gene." These included (i) gene interrup- Although the potential effects of gene dosage tion at the duplication junction (most favored by imbalance in chromosomal syndromes had al- this author), (ii) a position effect, and (iii) a gene ready been appreciated (33), the concept of dosage effect due to a dosage-sensitive gene lo- "gene dosage" or gene copy number effects was cated within the duplicated region. The identifi- crystallized by findings at the CMT1A locus. A cation of large, cytogenetically visible chromo- submicroscopic DNA duplication was passed somal duplications of chromosome 17p that through generations as a dominant trait and was contained the CMT1A locus in patients whose responsible for the segregation of the CMT1A phenotype included slowed motor NCV sup- neuropathy phenotype observed by electro- ported the gene dosage model (13,14) (Fig. 1). physiologic studies revealing reduced motor NCV J. R. Lupski: Charcot-Marie-Tooth Disease 5

CHROMOSOMAL 17p SUBMICROSCOPIC C,) ! * CMTlA-REP z 13.3 0 If 13.2 J. 13.1 .1 1* CD 12 J, I CM 11.2 * PMP22 I

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I I CMTlA-REP T 13.3 .1 I .4 CD 13.2 1* I z 0 0 13.1 t I J, I T uJ 12 0 -j I I PMP22 - * LU 11.2 T I I A I CMTlA-REP 100kb HNPP I deletion

Fig. 1. Chromosomal syndrome versus Mende- karyogram is an expansion of the submicroscopic lian disorders. In the middle of the figure the G- 17pl2 region with the PMP22 gene (hatched box) banded ideogram for the short arm of chromosome flanked by CMT1A-REP repeats (closed box). The 17-17p is shown; to the left are cytogenetically visi- region duplicated is shown by a bold vertical line ble chromosomal DNA rearrangements, while to the (top right) whereas that deleted is shown by a right are submicroscopic rearrangements. Bold verti- dashed vertical line (bottom right). The asterisk rep- cal rectangles represent the 17p region duplicated resents the point mutation in PMP22 that can be as- (top left), open vertical rectangles show the region sociated with HNPP. deleted (bottom left). Shown to the right of the

