Chapter 19 Genetic basis of peripheral neuropathies

F.W. Van Leeuwen, A. Salehi. R.J. Giger, A.J.G.D. Holtmaat and J. Verhaagen (Eds.) Progress in Brain Research, Vol I17 0 1998 Elsevier Science BV. All rights reserved.

CHAPTER 19

Genetic basis of peripheral neuropathies Linda J. Valentijn and Frank Baas* Department of Neurology, K2-214 Academic Medical Center, PO Box 22660, 1100 DD Amsterdam, The Netherlands

The conduction of signals through nerves (nerve conduction velocity, NCV) is fast due to the electric isolation of axons by myelin sheaths. In the peripheral nerves the myelin sheats are formed by the Schwann cells by the formation of multiple membrane layers around the axons. The major proteins of myelin are myelin protein PO (over 50% of total protein) and myelin basic protein (MBP, 5-1 5%). Disturbances in myelin formation can lead to a progressive loss of myelin, demyelination or even an incomplete formation of the myelin structure (amyelination), which result in a decreased NCV and is the basis of several neuropathies. The identification of the genetic basis of these neuropathies has led to identification of novel myelin proteins and to a better understanding of the pathogenesis of Schwann cell disorders.

Hereditary peripheral neuropathies Charcot-Marie-Tooth disease type 1 (CMT) or hereditary motor and sensory neuropathy type I (HMSNI) is the most common hereditary neuropathy. CMTl is one of seven subtypes of HMSN as classified by Dyck (1984). The classification was based on clinical, electrophysiological, histopathological features and mode of inheritance (Table I).

* Corresponding author. e-mail: [email protected]

CMTl is a hypertrophic demyelinating neuropathy with reduced NCVs. Teased nerve fibre studies show that most myelinated fibres have demyelinated segments and are frequently surrounded by concentric, loose Schwann cell membrane structures, called onion bulbs (Dyck, 1984, GabreelsFesten et al., 1992; 1993). This suggests that the pathology is not due to a defect in myelin formation, but more likely due to a defect in maintaining the integrety of a myelin sheet. CMTX is a subtype of CMTl and was classified separately for its X-linked inheritance. HMSNII/ CMT2 is the axonal type. The NCVs are in the (near) normal range (50-70 mjsec) and no myelin abnormalities are observed. Other less common subtypes were classified of which HSMNIII or Dejerine-Sottas Syndrome (DSS) is a severe demyelinating neuropathy starting in early childhood (Dyck, 1984). The NCVs are extremely low and the nerve biopsies show demyelination, hypomyelination and even amyelination (Gabreels-Festen et al., 1993). A link between DSS and CMTl was presented in an extensive CMTl pedigree, suggesting that these two diseases might be allelic. A few patients inferred to be homozygous for the CMTl mutation, presented DSS phenotype (Killian and Koepfler, 1979). Hereditary neuropathy with liability to pressure palsies (HNPP) or tomaculous neuropathy is characterized by acute or recurrent transient muscle palsies in response to microtrauma (Davies, 1954; Earl et al., 1964). The peripheral nerves

250

TABLE I Features of CMTl and related neuropathies.

A D CMTliHMSNI CMTX

A D CMTZ/HMSNII AR Dejerine-Sottds neuropathy/HMSNIIi HNPP

Clinical features

Median MNCV (mjsec)

neuropathology

distal muscular atrophy and weakness hollow feet claw hands males more severely affected than CMTl distal muscular atrophy and weakness hollow feet Severe form of CMT 1 early onset recurrent muscle palsies and paralysis

638

segmental demyelination and remyelination (onion bulbs)

males 30 to normal females 44 to normal near normal

distal neuro-axonal degeneration loss of myelinated fibres and marked regeneration hypomyelination with onion bulbs, o r even amyelination tomacula

< 6 decreased'

'MNCV not uniformly reduced. At the site of trauma, the MNCV is more reduced. A D recessive

=

autosomal dominant, AR

=

autosomal

show localized myeline thickenings, named tomacula (Madrid and Bradly, 1975). In this paper we will discuss the research that has led to the identification of the genes for CMT1, HNPP and DSS and speculate about possible pathogenetic mechanisms.

et al., 1990; Middleton-Price et al., 1990; Timmerman et al., 1990). The CMTlA locus was narrowed down to chromosome 17 band pl1.2 (Pate1 et al., 1990; Vance et al., 1991).

