Principles of pathology and nerve biopsy

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Introduction

Pathologic examination of peripheral nerves was the only valuable diagnostic tool for hereditary neuropathies, until molecular genetic diagnosis became available. In fact, after the original description of a duplication of chromosome 17p11.2, containing the gene coding for peripheral myelin protein 22 (PMP22), in Charcot-Marie-Tooth (CMT) neuropathy type 1A [59, 76], several other genes involved in the pathogenesis of the different types of CMT and of other inherited neuropathies have been discovered. The diagnosis of hereditary neuropathies now mostly depends upon the search for mutations in all the known genes. The diagnostic biopsy of a peripheral nerve is, therefore, limited to selected cases of unknown origin, in spite of a wide search for mutations in the most commonly involved genes. Typically, pathologic examination of peripheral nerves is reserved for sporadic cases where an inherited neuropathy is strongly suspected, but acquired neuropathies like chronic inflammatory demyelinating polyneuropathy (CIDP) need to be excluded.

In general terms, a peripheral nerve has to be sampled in carefully selected patients only, because of the potential neurologic deficits following a nerve biopsy. Moreover, the diagnostic value of a nerve biopsy is influ- enced by:

z the small amount of tissue removed;

z the fact that very few nerves in the body are suitable for sampling;

z the availability of a specialized laboratory to process and analyze the tis- z sue;the need of relating histologic measures with physiologic, biochemical

and pharmacological results [26].

The nerve to biopsy has to be accurately chosen. It should be a cutaneous nerve affected by the neuropathic process, easily accessible to neurophysio- logic studies prior to biopsy, constant and superficial in location. The sural nerve meets all these criteria [26]. Alternatively, the radial cutaneous nerve in the arms and the superficial peroneal nerve in the legs may be selected [78]. The sural nerve is a sensory nerve and contains, in the endoneurial space, the axonal processes of neurons located in the dorsal root ganglia,

Principles of pathology and nerve biopsy

A. Schenone

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surrounded by myelinating and non-myelinating Schwann cells, and by fi- broblasts (Fig. 4.1). Present in the epineurium, perineurium and endoneu- rium are also several blood vessels, whose examination may be useful in case of a suspected vasculitis. Since hereditary neuropathies are normally diffuse processes, two or three nerve fascicles (fascicular biopsy) may be sufficient to provide enough tissue to perform a pathological examination.

However, a careful comparison between fascicular and whole sural nerve biopsies showed no differences in residual deficits between the two surgical procedures [73]. Moreover, when hereditary disorders like familial amyloid polyneuropathy (FAP) are suspected, a whole sural nerve biopsy is necessary to detect the amyloidotic deposits.

Postoperative symptoms after sural nerve biopsy are usually mild and include sensory deficit, dysesthesia and pain in the cutaneous distribution of the nerve. Among patients with neuropathy, 93% reported sensory symptoms however, only 33% had mild persistent pain and 19% persistent dysesthesia [34]. Over time, dysesthesia tends to improve and pain to com- pletely subside [34]. The sural nerve is removed under sterile conditions in the operating room by a specialized surgical team. After local anesthesia, the nerve is exposed at the distal calf level, where it lies adjacent to the sa- phenous vein, which is the optimal anatomical landmark for locating the sural nerve. After removal, the nerve is processed for the morphological studies. Normally, the specimen is divided into three segments, each one 2±2.5cm long, which are suspended in glutaraldehyde 2.5%, paraformalde- hyde 4% and frozen in liquid nitrogen, respectively. After fixation, speci- mens are embedded in paraffin or in epoxy resin for examination at the light and electron microscopic levels. One segment of the nerve, fixed in glutaraldehyde and osmium tetroxide, is used for teased fiber preparations Fig. 4.1. Normal sural nerve. Semithin section showing a normal number of myelinated and unmyelinated fibers. The distribution of myelinated fibers is typically bimodal. Toluidine blue;

bar: 15 lm

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(Fig. 4.2). The examination of teased fibers allows the study of a single myelinated fiber over a distance of 3±15internodes and is useful to identify the incidence of fibers undergoing Wallerian degeneration and segmental demyelination or remyelination. Sometimes specific changes, such as the sausage-like thickenings of the myelin sheath, which are typical of the he-

4 Principles of pathologyand nerve biopsy z 43

Fig. 4.2. Normal sural nerve. Teased fiber preparation. Several normallymyelinated internodes and nodes of Ranvier (arrows) maybe seen along a single fiber (A through D). O.T. 2%

Fig. 4.3. Normal human sural nerve. A normal myelinated fiber and several unmyelinated fibers (arrows) are present. Electron microscopy. Lead citrate and uranyl acetate; bar: 0.5 lm

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reditary neuropathy with liability to pressure palsy (HNPP), are easier to detect in teased fibers than in paraffin- or in epoxy-embedded sections.

Electron microscopic examination (Fig. 4.3) is useful when the study of unmyelinated fibers or a search for specific inclusions in Schwann cells, as observed in hereditary storage disorders, are necessary. Immunohistochem- ical techniques may be performed, mainly in acquired neuropathies, to detect and characterize inflammatory infiltrates. On paraffin embedded sections, specific stains, like Congo red or immunolabeling with antitransthy- retin or anti-light chain antibodies are useful to detect endoneurial amyloid deposits and to distinguish between FAP and neuropathy due to a monoclonal gammopathy.

Morphometry of sural nerve biopsies can be used to determine the number, density, diameter distribution and shape of myelinated and unmyelinated fibers. In the past, morphometric studies were helpful in correlating morphological and electrophysiological findings in hereditary motor and sensory neuropathies (HMSN) [42] and to separate CMT1 (also called HMSN 1) from Djerine-Sottas syndrome (DSS, also called HMSN 3) [70].

The ratio of axon diameter to fiber diameter (g-ratio) is a useful measurement to grade hypomyelination and to detect signs of axonal atrophy in sural nerve biopsies. In case of hypomyelination the g-ratio is higher (> 0.75), whereas axonal atrophy is suspected when g-ratios are <0.4.