(34). Likewise, cytogenetically visible chromo- tion that appears mechanistically to occur by somal duplications involving the same region homologous recombination of a flanking repeat presented with the motor NCV abnormality as a gene cluster (39). However, some rare patients distinct part of their more complex phenotype have larger deletions which can include the (35). CMT1A/HNPP genomic region in 17pl2 (40,41). The HNPP deletion is a 1.5 Mb submicro- When SMS patients have a larger deletion that scopic DNA rearrangement. Although as many as includes PMP22, they exhibit eletrophysiologic 30 to 50 genes are likely deleted, only one gene, features consistent with HNPP (40,41). Thus, PMP22, appears to be dosage-sensitive, resulting manifesting a single Mendelian disorder versus a in a haploinsufficiency phenotype. The proof for chromosomal syndrome may be reflective of the this concept is provided by the identification of size of the DNA rearrangements and the number frameshift mutations in PMP22, presumably re- of dosage-sensitive genes involved. sulting in null alleles, in some nondeletion HNPP patients (36,37). Chromosomal Duplication Can Be a Cytogenetically visible deletion of 17pll.2 High-Frequency Mutation results in the Smith-Magenis syndrome (SMS) Chromosomal duplications have been known for (38). Most SMS patients have a common dele- decades in fruit flies and have been extensively 6 Molecular Medicine, Volume 4, Number 1, January 1998 analyzed both genetically and physically in bac- Proximal Distal teria during the last 20 years (42). They have CMT1 A-REP CMT1 A-REP been demonstrated to occur at a surprisingly high frequency. As there is no net loss of genetic W\\\\\\\\\\\\H information, duplications are essentially unre- I stricted in size and location on the chromosome. 1\i \\\\\ 0 k- To date, at least seven loci have been associ- , v ated with a CMTI phenotype, yet 70-90% of all patients have the CMT1A duplication (43). These EcoRl I Distal observations suggest that duplication may be ten U 7 CMT1A-REP times or more likely to cause CMT1 than other mutational mechanisms. Furthermore, CMT1 is 7 Proximal one of the most common inherited disorders 7 CMT1A-REP 5' 3, Ns! I with an estimated prevalence of 1/2500 individ- MITE uals. One study estimates that 10% of the Hotspot CMT1A duplication cases result from de novo events (44), suggesting a mutation frequency on Fig. 2. Mariner transposon mediated recombi- the order of 10-4. This is 2 to 4 orders of mag- nation hot spot. The unequal crossing-over event with the distal CMTlA-REP aligning with homolo- nitude greater than the spontaneous mutation gous proximal CMTIA-REP is shown at the top of frequency usually associated with single-gene the figure; the bottom section shows an enlargement disorders. of the recombination hotspot region. The mariner insect transposon-like element (MITE) is depicted with four hypothetical transposase molecules (filled circles) at the ends. The model consist of three parts: A Recombination Hotspot Associated with Reciprocal (1) trans-acting transposase initiating a double-strand Recombination break at MITE, (2) CMTlA-REP providing 24 Kb of Homologous -99% homology for recombination mediated repair, The CMTIA-REP repeats flanking the CMT1A and (3) the hotspot reflecting resolution of the Holli- duplication/HNPP deletion (45) are -99% iden- day junction. tical across a 24,011 bp region. This provides a substantial region of homology for crossover events to occur, yet the majority of crossover events resulting in these DNA rearrangements Genome Evolution and Consequences of DNA occur within an approximately 1.7 Kb region Rearrangements in the CMT1A/HNPP Region (46,47). Analysis of the DNA sequence sur- The nucleotide sequence of CMTlA-REP has re- rounding the crossover hotspot region reveals no vealed one other coding exon in addition to that significant increase in sequence identity between for the putative transposase of MITE. This exon is the proximal and distal CMT1A-REPs when part of the COX1O gene encoding heme A: farne- compared with surrounding sequence (46). syltransferase which farnesylates the heme A These data suggest that some other signal must moiety incorporated into cytochrome oxidase be present at or near the site of strand exchange (45). The human COXIO gene was cloned by to generate the hotspot. Intriguingly, a mariner human cDNA complementation of a cytochrome transposon-like element, termed MITE, maps oxidase-deficient yeast but was never mapped in near the hotspot (46). We have proposed that the human genome. Subsequent analysis re- MITE may stimulate homologous recombination vealed that the entire COX10 gene spans the dis- between CMT1A-REPs by providing a target for tal CMTlA-REP (45,49). Exon VI of COX10 and double-strand breaks (46) (Fig. 2). The analysis 24 Kb of surrounding sequence appear to have of the DNA sequence of the recombinant been duplicated during genome evolution, at a CMTIA-REP in HNPP patients reveals homolo- time of divergence between gorilla and chimpan- gous recombination products consistent with a zees (45,50), as there are two copies present in double-strand break model (48). Furthermore, chimpanzees and only one in gorilla, and copied these DNA sequencing studies of recombinant 1.5 Mb proximally on chromosome 17p to yield CMTIA-REP elements have revealed what may the proximal CMTIA-REP (Fig. 3). Thus, the be minimal efficient processing segments (MEPS) HNPP deletion results in one null allele of COX10 required for human homologous meiotic recom- (45). These findings suggest a further complexity bination. to the consequences of DNA rearrangements in J. R. Lupski: Charcot-Marie-Tooth Disease 7

cox1o CO1 -I PMP22 .I cox1o

I~~~~~II/~~~~ % rr_ P - _1 Li//// ' I PMP22 II cox1o

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Fig. 3. HNPP deletion interrupts the COX1O end of COX10 is located outside the genomic region gene. The unequal crossing-over between distal duplicated in CMT1A and deleted in HNPP while the CMTIA-REP and proximal CMTlA-REP is shown at 3' end is within this genomic region. Note the un- the top of the figure. The COX10 gene (bold) spans equal crossover results in recombination products distal CMTlA-REP with one coding exon (exon VI) consisting of the CMT1A duplication, with the re- embedded within distal CMTlA-REP. The PMP22 combinant CMTIA-REP missing the 5' end of COXIO gene (stippled box) is located between the flanking but having one normal copy present, and the HNPP CMTIA-REPs. The direction of transcription for deletion with the recombinant CMTIA-REP missing COXIO is depicted by the horizontal arrow. The 5' the 3' end of COX10.