Linkage studies in CMTl show evidence for heterogeneity

The observation that markers of chromosome 17p detected three fragments on a DNA Southern blot of some CMTlA patients, instead of the normally expected one or two, started the search for DNA rearrangements in the CMTlA region (Lupski et al., 1991; Raeymaekers et al., 1991). A large DNA duplication on chromosome 17pl1.2 appeared to be associated with CMTlA. Based on analysis of a family with a CMTl patient carrying a de novo duplication, Raeymaekers et al. (1991) suggested that the duplication was the result of unequal crossing over between the two chromosomes 17 during meiosis. The CMTlA associated DNA duplication was identified in 9 out of 10 de novo CMTl patients (Hoogendijk et al., 1992). This observation presented strong evidence that the DNA duplication was the genetic defect resulting in the CMTlA phenotype.

Initial genetic studies revealed linkage of CMTl to the Duffy locus on chromosome 1 (Bird et al., 1982). However, in two large CMTl families evidence against linkage to chromosome 1 was found (Bird et al., 1983; Dyck et al., 1983). Other reports confirmed the existence of a second locus (Griffits et al., 1988; Middleton-Price et al., 1989; Raeymaekers et al., 1989a). In fact, the majority of the CMTl families did not show linkage to chromosome 1. These observations led to the subdivision in CMTl A (location unknown) and CMTlB (chromosome 1). In 1989, Vance et al. presented evidence for linkage of CMTlA to chromosome 17. These results were confirmed in other families (Raeymaekers et al., 1989b; Defesche et al., 1989; Chance et al., 1990; McAlpine

Duplication on chromosome 17pll.t in CMTlA

25 I

The identification of a duplication provided geneticists with a tool for mutation detection, but the mechanism by which the duplication would result in CMTlA was still unclear. Either a gene at the duplication border could be disrupted or the presence of additional copies or overexpression of one or more genes in the duplication could be the pathogenic mechanism. Gene disruption, which might result in expression of a novel fusion protein or altered expression of a normal protein, was initially considered the most likely mechanism. There were not many precedents for alterations in gene copy associated with disease, trisomies excluded. Therefore, research was focussed on identification of the breakpoints of the CMTlA duplication. However, after the description of a partial 17p trisomy by Lupski et al. (1992) and of a 17p trisomy due to a translocation [t(14; 17)] by Chance et al. (1992) in patients with typical CMTlA symptoms in addition to other defects, an increased gene copy number, and not gene disruption could be the pathogenetic mechanism of CMTlA. Therefore, any gene within the large duplication, which spanned at least 1.1 megabase (Hoogendijk et al., 1991) was a candidate gene for CMTlA. Identification of a novel myelin gene in the Trembler mouse, an animal model for CMTlA The identification of the gene responsible for CMTlA was greatly facilitated by the presence of an animal model for peripheral neuropathies, the Trembler mouse. The Trembler mouse is a ‘spontaneous’ neurological mutant with partial paralysis of the limbs and tremor (Falconer, 1951). Two independent strains, Trembler and TremblerJ, from independent mutation events were described (Henry et al., 1983). The affected nerves of Trembler mice show thin or absent myelin, onion bulbs and an increased number of Schwann cells (Ayers and Anderson, 1973). The NCV was below 10 m/sec, with the normal values ranging from 35 to 60 m/sec (Low and McLeod, 1975). This decrease in NCV and the observed Schwann cell defect with extensive demyelination and onion