4.1 Charcot-Marie-Tooth disease type 1 (CMT1)

The neuropathological phenotype of patients affected by CMT1 is characterized by diffuse demyelinating changes. This finding is in agreement with the neurophysiological studies showing reduced motor and sensory nerve conduction velocities (<38 m/s).

4.1.1 Charcot-Marie-Tooth disease type 1A (CMT1A)

Sural nerve biopsies of CMT1A patients, due to the 17p11.2 duplication, show rather stereotyped abnormalities. There is a reduction in myelinated fiber density, ranging from moderate to severe [44]. Unmyelinated fibers may be normal or slightly affected [44]. Compared to normal nerves, there is an increase in transverse fascicular area, which may also be demonstrated by non-invasive ultrasonographic techniques (Fig. 4.4) [64]. Onion bulbs, made up of thinly or near normally myelinated axons surrounded by concentric layers of Schwann cell cytoplasm, are frequently present in sural nerves of CMT1A patients (Fig. 4.5). Up to 85% of myelinated fibers may consist of onion bulbs [44]. Some onion bulbs may contain several myelinated or unmyelinated axons that are believed to be nerve sprouts.

The occasional presence of large axons devoid of a myelin sheath, some-

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times surrounded by onion bulb formation, confirms the demyelinating na- ture of the pathological process. This is even more evident in the teased fiber preparation, where most of the remaining fibers show segmental demyelination and/or remyelination (Fig. 4.6).

Morphometric studies show that larger fibers are more affected than smaller ones. The mean internodal length is lower than in normal nerves, meaning that remyelination occurs in nerve fibers of patients affected by

Fig. 4.4. Ultrasonographyof median nerve in a CMT1A patient. With high frequencytransdu- cers, peripheral nerves show a typical fascicular structure in transverse sections. In CMT1A (A) the nerve and the fascicles (arrows) appear enlarged compared to a normal control (B)

Fig. 4.5. Sural nerve biopsyfrom a CMT1A patient. At lower magnification (A) the densityof the myelinated fibers appears to be decreased from normal. At higher magnification (B) onion bulbs (arrows), made up of concentric proliferation of Schwann cell cytoplasm around normally or thinlymyelinated fibers, can be seen. Semithin section, toluidine blue; bars: 10 lm

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CMT1A. The observation of myelinated fibers with a reduced axon diameter as compared to myelin thickness suggests the presence of an associated axonal impairment [28]. The relevance of axonal atrophy in CMT1A nerves has been confirmed in xenografts of sural nerves from CMT1A patients into nude mice sciatic nerves [77].

CMT1A phenotypes caused by mutations in the PMP22 gene are rare. In these cases the neuropathological picture may be more severe than in typical duplication cases, but specific changes in sural nerve biopsies have not been detected.

Sometimes, inflammatory changes may be observed in sural nerve biopsies of CMT1A patients [38, 99]. This observation suggests the possibility of a genetic susceptibility to immune mediated demyelination in certain CMT families and explains previous reports on steroid-responsive inherited neuropathies [28].

4.1.2 Charcot-Marie-Tooth disease type 1B (CMT1B)

CMT1B, caused by point mutations in the MPZ gene, has a more severe clinical and neuropathological phenotype than CMT1A. In typical cases, the sural nerve biopsy shows a variable loss of myelinated fibers, onion bulbs made up of concentric layers of Schwann cell cytoplasm surrounding thinly myelinated fibers and segmental demyelination in teased fibers [44].

Focal thickenings of the myelin sheath have been observed in CMT1B patients (Fig. 4.7) [31, 36]. Although not specific, focally folded myelin seems to be particularly frequent in a subgroup of patients harboring mutations in the extracellular domain of MPZ [31]. Focally folded myelin is also a hallmark of some very rare forms of CMT4. In other families, ultrastruc- Fig. 4.6. Sural nerve biopsyfrom a CMT1A patient. Teased fiber preparation. Several internodes lacking of myelin (demyelinated internodes) can be seen along a single fiber (A through F).

O.T. 2%

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tural examination shows uncompacted myelin in several fibers (Fig. 4.8), thus, suggesting two divergent neuropathological phenotypes in CMT1B, the first dominated by myelin thickenings and the second by loosening of myelin lamellae [36].

Morphometric studies show, like in CMT1A, a preferential loss of larger fibers. Total transverse fascicular area is not as enlarged as in typical CMT1A.

Most of the remaining fibers, outside of the myelin thickenings, have high g- ratios, thus, confirming the severity of the demyelinating process [36].

Fig. 4.7. Sural nerve biopsyfrom a CMT1B patient. The densityof myelinated fibers is from moderatelyto severelydecreased from normal. Some fibers show focal thickenings of the myelin sheath (arrow). Toluidine blue; bar: 10 lm

Fig. 4.8. Sural nerve biopsyfrom a CMT1B patient. Electron microscopyshows, in some thinly myelinated fibers, uncompaction of myelin lamellae (arrows). Bar: 0.1 lm

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4.1.3 Charcot-Marie-Tooth disease type 1C (CMT1C)

CMT1C, due to mutations in the LITAF/SIMPLE gene, shares clinical and electrophysiological features with typical CMT1 [91]. The neuropathological phenotype is also suggestive of a hypertrophic demyelinating neuropathy. In particular, myelin loss and onion bulbs similar to those observed in CMT1A have been reported in a family with CMT1C [92].

4.1.4 Charcot-Marie-Tooth disease type 1D (CMT1D)

Mutations in the EGR2 zinc-finger transcription factor have been demonstrated in a few cases of CMT1, DSS and congenital hypomyelination (CH) phenotypes [100]. Patients with CMT1D show a typical CMT1 phenotype [81, 96] and display neuropathological changes ranging from severe to relatively mild fibers loss and demyelination. Onion bulbs, although present, are not as prominent as in CMT1A (Fig. 4.9). Fibers showing a reduced axon diameter compared to myelin thickness have also been observed, in a CMT1D family, and may suggest an associated axonal atrophy [96].