the CMTlA/HNPP region in addition to altering tional mechanism in demyelinating peripheral the copy number of the dosage-sensitive PMP22 neuropathy. However, this mechanism does not gene. The disruption of COX10 may have ramifi- constitute a mutation hotspot in these three cations for phenotype expression in individuals genes that is significantly different from other with the HNPP deletion (45). disease-associated genes (51). Mutations in MPZ and PMP22 have been identified in patients with the related peripheral Peripheral Neuropathies Represent a Spectrum of neuropathy Dejerine-Sottas syndrome, while Genotypically Related Entities MPZ mutations have also been observed in pa- While the majority of patients with CMT1 or tients with congenital hypomyelination (Fig. 4). HNPP have DNA rearrangements that are re- The crystal structure of the extracellular domain sponsible for their disease, the discovery of point of PO has recently been determined (52). This mutations in genes encoding major myelin pro- information, in conjunction with natural muta- teins have yielded new insights into the patho- tions observed in demyelinating neuropathy pa- genesis of these disorders. The three genes iden- tients, has enabled attempts at genotype/pheno- tified to date are PMP22, MPZ encoding myelin type correlations (53). Such analyses suggest that protein PO, and Cx32 encoding the gap junction these neuropathies represent a spectrum of clin- protein connexin 32 (51). The analysis of dis- ical severity and that the phenotype is dependent ease-associated sequence alterations in patients upon the nature of the mutation and its effect on reveals that point mutations via methylation- the gene and protein product. Furthermore, mediated deamination is an important muta- more severe phenotype associated with carboxy 8 Molecular Medicine, Volume 4, Number 1, January 1998

*I# Charcot-Marie-Tooth disease JC Nonsense mutation Al* .-A-. 9' Frameshift mutation A Amino acid deletion *0/Missense