bulb formation showed resemblance to DSS. Therefore Low and Mcleod (1975) suggested that Trembler was an animal model for DSS. The Trembler locus was located on murine chromosome 11 (Henry et al., 1983) in a region which is syntenic to the CMTlA region on human chromosome 17~11.2.In view of the chromosomal localization and the phenotype, Trembler was considered a model for CMTlA (Vance, 1991). The mutation causing Trembler was identified by Suter et al. (1992b). They mapped a novel gene, the peripheral myelin protein-22 (pmp22) gene, previously known as the growth arrest specific gene 3 (gas3) or rat SR13, to the murine chromosome 11 in the vicinity of the Trembler locus. Analysis of the coding region of pmp22 showed a point mutation resulting in an amino acid substitution (GlylSOAsp) in Trembler (Suter et al., 1992b) and the identification of a point mutation in pmp22 in Trembler-J (amino acid substitution Leul6Pro) provided more evidence of the involvement of pmp22 in neuropathies and showed that Trembler and Trembler-J are allelic (Suter et al., 1992a). Pmp22/gas3 was initially identified as one of six genes which were induced upon growth arrest in mouse fibroblasts (Schneider et al., 1988). The 1.8 kb mRNA encodes a 22 kDa protein. In vitro translation studies established that pmp22 is a glycosylated transmembrane protein (Manfioletti et al., 1990). Analysis of the expression pattern of pmp22 showed mRNA expression in most tissues tested and the levels in the peripheral nerves were very high. The regulation of pmp22 expression during sciatic nerve degeneration and regeneration appeared to be linked to proliferation and differentiation of Schwann cells, again compatible with a role of pmp22 in growth regulation (Spreyer et al., 1991). However, like myelin basic protein, pmp22 is only expressed in differentiated, quiescent Schwann cells, which is also compatible to a direct role of pmp22 in myelin formation. The expression of rat pmp22 protein (SR13) was found to be similar to major myelin protein PO, which is located in the myelin sheats surrounding the axons (Welcher et al., 1991).

252

The identification of two differentially regulated pmp22 mRNAs from rat peripheral nerves is compatible with a dual role for pmp22. The two transcripts, CD25 (Spreyer et al., 1991) and SR13 (Welcher et al., 1991), differ in the 5’-untranslated region (5’UTR) and show a different expression pattern during the development of the peripheral nerves (Bosse et al., 1994). CD25-mRNA is low at birth and increases to a maximal expression two weeks after birth, when myelin formation is at an advanced stage. SR13 expression is high at birth and rapidly declines after birth. In addition, SR13 expression was also observed in other tissues (colon, lung), whereas CD25 was expressed only in nerves. In vitro studies established that SR13 is probably related to cell growth and CD25 to myelination (Bosse et al., 1994). In summary, the pmp22 expression in Schwann cells, mutations in Trembler and Trembler-J, and chromosomal localization made PMP22 a good candidate for CMTlA. Alterations of PMP22 in CMTlA Many investigators provided evidence for the localization of PMP22 in the CMTl A-associated DNA duplication (Patel et al., 1992, Valentijn et al., 1992b; Timmerman et al., 1992; Matsunami et a]., 1992). Using pulse-field gel electrophoresis analysis, in situ hybridization and analysis of yeast artificial chromosomes from the CMT 1A region, the PMP22 gene was shown to be located in the middle of the duplicated region. Consequently, overexpression of PMP22 was an appealing hypothesis as cause for CMTlA, although involvement of other genes in the region could not be ruled out. The expression pattern in human, mouse and rat tissues is comparable: high in peripheral nerves and low in brain, heart and muscle (Patel et al., 1992). The increased PMP22 expression after growth arrest in human fibroblasts was similar to gas3/pmp22 expression in mouse fibroblasts (Valentijn et al., 1992b). Final evidence for PMP22 being the gene causing CMTlA came from a study in a family with linkage to chromosome 17~11.2,but without

the duplication (Hoogendijk et al., 1993). We have analysed the coding region of the PMP22 gene and identified a point mutation which segregated with CMTlA in the pedigree (Valentijn et al., 1992a). The mutation resulted in an amino acid substitution (LeulGPro) in the putative first transmembrane domain of PMP22. This mutation was identical to the mutation in the Trembler-J mouse. A de novo mutation in PMP22 presented confirmational evidence that PMP22 is the ‘CMT1A’gene (Roa et al., 1993b). This mutation, Ser79Cys, was also located in a transmembrane domain. Duplication versus deletion of the CMTlA region