4.1.5 Djerine-Sottas syndrome (DSS)

DSS may be considered, from the neuropathological point of view, as a severe variant of demyelinating CMT. Originally, the distinction between DSS (HMSN III) and CMT1 (HMSN I) was based on the presence of a significantly lower density of myelinated fibers > 8 lm in diameter, a greater frequency of

Fig. 4.9. Sural nerve biopsyfrom a CMT1D patient. The densityof myelinated fibers is moder- atelydecreased from normal. Several onion bulbs (arrows) maybe seen, sometimes surrounding thinlymyelinated fibers. Semithin section, toluidine blue; bar: 10 lm

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onion bulbs and higher g-ratios in DSS [70]. Molecular genetic studies made this distinction less important. In fact, mutations in the PMP22, MPZ, EGR2 and PRX genes may cause either a CMT1 or a DSS phenotype [11, 12, 45, 71, 96]. Sural nerve biopsies have been performed in most of these cases, but genotype-phenotype correlations based on neuropathological features are not possible, because specific changes for each genotype have not been observed. Patients with DSS show a diffuse loss of myelinated fibers (Fig. 4.10). Unmyelinated fiber density is also, but less severely, decreased.

Onion bulbs are made up of multiple layers of Schwann cell cytoplasm with or without a central axon (denervated onion bulbs). Several larger axons devoid of myelin may also be observed. Hypertrophy of peripheral nerves is a common feature and enlargement of nerve roots has been found in a family with a MPZ point mutation [89]. Focally folded myelin, especially at para- nodes, has been observed in a patient with mutations in the PRX gene [94].

4.1.6 Congenital hypomyelination (CH)

CH is characterized by a defect in myelination, probably due to a primary failure in myelin formation. It may be clinically undistinguishable from severe, early onset CMT1 and DSS. Sural nerve biopsy (Fig. 4.11), although not diagnostic, shows a severe loss of myelinated fibers with all the residual axons lacking a myelin sheath or surrounded by very thin myelin and onion bulbs made up of multiple laminae of double layered Schwann cell basement membranes (basal lamina onion bulbs). However, most authors considered CH as a more severe variant of DSS. Molecular genetic studies confirmed that mutations in the same genes known to cause DSS or severe

Fig. 4.10. Sural nerve biopsyfrom a DSS patient. The densityof myelinated fibers is severely decreased from normal. Onion bulbs formed bySchwann cell processes wrapping around thinly myelinated axons may be seen (arrow). Semithin section, toluidine blue; bar: 5 lm

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CMT1, like PMP22, MPZ and EGR2, can be found in CH families [32, 62, 100]. Neuropathological genotype-phenotype correlations are virtually im- possible in all these cases.

4.1.7 Hereditary neuropathy with liability to pressure palsy (HNPP) HNPP is clinically characterized by recurrent episodes of peripheral nerve palsies, due to mechanical compression of the nerve trunks. Various pathologic changes have been described in association with HNPP. However, the most common abnormality, in sural nerve biopsies, is constituted by focal thickenings of the myelin sheath, named tomacula by the sausage-like ap- pearance they give to the myelinated fiber in longitudinal sections or in teased fiber preparations (Fig. 4.12). Typical tomacula have been described as focal enlargements of the myelin sheath, in the internodal or paranodal segment of the fiber, which are between 40 and 250 lm long (Fig. 4.13) [101]. In transverse sections, tomacula are characterized by an extremely thickened myelin sheath wrapping around an axon of reduced diameter. It is not known whether this is due to a constriction of the axon by the thickened myelin or to an ongoing axonal atrophy. However, the observations that neurofilament density is increased in the narrowed area of the Fig. 4.11. Sural nerve biopsyfrom a patient with a CH phenotype and a mutation in the P0 gene. The densityof myelinated fibers is severelydecreased. Several axons are either demyelinated (thick arrows) or surrounded byan extremelythin myelin sheath (thin arrows). Toluidine blue; bar: 10 lm

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axon [101], lower g-ratios correlate to the levels of PMP22 mRNA in HNPP patients [82] and that xenografts of HNPP nerves in mice sciatic nerves result in axonal atrophy [77] suggest that axonal impairment does occur in HNPP. Rarely, tomacula may be absent in transverse semithin sections of HNPP sural nerves. Therefore, the analysis of longitudinal sections and teased fiber preparations is needed to identify these peculiar myelin abnormalities. Sometimes, focal thickenings of the myelin sheath have been observed in other hereditary neuropathies, particularly those due to P0 mutations [31]. However, in the author's experience, typical tomacula, as de- fined above, may be observed only in HNPP sural nerves.

The density of myelinated fibers may be normal or slightly reduced from normal. Onion bulbs are usually absent or observed occasionally. Rarely, HNPP sural nerves show a severe reduction of myelinated fibers and a large number of onion bulbs. These patients, although carrying the 17p11.2 deletion, develop a clinical and neurophysiological phenotype undistinguishable from CMT1 [61].

The teased fiber preparation shows a variable degree of segmental demyelination and remyelination. This corresponds to the electrophysiologic observation of slowing of conduction velocity in clinically unaffected

Fig. 4.12. Sural nerve biopsyfrom a HNPP patient. The densityof myelinated fibers is within normal limits. Several focal thickenings of the myelin sheath (tomacula) (arrows) may be seen.

Toluidine blue; bar: 10 lm

Fig. 4.13. Sural nerve biopsyfrom an HNPP patient. Teased fiber preparation. Several thickenings of the myelin sheath (tomacula) may be seen along the fibers. Sometimes tomacula are up to 250 lm long (arrows). O.T. 2%

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nerves [101]. At the electron microscopic level, tomacula look like redundant loops of myelin with irregularly folded lamellae, which are also enor- mously increased in number.