PMP22 PO Cx32 Fig. 4. Myelin gene mutations and myelinopa- the PMP22 mutations are located in the predicted thies. Protein structure models for myelin gene transmembrane domain whereas PO mutations are products associated with myelinopathies. The upper predominantly in the extracellular domain. The Cx32 left portion of the figure shows the symbols for the mutations associated with X-linked CMT are located specified myelinopathy phenotypes while the upper throughout the molecule. right gives the key to the mutation types. Note that terminal mutations suggests an important func- duplicated segment of the genome provides an tion for the PO intracellular domain (53). even larger region for homologous recombination and reversion of the duplication mutation. At least one such case has been suggested by the identification of a patient mosaic for the CMT1A What Does It Mean? duplication who displayed a milder phenotype The identification of the CMT1A duplication sug- than that in the affected parent with the dupli- gests that other clinical phenotypes may result cation (54,55). However, if this were occurring from submicroscopic DNA rearrangements as op- to a significant extent, one might expect to ob- posed to mutations within a gene classically serve segregation distortion, or less than the ex- thought of for Mendelian diseases. These rear- pected 50% affected individuals, in large autoso- rangements may reflect structural features of the mal dominant pedigrees. human genome. The mutation frequencies for The CMT1A duplication frequency also DNA rearrangements, and thus de novo muta- likely reflects the recombination hotspot in tion in sporadic cases, may be quite high when CMTIA-REP. Whether DNA transposons such as compared with point mutations. Since these re- MITE or other mariner-like elements (MLEs) are arrangements may reflect intrinsic structural involved in initiating double strand breaks that properties of the genome, the mutation and thus stimulate homologous recombination events the disease frequency may be similar in different elsewhere in the human genome remains to be populations. determined. The high frequency of new mutations sug- DNA duplications can be quite large since gests that the disease prevalence could be in- there is no net loss of genetic information. Given creasing unless reversions are in equilibrium. Af- the potential for large size, a duplication may ter the CMT1A duplication occurs, the 1.5 Mb encompass several genes, but it appears from J. R. Lupski: Charcot-Marie-Tooth Disease 9 studies of CMT1A and HNPP that only a subset of References genes are dosage-sensitive. The PMP22 dosage 1. Charcot J-M, Marie P. (1886) Sur une forme par- phenomenon also suggests that the stoichiome- ticulaiere d'atrophie musculaire progressive sou- try of this protein is important in maintaining vent familiale debutant par les pied et les jambes normal myelin structure and function. The find- et atteignant plus tard les mains. Rev. Med. 6: 97- ing of only one dosage-sensitive gene in a rela- 138. 2. Tooth H. (1886) The Peroneal Type of Progressive tively large 1.5 Mb genomic region suggests that Muscular Atrophy. H. K. Lewis, London. the phenotypic manifestations of chromosomal 3. Allan W. (1939) Relation of hereditary pattern to aneuploidy syndromes may represent the effects clinical severity as illustrated by peroneal atrophy. of a small subset of dosage-sensitive genes. Fur- Arch. Intern. Med. 63: 1123-1131. thermore, the identification of specific clinical 4. Dyck PJ, Chance P, Lebo R, Carney JA. (1993) features that can sometimes be found as domi- Hereditary motor and sensory neuropathies. In: nant traits (e.g., syndactyly, brachydactyly, mi- Dyck PJ, Thomas PK, Griffin JW, Low PA, Poduslo crocornea, etc.) in patients with cytogenetically JF (eds). Peripheral Neuropathy. W. B. Saunders, Philadelphia, pp. 1094-1136. visible duplications may suggest a potential lo- 5. Bird TD, Ott J, Giblett ER. (1982) Evidence for calization for a dosage-sensitive gene associated linkage of Charcot-Marie-Tooth neuropathy to with the trait present within the region of seg- the Duffy locus on chromosome 1. Am. J. Hum. mental aneuploidy. Genet. 34: 388-394. In summary, there recently has been re- 6. Gal A, Mucke J, Theile H, Wieacker PF, Ropers markable progress in elucidating the molecular H-H, Wienker TF. (1985) X-linked dominant genetic bases of inherited peripheral neuropa- Charcot-Marie-Tooth disease: Suggestions of link- thies. These investigations confirm the long-held age with a cloned DNA sequence from the proximal Xq. Hum. Genet. 70: 38-42. suspicion that inherited peripheral neuropathies 7. Vance JM, Nicholson GA, Yamaoka LH, et al. are the clinical manifestations of peripheral (1989) Linkage of Charcot-Marie-Tooth neuropa- nerve dysfunction resulting from abnormalities thy type la to chromosome 17. Exp. Neurol. 104: in Schwann cells and their myelin sheath. These 186-189. studies have also revealed much about the biol- 8. Lupski JR, Montes de Oca-Luna R, Slaugenhaupt ogy and structure of the peripheral nerve and S, et al. (1991) DNA duplication associated with uncovered novel genetic mechanisms. In addi- Charcot-Marie-Tooth disease type 1A. Cell 66: tion, the new molecular knowledge has ramifi- 219-232. care. The the 9. Raeymaekers P, Timmerman V, Nelis E, et al. cations for patient findings provide (1991) Duplication in chromosome 17plI.2 in clinician with diagnostic tools to enable a precise Charcot-Marie-Tooth neuropathy type la and secure diagnosis (56-58), enable accurate (CMTla). Neuromuscul. Disord. 1: 93-97. recurrence risk estimates, and provide prognostic 10. Pentao L, Wise CA, Chinault AC, Patel PI, Lupski information (59) and the potential to design ra- JR. (1992) Charcot-Marie-Tooth type 1A duplica- tional therapeutic approaches. However, perhaps tion appears to arise from recombination at repeat most importantly, genetic findings in this group sequences flanking the 1.5 Mb monomer unit. of disorders has widespread implications for hu- Nat. Genet. 2: 292-300. man and the molecular medicine of 11. Chance PF, Alderson MK, Leppig KA, et al. (1993) genetics DNA deletion associated with hereditary neurop- other disease phenotypes. athy with liability to pressure palsies. Cell 72: 143- 151. 12. Chance PF, Abbas N, Lensch MW, et al. (1994) Two autosomal dominant neuropathies result from reciprocal DNA duplication/deletion of a re- Acknowledgments gion on chromosome 17. Hum. Mol. Genet. 3: 223- I thank the members of my laboratory for their 228. critical reviews, for providing a stimulating envi- 13. Lupski JR, Wise CA, Kuwano A, et al. (1992) ronment, and for continuing to make doing sci- Gene dosage is a mechanism for Charcot-Marie- Tooth disease type IA. Nat. Genet. 1: 29-33. ence fun. The work described here has been 14. Chance PF, Bird TD, Matsunami N, Lensch MW, generously supported by the National Institute of Brothman AR, Feldman GM. (1992) Trisomy 17p Neurological Disorders and Stroke (NIH), the associated with Charcot-Marie-Tooth neuropathy Muscular Dystrophy Association, and the Pew type IA phenotype: Evidence for gene dosage as a Scholars Program in Biomedical Sciences. mechanism in CMT1A. Neurology 42: 2295-2299. 10 Molecular Medicine, Volume 4, Number 1, January 1998

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