In most patients the duplication is about 1.5 Mb, which suggests that a common mechanism is responsible for the duplication. With two duplicated probes used in fluorescent in situ hybridization, we showed that the duplication is a direct repeat (Valentijn et al., 1992b). The identification of a low copy repeat (CMTlA-REP) flanking the duplication provided an explanation for the fact that the majority of duplications are of similar size (Pentao et al., 1992). The CMTlA-REP units cover at least 17 kb. Unequal crossing-over due to misalignment at the site of the CMTlA-REP would result in one chromosome carrying a duplication and one with a deletion (Fig. 1). This model predicted that not only duplications of the CMTlA region, but also deletions in this region could occur. The identification of the predicted reciprocal deletion event in three HNPP families by Chance et al. (1993) supported the model of unequal cross-over. Subsequently, the deletion was identified in other HNPP families (Mariman et al., 1993/1994; LeGuern et al., 1994; Verhalle 1994). The reciprocal event of the duplication versus deletion was shown by analysis of the CMTlA-REP in CMTlA and HNPP samples. Using a probe which detects the separate repeats as two different sized fragments, it was demonstrated that the unequal crossing over event occurs within a limited area of the large repeat (Chance et al., 1994). These findings suggest that the prevalence of CMTlA and HNPP could be

253

Fig. 1. Proposed mechanism of recombination resulting in chromosomes duplicated and deleted for PMP22. The PMP22 gene is indicated with a black circle, the CMTlA-REP with triangles. (Baas et al. 1994).

similar. However the prevalence of HNPP is unknown possibly due to the comparatively large number of subclinical cases, which are only identified if other relatives develop a clinical phenotype. The misalignment event resulting in duplication or deletion mainly seems to take place during spermatogenesis. The paternal origin of the 1.5 Mb duplication or deletion is the result of unequal sister chromosomal exchange (interchromosomal exchange) as has been shown in de novo patients (Chance et al., 1993; Raeymaeckers et al., 1991; Palau et al., 1993; Verhalle et al., 1994; Wise et al., 1993; Blair et al., 1996; Lopes et al., 1997). Five cases of maternal origin have been reported so far (Blair et al., 1996; Lopes et al., 1997). The origin of the maternally derived duplication or deletion is different from the paternal cases. This event is the result of unequal crossing over between sister chromatids (intrachromosomal exchange) (Lopes et al., 1997). The estimated number of genes in the 1.5 Mb duplication is 30, based upon an average of one gene per 50 kb (Fields et al., 1994). Therefore it can not be excluded that other genes in the duplication are involved in modulation of the CMTlA phenotype. So far three other duplicated genes have been identified (Murakami et al., 1997). Three cases of smaller duplications including PMP22 were reported (Ionasescu et al., 1993;

Palau et al., 1993; Valentijn et al., 1993). One of these small duplications was mapped in detail and involved a region of 460 kb including PMP22 (Valentijn et al., 1993). The 1 Mb region telomeric of PMP22 was not duplicated. Consequently, genes in this region are not required for the CMTlA phenotype. Mutations in PMP22 Extensive studies of unrelated CMTlA and HNPP patients showed the presence of the duplication or deletion in 75% of the cases (Schiavon et a1.,1994; Ohnishi et al., 1995; Nelis et al.; 1996; Timmerman et al., 1997). In non-duplicated CMTlA multiple mutations have been described, which are all located in the transmembrane regions (Table 11). Direct evidence for the involvement of PMP22 in HNPP was shown in a family lacking the 1.5 Mb deletion (Nicholson et al., 1994). We identified a two base pair deletion in codon Ser7 of PMP22. The mutation results in a frameshift and premature stopcodon at codon 36 and has therefore an effect similar to deletion of the PMP22 gene. Additional mutations in HNPP were identified which always involved frameshifts or splice mutations. (Table 11). One exception has been described by Nelis et al. (1994a): a splice site mutation resulting in a putative frame shift mutation in a case of CMTlA. In this case a protein with

254 TABLE I1 Structural domains and mutations of PMP22. PMP22 domains: TM, transmembrane; EC, extracellulair; IC, intracellulair; GS, Nglycosylation site. PMP22 domain

Mutation

Disease

Reference

T M l (2-31)

frame shift at Ser7 HislZGln Leu 16Pro

HNPP DSS CMTlA trembler-J

Nicholson et al. 1994 Valentijn et al. 1994 Valentijn et al. 1992a Suter et al. 1992b