Madrid and Bradley identified several mechanisms by which focal thickenings are formed, several years before the discovery of the genetic abnormality underlying HNPP [60]. These include branching and duplication of the mesaxon (the Schwann cell membrane adjacent to the axon), trans- nodal myelination, the participation of two or more Schwann cells in the formation of the myelin sheath, and the degeneration of myelin in the adaxonal or intramyelin regions between an intact outer layer of the myelin sheath and the axon. The knowledge that a reduced dosage of the PMP22 gene is present in HNPP patients [83, 97] is not sufficient to understand the mechanisms of tomacula formation and the relationship between these structural changes and mechanic compression. However, it is possible that low levels of PMP22 affect the structural integrity of the myelin sheath and make it more susceptible to damage from external trauma [88].

4.2 Charcot-Marie-Tooth disease type 4(CMT4)

The autosomal recessive forms of CMT1 are traditionally called CMT4. The different forms of CMT4 are extremely rare. They are neuropathies characterized by a neuropathological phenotype, which in some cases, like CMT4B1, is characterized by extremely peculiar changes.

4.2.1 Charcot-Marie-Tooth disease type 4A (CMT4A)

CMT4A is caused by mutations in the ganglioside-induced differentiation- associated protein-1 gene (GDAP1). Neuropathological findings in CMT4A range from an axonal to a demyelinating phenotype [7, 20]. Irrespective of the prevalence of axonal or demyelinating changes, a severe loss of myelinated fibers, especially affecting the larger fibers has been found in all sural nerve biopsies of CMT4A patients. Onion bulbs have been described, but in most cases they are scattered and atypical, since they enclose regenerating axons and show only few concentric layers of Schwann cell cytoplasm [10, 87]. Demyelinated axons and abundant onion bulbs have been observed in only a few affected individuals [7]. In most families, neither demyelinated axons nor abnormalities in myelin compaction could be found [10, 67, 87]. However, some cases do show signs of de-remyelination along with axonal changes [67]. Morphometric studies confirm the morphological observations and show that larger fibers (>8 lm) are always lacking, whereas there are more smaller fibers (<3 lm) [67, 87]. These observations may suggest that axonal regeneration is a prominent feature of CMT4A.

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The g-ratio ranges from normal (0.7) to low values (<0.4), suggesting that an axonal atrophy is present in some fibers [10, 87].

In agreement with these observations, we found in sural nerves of patients carrying a M116R mutation at exon 4 of the GDAP1 gene [E. Di Maria, per- sonal communication] a loss of myelinated axons, especially affecting larger fibers. Several of the remaining fibers showed a reduction in myelin thickness. Sometimes, thinly myelinated axons surrounded by Schwann cell processes concentrically organized to form small onion bulbs are also observed (Fig. 4.14). Occasionally, clusters of small regenerating fibers may be found.

An association of axonal and demyelinating features has also been recently reported in other CMT4A families [85]. Therefore, although the number of mutations in the GDAP1 gene described so far is too low for reliable genotype-phenotype correlations, it is possible that CMT4A, similarly to CMTX, combines axonal and demyelinating changes. This may be due to a combined, negative effect of GDAP1 mutations on neurons and Schwann cells [93]. How- ever, further studies on the function of this protein are needed to explain these mixed changes.

4.2.2 Charcot-Marie-Tooth disease type 4B1 and 4B2 (CMT4B1, CMT4B2)

Mutations in different members of the myotubularin-related gene family cause CMT4B [5, 13]. In particular, CMT4B1 is due to mutations in the myotubularin related protein-2 (MTMR2) [13] and CMT4B2 to mutations in the MTMR13 gene [5]. However, sural nerve biopsy findings do not dif-

Fig. 4.14. Sural nerve biopsyfrom a CMT4A patient. The densityof myelinated fibers is decreased from normal. Several thinlymyelinated fibers maybe seen, occasionallysurrounded by concentric layers of the Schwann cell cytoplasm forming a small onion bulb (arrow). Semithin section, toluidine blue; bar: 10 lm

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Fig. 4.15. Sural nerve biopsyfrom a CMT4B patient. The densityof myelinated fibers is severelydecreased from normal. The majorityof fibers show the typical abnormalities of the myelin sheath, which is redundant and irregularly folded (arrows). Semithin section, toluidine blue; bar: 10 lm

Fig. 4.16. Sural nerve biopsyfrom a CMT4B patient. Electron microscopyshows complex outfoldings of the myelin sheath surrounding the axon (asterisk). Bar: 1 lm

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fer between the two types of CMT4B and are characterized by typical abnormalities of the myelin sheath, which is redundant and irregularly folded (Figs. 4.15, 4.16). These changes are called myelin outfoldings. Myelin outfoldings were first related to autosomal recessive hereditary motor and sensory neuropathies and described in detail by Onishi et al. in 1989 [69]. In CMT4B up to 90% of the fibers presents myelin outfoldings, discriminating this disorder from all the other autosomal dominant or recessive forms of CMT [75, 80]. A profound loss of myelinated fibers is also observed. Occa- sionally, small onion bulbs may be seen [80].

4.2.3 Charcot-Marie-Tooth disease type 4C (CMT4C)

The neuropathological features of CMT4C, which has been recently related to mutations in a gene encoding a SH3/TPR domain protein [86], have been known since 1999, when the phenotypic characterization of an autosomal recessive demyelinating CMT linked to chromosome 5q23-q33 was described [35]. The sural nerve biopsy shows a predominantly demyelinating neuropathy with a severe loss of large myelinated fibers (>8 lm), ab- normally thin myelin sheaths in the remaining fibers and extensive onion bulbs formation [53]. Onion bulbs made up of concentric, basal membrane layers surrounding a demyelinated axon are frequently seen (basal lamina onion bulbs).

4.2.4 Charcot-Marie-Tooth disease type 4D (CMT4D)

Sural nerve biopsy findings, in CMT4D, also known as hereditary motor and sensory neuropathy of the Lom type (HMSN-L), are suggestive of a severe demyelinating neuropathic process [52]. In fact, a profound loss of myelinated fibers and prominent hypertrophic changes, characterized by frequent onion bulbs consisting of multiple layers of Schwann cell cytoplasm and basal lamina around thinly myelinated or unmyelinated axons, have been described [17, 52, 54]. Interestingly, onion bulbs are more evident in younger patients and seem to subside with aging [52]. Why there is a regression of the onion bulbs in CMT4D is unknown; however, it may be related to the axonal loss which is striking in this hereditary neuropathy [54]. With electron microscopy, various abnormalities both in the axons and in the Schwann cells of CMT4D patients have been shown, like axonal inclusions consisting of tubular or small curvilinear profiles, pleomorphic material in the adaxonal Schwann cell cytoplasm and uncompaction of the myelin lamellae adjacent to the axon [54]. However, none of these ultrastructural abnormalities seem to be specific of CMT4D.

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4.2.5 Charcot-Marie-Tooth disease type 4E (CMT4E)

Point mutations in the EGR2 gene, besides causing autosomal dominant CMT1, DSS and CH phenotypes, may also be responsible for severe demyelinating autosomal recessive CMT, now classified as CMT4E [55]. However, the neuropathological phenotype does not allow clear differentiation of CMT4E from autosomal dominant CH. In fact, the sural nerve biopsy shows a diffuse loss of myelinated fibers with absence of myelin in virtually all the remaining fibers [100].

4.2.6 Charcot-Marie-Tooth disease type 4F (CMT4F)

CMT4F is due to mutations in the PRX gene [94]. PRX encodes two PDZ domain proteins, L- and S-periaxin, which are required for the maintenance of peripheral nerve myelin. To date, most of the mutations responsible for an autosomal recessive demyelinating neuropathy affect L-periaxin and only one family shows a homozygous mutation in the region common to both forms and therefore affecting both L- and S-periaxin [94]. However, the neuropathological findings do not allow a distinction between mutations of the L- or S-periaxin.

The sural nerve biopsies in patients carrying mutations in the PRX gene, even if the clinical phenotype may vary from a severe demyelinating sensory motor neuropathy to a milder, mainly sensory phenotype, show similar features. A severe loss of myelinated fibers accompanied by prominent onion bulb formation is always present [12, 23, 40, 94]. Onion bulbs are made up of Schwann cell cytoplasm and basal lamina. Hypermyelination and tomacula are also frequently observed in CMT4F [40, 94]. The paranodal region shows incomplete myelination and separation of multiple terminal myelin loops from the axon, suggesting that PRX may play an important role in mediating Schwann cell- axon adhesion at the node of Ranvier [94]. Finally, it is important to underscore that the PRX null mice develop a neuropathic process which is strikingly similar to the human pathology, supporting the view that this animal model may lead to a better understanding of the human disease [37].

4.3 X-linked Charcot-Marie-Tooth disease (CMTX)

CMTX is in nearly all cases due to mutations in the GJB1 gene, coding for Cx32, a protein belonging to the family of gap junction proteins and highly expressed in the Schwann cells at the nodes of Ranvier and Schmidt-Lanter- man incisures. Cx32 mediates the transport of low molecular weight sub- stances from the adaxonal to the outer myelin lamellae [6]. This type of CMT is also known as CMTX1 [55]. In fact, neurophysiological studies reveal a slowing of conduction velocities intermediate between a demyelinating and an axonal CMT. However, given the peculiar localization and function of

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Cx32, the relationship between Schwann cells and axon should be particularly affected in CMTX1 patients and the compact myelin relatively preserved [88].

Sural nerve biopsies show that axonal features are predominant in CMTX1 patients [44]; however, concomitant demyelination has been described [41, 79, 84]. Myelinated fiber density is reduced, unmyelinated fibers are relatively well preserved and the density of small myelinated fibers is less affected than that of larger ones, due to the frequency of axonal sprouting (Fig. 4.17) [84]. Onion bulbs, sometimes surrounding normally myelinated fibers, may be seen. Teased fiber preparations show an increased rate of remyelinating fibers, mainly secondary to axonal damage.

Rarely, fibers undergoing segmental demyelination have been described [44, 79]. Due to the pattern of inheritance, males are normally more affected than females and this is also evident in sural nerve biopsies [41].

According to the distribution of Cx32, electron microscopy analysis reveals unusual findings at the nodal and paranodal regions. Widening of the Schmidt-Lanterman incisures and of the nodes of Ranvier have been described along with a separation of the myelin sheath from the axon leaving a clear periaxonal space that appeared either empty or contained vesicular material and whose significance is still unknown [41].

An extremely rare form of CMTX is linked to chromosome Xq24-q26.1 (CMTX2) [74]. In the original report this type of CMTX was described as an axonal HMSN II with additional clinical features (deafness and mental retardation) [18]. The sural nerve biopsy shows typical axonal changes, like a slight reduction of myelinated fibers density with axonal loss and sprouting, but no onion bulb formation or segmental demyelination.

Fig. 4.17. Sural nerve biopsyfrom a CMTX1 patient. The densityof myelinated fibers is moder- atelydecreased from normal. Several clusters of thinlymyelinated fibers (arrows) maybe seen.

Fibers with thin myelin sheath occasionally surrounded by concentric layers of Schwann cell cytoplasm to form small onion bulbs (arrow heads) are occasionallypresent. Semithin section, toluidine blue; bar: 10 lm

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4.4 Charcot-Marie-Tooth disease type 2 (CMT2)

CMT2 is the axonal form of CMT. Classically, conduction velocity studies allow a distinction between CMT2 and CMT1 [42]. However, in the past, neuropathological studies have also been performed to distinguish axonal from demyelinating CMT [28]. Typical CMT2 is autosomal dominant and in most cases the genetic defect is unknown. Point mutations in a few genes have been now identified to cause CMT2, e.g. GTPase mitofusin 2 and the kinesin motor protein-1-b (KIFBb) (CMT2A), the neurofilament light chain (NEFL) (CMT2E), the RAS-related GTP binding protein 7 (RAB7) (CMT2B), the glycyl-tRNA synthetase (GARS) (CMT2D), the MPZ, the heat shock protein 27 (HSP27) genes [3, 4, 30, 63, 66, 98, 102, 103].