Met69Lys Ser72Leu

DSS DSS

Ser72Trp Ser7911e Ser79Cys Leu80Arg Leu8OPro frame shift at Gly94 frame shift at Arg95 LeulOSArg GlylO7Val’ intron base g( + 1) to a at splice donor exon 3 intron base g(-1) to a at splice acceptor exon 4 Thrl18Met’

DSS DSS CMTlA DSS DSS HNPP HNPP CMTlA CMTlA CMTlA

Roa et al. 1993a Roe et al. 1993a lonasescu et al. 1996 Tyson et al. 1997 Tyson et al. 1997 Roe et al. 1993c

Leul47Arg Glyl SOAsp

EC (32-64) (GS Asn41) TM2 (65-91)

IC (92-95) TM3 (96-119)

EC (12C133) TM3 (134-156) IC (157-160)

Tyson et al. 1997 Young et al. 1997 unpublished results’ Marrosu et al. 1997 Nelis et al. 1994a

HNPP

unpublished results3

arCMT HNPP

Roa et al. 1993b unpublished results3

CMTlA trembler DSS

Navon et al. 1996 Suter et al. 1992a lonasescu et al. 1997

‘Only if no alternative splice site is used, mutation located at splice acceptor exon 4. *phenotype in combination with deleted PMP22. ’AAWM Gabreels-Festen, ECM Mariman, LJ Valentijn, NHA van den Bosch

different aminoacid composition, starting in the third transmembrane segment is to be expected. A possible explanation for this apparent discrepancy is that this patient might have been misdiagnosed, since CMTl features have been described in HNPP (Barbieri et al., 1990). The CMTlA patients in the families with point mutations show variable phenotypes. In the family carrying the Trembler-J mutation, 3 of the 11 patients had a severe phenotype, which might have been diagnosed as DSS, if they were sporadic cases (Hoogendijk et al., 1993; Valentijn et al., 1992a). A

similar severe phenotype was observed in 2 of the 3 affected individuals in a family described by Roa et al. (1993~).Analysis of PMP22 in DSS patients resulted in the identification of additional point mutations (Table 11). All mutations were located in the transmembrane domain. The mutations were heterozygous and showed dominant inheritance, or were de novo. Inheritance from mother to son was also demonstrated in three DSS families with the Glyl 5OAsp (Trembler) mutation (Ionasescu et al., 1997) and Ser72Leu mutation (Roa et al., 1993a; Tyson et al., 1997). Thus, the

255

Dejerine-Sottas phenotype, originally defined as autosomal recessive, can be the result of dominant mutations.

autosomal dominant mutations result in phenotypes with differences in severity, depending on the nature of the mutation.

Identification of the CMTlB gene

Connexin 32 mutations in CMTX

Genetic studies for the chromosome 1 linked form of CMTIB placed the CMTlB locus near the FCG2 locus (immunoglobulin G Fc receptor I1 gene) on lq21-q23 (Lebo et al., 1991; Ionasescu et al., 1992). Comparison of the murine and human genetic map showed that a large region of the murine chromosome 1 was syntenic to human chromosome 1 (Oakey et al., 1992). The PO gene for the major myelin protein PO was located close to FCG2 and therefore an obvious candidate gene for CMTlB. PO is a transmembrane protein which is the major component ( > 50%) of peripheral myelin. PO is involved in the formation of myelin through homophilic interactions of the immunoglobulin-like extracellular domain (Lemke and Axel, 1985). The glycosylation site, the cytoplasmic domain, and the Cys-Cys disulphide bond are all necessary for homophilic interactions (Filbin and Tennekoon, 1993; Wong and Filbin, 1994; Zhang and Filbin, 1994). The coding sequence of the human cDNA (Hayasaka et al., 1991) was analyzed in CMTlB patients. The first mutations identified in CMT 1 B families were two extracellular amino acid substitutions, Lys96Glu and Asp90Glu (Hayasaka et al., 1993a) and deletion of the Ser34 codon (Kulkens et al., 1993). The analysis in the other CMTlB families resulted in additional mutations (Table 111). The mutations are mainly extracellular, and might result in unstable homophilic interactions between the PO molecules. The PO gene was also analyzed in DSS patients. The first two de novo mutations causing DSS were Ser63Cys (Hayasaka et al., 1993b) and a frame shift mutation due to a two base pair insertion (Rautenstrauss et al., 1994). Subsequently other mutations were identified (Table 111) . The mutations identified in CMTlB and DSS confirm the importance of PO in the compaction of myelin. The