Since the neuropathological phenotype of CMT2 patients is similar, irrespective of the different genotype, the sural nerve biopsy changes will be described as a whole. The number of myelinated fibers is slightly decreased, the larger fibers being more affected than the smaller ones (Fig. 4.18). The reduced density of myelinated fibers is more severe in distal than in proximal nerves. No abnormalities in the distribution of unmyelinated fibers have been described [28]. Occasionally, clusters of small regenerating fibers or small onion bulbs may be seen. Sometimes, irregular foldings of the myelin sheath have been described, particularly in biopsies of patients with MPZ and NEFL mutations [22, 50]. Teased fiber preparations frequently show fibers with short internodes, but in the absence of clear segmental demyelination. An autopsy report demonstrated a distal to proximal gradient of atrophy and fiber loss in the peripheral nerves, depletion of anterior horn and dorsal root gan- glion cells maximal in the lumbosacral region and secondary axonal loss in

Fig. 4.18. Sural nerve biopsyfrom a CMT2 patient. The densityof myelinated fibers is slightly decreased from normal, particularlyfor large fibers. Several fibers show small axonal caliber compared to myelin thickness (arrows). Occasionally small onion bulbs (arrow heads) and clusters of regenerating fibers maybe seen. Semithin section, toluidine blue; bar: 10 lm

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the gracile fasciculi [9]. All these findings have been interpreted as the result of neuronal atrophy and degeneration of peripheral motor and sensory neurons [28]. The genetic studies demonstrating mutations in genes related to the axonal cytoskeleton (NEFL) and transport (KIF1Bb) in some CMT2 families fully confirmed these observations made only on a neuropathological basis.

Although specific genotype-phenotype correlations are not possible based on sural nerve biopsy findings alone, a few peculiar changes have been found in different types of CMT2. For example, in patients carrying mutations in the NEFL gene, sural nerve biopsies reveal a relatively high frequency of thin onion bulbs, which could be suggestive of a demyelinating process secondary to the axonal impairment [50]. The recent observation of giant axons in the sural nerve biopsy of a family with a Pro22Ser mutation of NEFL, strengthen the concept of CMT2E as a primary axonopathy with secondary demyelination [33]. As giant axons have been previously observed, although rarely, in other hereditary motor and sensory neuropathies [9, 104], it is still unclear whether they are a specific neuropathological marker of CMT2E or not [33].

Autosomal recessive forms of CMT2 (AR-CMT2) have been also described. As expected, the neuropathological phenotype seems to be more severe in AR-CMT2 than in the autosomal dominant types. In AR-CMT2 due to mutations in the LaMiN A/C (LMNA) gene, a profound loss of myelinated and unmyelinated fibers was observed in the absence of onion bulbs or regenerating clusters [16]. However, in AR-CMT genotype-phenotype correlations based on sural nerve biopsy findings are also very diffi- cult. In fact, similar to AR-CMT2 due to mutations in the LMNA gene, patients carrying GDAP-1 mutations may show a severe axonal phenotype with myelin loss and no signs of demyelination and remyelination [20].

4.5 Hereditary sensory and autonomic neuropathies (HSAN)

HSAN are clinically characterized by predominant peripheral sensory and autonomic disturbances. The neuropathological findings, represented by a prevailing degeneration and loss of fibers in the sensory nerves and in the posterior columns, posterior roots and spinal ganglia, mirror the clinical picture [27]. As for CMT disease and related neuropathies, different varieties of HSAN have been identified based on mode of inheritance, natural history, neurophysiological and neuropathological abnormalities [27]. Only recently have mutations in known genes been related to different variants of HSAN [55].

4.5.1 Hereditary sensory and autonomic neuropathy type 1 (HSAN1) In patients affected by HSAN1, mutations in the serine palmitoyltransferase-1 (SPTLC1) gene have been recently found [8, 21]. Classically, the neuropathological studies have focused on the morphological abnormalities

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and the class of fibers affected in the sural nerve. However, a degeneration of nerve fibers and a loss of neuronal cells were also observed in posterior columns and dorsal root ganglia [8, 27]. Unfortunately, due to the relatively low number of HSAN1 patients with SPTLC1 mutations and the overall rar- ity of the disease, studies addressing phenotype-genotype correlations in this hereditary neuropathy are not available.

In typical HSAN1 patients, the sural nerve biopsy shows a loss of myelinated and unmyelinated fibers. Dyck and coauthors found a lower fiber density at the ankle than at the midcalf level and a predominant axonal degeneration in the teased fiber preparation [25]. Unmyelinated fibers, however, were not markedly decreased at the ankle level. These results may suggest that small fibers are slightly more affected that larger ones, and axonal atrophy and degeneration occur in HSAN1 [27]. Whether this is due to a neuronopathy affecting sensory neurons or to a distal axonopathy in sensory fibers is still unclear. The function of SPTLC1 as an enzyme of sphingolipid synthesis and the elevated concentrations of glucosyl-cera- mide, a molecule inducing cells apoptosis [46], within the lymphoblasts of HSAN1 patients may suggest pathogenetic mechanisms involving neuronal death in sensory ganglia [51, 55].

4.5.2 Hereditary sensory and autonomic neuropathy type 2 (HSAN2) HSAN2 is characterized by autosomal recessive inheritance and is caused by mutations in a novel gene of unknown function [56]. The sural nerve biopsy shows an almost complete absence of myelinated fibers with a relative preservation of unmyelinated fibers [27] (Fig. 4.19). Nerve fascicles

Fig. 4.19. Sural nerve biopsyfrom an HSAN type 2 patient. There is a severe loss of myelinated fibers with relative preservation of unmyelinated fibers. Semithin section, toluidine blue;

bar: 1 lm

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also show a decrease in transverse fascicular area. Ohta and coauthors found a gradient of fiber loss from midcalf to the ankle level and, in the deep peroneal nerve, a low percentage of fibers undergoing axonal degeneration along with segmental demyelination and remyelination, suggesting a degenerative process beginning in utero or in infancy [24, 27, 68].