In a few CMT families males appeared to be more severely affected than females. In these families no male to male inheritance was found and an Xchromosomal form (CMTX) was suspected. Linkage analysis showed weak lod scores and possible evidence for genetic heterogeneity. Analysis of Xlinked dominant and X-linked recessive families indicated the existence of multiple loci on the proximal long arm and the proximal short arm of the X chromosome (Fischbeck et al., 1986). Three loci on chromosome X were assigned: dominant CMTX between Xq13 and Xq21.1 (Ionasescu et al., 1988; Bergoffen et al., 1993b; Cochrane et al., 1994) and recessive CMTX on Xp22.2 and Xq26-28 (Ionasescu et al., 1991). The dominant CMTX region encompasses the connexin 32 (Cx32, GJBl) gene (Corcos et al., 1992) and was therefore considered a candidate gene (Bergoffen et al., 1993a). Connexins are membrane spanning proteins which assemble into hexamers and form the gap junctions responsible for transport of small molecules from cell to cell. Northern blot analysis of rat tissues showed connexin 32 expression in liver, the sciatic nerve and the spleen (Bergoffen et al., 1993a). Immunofluorescent localization of the Cx32 protein showed that the distribution of Cx32 in Schwann cells is limited to the nodes of Ranvier and Schmidt-Lanterman incisures. The localization suggests that Cx32 might be, involved in the transport of small molecules between the different Schwann cell layers. Analysis of the connexin 32 gene showed mutations in 8 out of 9 CMTX families (Bergoffen et al., 1993a). The Cx32 mutations identified in CMTX include amino acid substitutions, a frame shift and a premature stop codon (Bergoffen et al., 1993a). Since CMT patients show nerve defects only, the function of connexin 32 must be rescued by other connexins expressed in liver and spleen. Since the first

256 TABLE I11 Structural domains and mutations of PO. Amino acid numbering according to the mature protein (= 219 aa; without the 29 aa from the signal peptide). Abbrevations: ht, heterozygous; hm, homozygous; PO domains see Table 11. PO domain

Mutation

Disease

Reference

EC 1-124 C Y S1-Cys98 ~ (Gs Asn93)

IlelMet Thr5lle Ser34 Ser34Cys Ser34Phe Ser49Val Try53Cys Asp61Glu Lys67glu

CMTlB CMTIB CMTIB DSS CMTlB CMTlB CMTlB CMTIB CMTIB

Arg69His

CMTl B

Arg69Ser Arg69Cys

CMT 1B severe CMTIB DSS ht: CMTIB hm: DSS DSS

Hayasaka et al. 1993d Gabreels-Festen et al. 1996 Kulkens et al. 1993 Hayasaka et al. 1993b Blanquet-Grossard et al. 1995 Nelis et al. 1994b Himoro et al. 1993 Hayasaka et al. 1993a Hayasaka et al. 1993a Su et al. 1993 Hayasaka et al. 1993c Meijerink et al. 1996 Warner et al. 1996 Meijerink et al. 1996 Warner et al. 1996 Warner et al. 1996 Warner et al. 1997

CMTIB CMTIB CMTlB CMT 1B CMTlB CMT 1B CMTIB DSS DSS DSS CMTlB CMTlB DSS

Blanquet-Grossard et al. 1996 Gabreels-Festen et al. 1996 Nelis et al. 1994b Nelis et al. 1994c Gabreels-Festen et al. 1996 Gabreels-Festen et al. I996 Nelis et al. 1994c Tyson et al. 1997 Hayasaka et al. 3993b Warner et al. 1996 Nelis et al. 1994c Su et al. 1993 Rautenstrauss et al. 1994

TM 125-150

IC 151-219

frame-shift at Gly74 lle85Thr + Asn87His Asp99Asn Asn93Ser Lysl 01Arg AsplO5Glu AsplO5Asn Ilel06Leu Vall07Glu Tyrl25Stop lle134Thr Gly138Arg frame-shift at Leu145 Tyr 152Stop Thrl87GluArg frame shift at Met193

'

+

'Predicted amino acid substitution from putative new splice site

mutation report, many other mutations were identified (Orth et al., 1994 Ionasescu et al., 1994; Janssen et al., 1997; Ressot et al., 1997). Functional analysis of myelin proteins The biological function of normal proteins can sometimes be derived from phenotypic changes due to mutations. The crystal structure of PO (Shapiro et al., 1996), the mutations in PO (Table

111) in combination with the homophilic interaction studies by Filbin and co-workers (Filbin and Tennekoon, 1993; Wong and Filbin, 1994; Zhang and Filbin, 1994) confirm the structural role of PO in myelin and provide a mechanism for diseases due to PO mutations. In fact, histological analysis of nerve biopsies from patients with PO mutations show that compaction of myelin is reduced (Meijerink et al., 1996), which is in line with the function of PO.

257

The correlation of the genotype and phenotype for PO mutations is obvious. For example, introduction of an additional cysteine aminoacid (Arg69Cys) in the extracellular PO domain results in a more severe phenotype (DSS) than mutation to a different aminoacid (Arg69Ser or Arg69His; Table 111). Warner et al. (1996) proposed an effect of the cysteines based on the position in the cristal structure. The outward directed cysteine side chain might form additional disulfide bonds within myeline which disturb normal myelin formation. A frame shift at codon Gly74 described by Warner et al. (1996) resulted in the CMTl phenotype, probably the result of reduced PO levels due to loss of function. The homozygous presence of the same mutation resulted in the severe DSS phenotype. The severe phenotype is most likely the result of absence of PO protein, and showed strong resemblance to the mouse model without PO expression (Giese et al., 1992). Despite the fact that duplication, deletion and mutations of PMP22 have been identified in CMTlA, HNPP, DSS and the Trembler mouse, its function remains unknown. PMP22 encodes a glycosylated transmembrane protein and accounts for about 5% of the myelin. In view of the observed alterations in the compaction of myelin, PMP22 might be a structural protein. However, since PMP22/gas3 was initially identified as a growth arrest specific protein in fibroblasts (Manfioletti et al., 1990), a role in proliferation can not be excluded and this has been put forward as the pathogenetic mechanism (Haneman et al., 1996). In case of only a structural function of PMP22, the gene dosage effect suggests a stoichiometric interaction with other factors. Transgenic mice carrying a pmp22 transgene established a dose response relation between pmp22 copynumber and disease severity. Unequal stoichiometry due to increased amounts of PMP22 (CMTlA) might disrupt the normal compaction of myelin sheaths and promote onion bulb formation. This disturbance can also be caused by mutations in the transmembrane region, resulting in an improper incorporation in the membrane. However, the observed increased myelin formation observed in the tomacula in

HNPP suggest a more complex disease mechanism. In order to reveal the function of PMP22 in the Schwann cells, animal models were constructed with increased (CMTlA model) and decreased (HNPP model) expression of PMP22 and in vitro studies were performed. Schwann cell cultures showed differences in BrdU incorporation after infection with retroviral expression vectors containing sense or antisense pmp22 (Zoidle et al., 1995). Reduced pmp22 expression due to antisense pmp22 resulted in enhanced DNA synthesis (150%), whereas enhanced expression of pmp22 decreased the DNA synthesis. These results are compatible with a role of PMP22 as negative regulator of Schwann cell growth, which is in agreement with the initial function of pmp22/ gas3, a growth arrest specific gene. Pronuclear injection of YACs from the CMTlA region, of which 8 copies were integrated, resulted in a mouse with a severe neuropathy with demyelination and onion bulb formation (Huxley et al., 1996). The severe neuropathy due to more than three copies of PMP22 is in line with the observation that homozygosity of the CMTl Aassociated duplication in a CMTlA family resulted in a DSS-like phenotype (Lupski et al., 1991). Increasing copies of PMP22 are the cause of more severe phenotypes as was also demonstrated in a mouse with 16 to 30 copies of pmp22 (Magyar et al., 1996). These mice sh0wed.a severe congenital hypomyeliniating neuropathy, with almost a complete lack of myelin. The nerves of the transgenic mice have an increased number of Schwann cells, as observed in a milder form in the Trembler mice, but seem to stay in a ‘premyelination’ state with continued proliferation in adulthood. Although pmp22 mRNA was present, almost no protein could be detected. The PO mRNA and protein was completely absent. Thus, overexpression of PMP22 during development of Schwann cells inhibits further differentiation. A pmp22 rat model with one additional pmp22 gene showed the CMTlA phenotype with gait abnormalities, reduced NCVs, peripheral hypomyelination and onion bulbs (Sereda et al., 1996). The