4.5.3 Hereditary sensory and autonomic neuropathy type 3 (HSAN3) HSAN3, also known as familial dysautonomia or Riley-Day syndrome, is an autosomal recessive disease, affecting Ashkenazi Jews, recently related to mutations in the inhibitor of kappa light polypeptide (IKBKAP) gene [2, 27, 90]. Interestingly, genetic tests allowed the identification of an IKBKAP mutation in a patient with typical HSAN3, but no Ashkenazi Jewish ances- try [57].

In typical HSAN3 patients, examination of the sural nerve shows an ap- proximately normal density of myelinated fibers and a striking loss of unmyelinated fibers, no active axonal degeneration and a decreased number of Schwann cell cytoplasmic clusters compared to other neuropathies [1].

A marked reduction of autonomic and spinal ganglia volume, and a decreased density of sympathetic and sensory neurons were also observed in HSAN3 [27]. These findings may suggest an arrest of embryogenesis in the neural crest [27]. However, although the function of the protein coded by the IKBKAP gene in the transcription control is known, the pathomechani- sms of HSAN3 remain to be elucidated [90].

4.5.4 Hereditary sensory and autonomic neuropathy type 4and type 5 (HSAN4and HSAN5)

These two rare and clinically similar disorders of the sensory system have been recently related to mutations in genes related to neurotrophic factors, namely the neurotrophin receptor tyrosin kinase-1 (NTRK1) gene [48, 49], which encodes for the nerve growth factor (NGF) high affinity receptor TRK1 and the NGF beta (NGFB) gene [29], which encodes for NGFB itself.

The neuropathological picture, however, is somehow different in these two diseases. In fact, the sural nerve biopsy, in the previously described cases of HSAN4, showed a nearly normal density of myelinated fibers and a virtual absence of unmyelinated fibers [39, 72]. Instead, in HSAN5, a se- lective decrease of the small myelinated and of the unmyelinated fibers was found in the sural nerve biopsy [58]. However, too few patients have been described with these disorders and NTRK1 or NGFB mutations to correctly describe genotype-phenotype correlations from the neuropathological point of view. Finding mutations in genes involved in the NGF pathway may explain why these patients have a loss of small myelinated and unmyelinated fibers, which are sustained by NGF in their embryonic development.

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4.6 Hereditary motor neuropathies (HMN)

Distal HMN are genetically heterogeneous disorders, which normally present as a classical peroneal muscular atrophy without sensory symptoms [43]. Therefore, the neurophysiology and neuropathology of sensory nerves is, by definition, normal. In fact, although rarely performed, the peripheral nerve biopsy does not show abnormalities in HMN patients [19, 65, 95].

The muscle biopsy, which has been more frequently carried out in HMN patients in the past, shows mainly denervation atrophy. Autopsy studies showed atrophy of spinal roots, loss of anterior horn cells and chromatoli- sis of the motor neurons [15].

4.7 Giant axonal neuropathy (GAN)

GAN is a rare autosomal recessive disorder commonly characterized by a progressive motor and sensory neuropathy with early onset of an associated involvement of the central nervous system [28]. Mutations in the gigaxonin gene, located on chromosome 16q24.1 have been recently found in GAN patients [14]. The neuropathological hallmark of the disease is the presence, in the sural nerve biopsy, of giant axonal swellings due to a massive accumulation of neurofilaments in axons (Fig. 4.20). Similar changes have been also described in the central nervous system [28]. As suggested by the neuropathological features, a generalized impairment of intermediate filaments or- ganization and transport may be responsible for the clinical phenotype.

Fig. 4.20. Sural nerve biopsyfrom a giant axonal neuropathypatient, carrying a gigaxonin mutation. Several axons are severelyenlarged (giant axons) and show an abnormallythin myelin sheath (asterisks). Semithin section, toluidine blue; bar: 10 lm

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This hypothesis is also supported by the observation that giant axons may be found in toxic neuropathies induced by iminodipropionitrile and 2,5-hex- anedione, which are known to affect the slow axonal transport of neurofilaments [47]. Moreover, as reported in the CMT2 section, giant axons have been also found in patients with CMT2E neuropathy, carrying a mutation in the NEFL gene [33]. However, as the number of giant axons is higher in the sural nerve of GAN patients than in any other known hereditary neuropathy, these neuropathological features may be still considered diagnostic for GAN. Nevertheless, since a genetic test is now available for the disease, per- forming a diagnostic sural nerve biopsy should be considered with caution.

4.8 Hereditary neuralgic amyotrophy (HNA)

HNA is a rare, autosomal-dominant, recurrent focal neuropathy whose genetic basis has yet to be identified [55, 101]. Descriptions of the pathologic changes in this condition is very limited. A motor fascicle from an affected nerve was examined in the past, but specific neuropathological changes were not found [101]. A sural nerve biopsy is useless because in most cases there is no evidence of a generalized neuropathy. In the past, sausage- shaped thickenings of the myelin sheath (tomacula) were occasionally reported in the sural nerve of patients suffering of a transient brachial plexo- pathy, but because the genetic test was not yet available, these were probably cases of HNPP than HNA, which are now known to be genetically different diseases [55].