258

protein analysis showed an additional 44 kDa fragment detected with pmp22 antibody, which might indicate that pmp22 forms dimers. The homozygous animal showed a severe phenotype without myelin. Two models for HNPP have been constructed: pmp22 knock-out mice (Adlkofer et al., 1995), and transgenic mice with antisense pmp22 (Maycox et al., 1997). The heterozygous mice show a clear HNPP phenotype with tomacula surrounding the axons. The homozygous animals were severely affected due to absence of pmp22. The tomacula were present after 24 days in -/- mice and no longer observered after 10 weeks, probably due to degeneration (Adlkofer et al., 1995). The myelin of the +/- mice appeared almost normal after day 24, but tomacula were present at 10 weeks. Thus pmp22 copynumber affects the initial stages of myelination by controlling the myelin thickness and in more advanced stages pmp22 controles the stability. Unstable myelin (tomacula) is most likely degraded. Eventhough all these different animal models show neuropathy, with a severity that correlates with pmp22 copynumber, the function of PMP22 remains unknown. Recent experiments with Trembler, and Trembler-J mice suggest that protein trafficking is altered in pmp22 mutants. In Trembler mice, pmp22 was not only found in myelin, but considerable immunoreactivity was found in the cytoplasm of the Schwann cells as if transport or processing of pmp22 was altered in these mice. Transfection studies of pmp22 in COS cells showed that mutated pmp22 (Trembler-pmp22) in COS cells inhibited transport of transfected wildtype pmp22 to the cell surface, whereas Tremblerpmp22 alone never reached the cell surface (Naef et al., 1997). This suggests a dominant effect of Trembler-pmp22 on transport of pmp22. This dominant effect could be due to the formation of complexes containing different pmp22 molecules and possibly other proteins in the ER. Mutations affecting folding of pmp22 might inhibit transport because they are recognized as improperly folded proteins, inducing a

“misfolded protein response”. This model can explain some of the findings in CMTlA patients and mice with point mutations, but the issue of gene dose is not resolved yet. In order to fit this also in the abnormal protein trafficking model, one has to propose that the complex that is formed in the E R depends on the stochiometric interaction between several components. one of which is pmp22. In view of this hypothesis, it is of interest that over-expression of MDR3 a non-myelin related membrane protein in Schwann cells of transgenic mice also resulted in a peripheral neuropathy (Smit et al., 1996). In this case abberant expression of a membrane protein in Schwann cells might affect protein trafficking. Naef et al. (1997) even suggest that in view of the structural similarities of Cx32 with PMP22 a similar model could apply to Cx32. This protein forms hexameric channels, which are assembled intracellularly. If mutated Cx32 proteins are not incorporated in myeline, the identical phenotypes of mutations and premature stops (i.e. Arg22stop; Ionasescu et al., 1994) are obvious. Alternatively, the response of Schwann cells to under- or over-expression of PMP22 might be different from the response to mutations. In view of the involvement of PMP22 in both proliferation and myelin stability, the influence of different amounts of PMP22 could be different. A high level of PMP22 due to the duplication might result in premature stop of the proliferation and in a premature expression of other myelin genes. The result would be an incomplete myelin sheath structure and onion bulb formation. The reduced expression would result in a n extended proliferation and in excess of myelination and thus the formation of hypermyelinated tomacula as observed in HNPP. The observation of two Schwann cells forming one myelin sheath in HNPP (Madrid and Bradley, 1975) is in line with this theory. The hypomyelinated regions in CMTl A, HNPP and DSS might be the result of the Schwann cells reacting to the improperly formed myelin resulting in demyelination.

259

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