References

1. Aguayo AJ, Nair CP, Bray GM (1971) Peripheral nerve abnormalities in the Riley- Day syndrome. Findings in a sural nerve biopsy. Arch Neurol 24:106±116

2. Anderson SL, Coli R, Daly IW, Kichula EA, Rork MJ, Volpi SA, Ekstein J, Rubin BY (2001) Familial dysautonomia is caused by mutations of the IKAP gene. Am J Hum Genet 68:753±758

3. Antonellis A, Ellsworth RE, Sambuughin N, Puls I, Abel A, Lee-Lin SQ, Jordanova A, Kremensky I, Christodoulou K, Middleton LT, Sivakumar K, Ionasescu V, Funa- lot B, Vance JM, Goldfarb LG, Fischbeck KH, Green ED (2003) Glycyl tRNA synthetase mutations in Charcot-Marie-Tooth disease type 2D and distal spinal muscular atrophy type V. Am J Hum Genet 72:1293±1299

4. Auer-Grumbach M, Strasser-Fuchs S, Robl T, Windpassinger C, Wagner K (2003) Late onset Charcot-Marie-Tooth 2 syndrome caused by two novel mutations in the MPZ gene. Neurology 61:1435±1437

5. Azzedine H, Bolino A, Taieb T, Birouk N, Di Duca M, Bouhouche A, Benamou S, Mrabet A, Hammadouche T, Chkili T, Gouider R, Ravazzolo R, Brice A, Laporte J, LeGuern E (2003) Mutations in MTMR13, a new pseudophosphatase homologue

(24)

of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma. Am J Hum Genet 72:1141±1153

6. Balice-Gordon RJ, Bone LJ, Scherer SS (1998) Functional gap junctions in the Schwann cell myelin sheath. J Cell Biol 142:1095±1104

7. Baxter RV, Ben Othmane K, Rochelle JM, Stajich JE, Hulette C, Dew-Knight S, Hentati F, Ben Hamida M, Bel S, Stenger JE, Gilbert JR, Pericak-Vance MA, Vance JM (2002) Ganglioside-induced differentiation-associated protein-1 is mutant in Charcot-Marie-Tooth disease type 4A/8q21. Nat Genet 30:21±22

8. Bejaoui K, Wu C, Scheffler MD, Haan G, Ashby P, Wu L, de Jong P, Brown RH Jr (2001) SPTLC1 is mutated in hereditary sensory neuropathy, type 1. Nat Genet 27:261±262

9. Berciano J, Combarros O, Figols J, Calleja J, Cabello A, Silos I, Coria F (1986) He- reditary motor and sensory neuropathy type II. Clinicopathological study of a family. Brain 109:897±914

10. Birouk N, Azzedine H, Dubourg O, Muriel MP, Benomar A, Hamadouche T, Mai- sonobe T, Ouazzani R, Brice A, Yahyaoui M, Chkili T, Le Guern E (2003) Phenotypical features of a Moroccan family with autosomal recessive Charcot- Marie-Tooth disease associated with the S194X mutation in the GDAP1 gene. Arch Neurol 60:598±604

11. Boerkoel CF, Takashima H, Bacino CA, Daentl D, Lupski JR (2001) EGR2 mutation R359W causes a spectrum of Dejerine-Sottas neuropathy. Neurogenetics 3:153±

12. Boerkoel CF, Takashima H, Stankiewicz P, Garcia CA, Leber SM, Rhee-Morris L,157 Lupski JR (2001) Periaxin mutations cause recessive Dejerine-Sottas neuropathy.

Am J Hum Genet 68:325±333

13. Bolino A, Muglia M, Conforti FL, LeGuern E, Salih MA, Georgiou DM, Christo- doulou K, Hausmanowa-Petrusewicz I, Mandich P, Schenone A, Gambardella A, Bono F, Quattrone A, Devoto M, Monaco AP (2000) Charcot-Marie-Tooth type 4B is caused by mutations in the gene encoding myotubularin-related protein-2. Nat Genet 25:17±19

14. Bomont P, Cavalier L, Blondeau F, Ben Hamida C, Belal S, Tazir M, Demir E, To- paloglu H, Korinthenberg R, Tuysuz B, Landrieu P, Hentati F, Koenig M (2000) The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy. Nat Genet 26:370±374

15. Chance PF, Rabin BA, Ryan SG, Ding Y, Scavina M, Crain B, Griffin JW, Cornblath DR (1998) Linkage of the gene for an autosomal dominant form of juvenile amyo- trophic lateral sclerosis to chromosome 9q34. Am J Hum Genet 62:633±640 16. Chaouch M, Allal Y, De Sandre-Giovannoli A, Vallat JM, Amer-el-Khedoud A, Kas-

souri N, Chaouch A, Sindou P, Hammadouche T, Tazir M, Levy N, Grid D (2003) The phenotypic manifestations of autosomal recessive axonal Charcot-Marie-Tooth due to a mutation in Lamin A/C gene. Neuromuscul Disord 13:60±67

17. Colomer J, Iturriaga C, Kalaydjieva L, Angelicheva D, King RH, Thomas PK (2000) Hereditary motor and sensory neuropathy-Lom (HMSNL) in a Spanish family: clinical, electrophysiological, pathological and genetic studies. Neuromus- cul Disord 10:578±583

18. Cowchock FS, Duckett SW, Streletz LJ, Graziani LJ, Jackson LG (1985) X-linked motor-sensory neuropathy type-II with deafness and mental retardation: a new disorder. Am J Med Genet 20:307±315

19. Christodoulou K, Zamba E, Tsingis M, Mubaidin A, Horani K, Abu-Sheik S, El- Khateeb M, Kyriacou K, Kyriakides T, Al-Qudah AK, Middleton L (2000) A novel form of distal hereditary motor neuronopathy maps to chromosome 9p21.1-p12.

Ann Neurol 48:877±884

(25)

20. Cuesta A, Pedrola L, Sevilla T, Garcia-Planells J, Chumillas MJ, Mayordomo F, Le- Guern E, Marin I, Vilchez JJ, Palau F (2002) The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal Charcot-Marie- Tooth type 4A disease. Nat Genet 30:22±25

21. Dawkins JL, Hulme DJ, Brahmbhatt SB, Auer-Grumbach M, Nicholson GA (2001) Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base sub- unit-1, cause hereditary sensory neuropathy type I. Nat Genet 27:309±312 22. De Jonghe P, Timmerman V, Ceuterick C, Nelis E, De Vriendt E, Lofgren A, Ver-

cruyssen A, Verellen C, Van Maldergem L, Martin JJ, Van Broeckhoven C (1999) The Thr124Met mutation in the peripheral myelin protein zero (MPZ) gene is associated with a clinically distinct Charcot-Marie-Tooth phenotype. Brain 122:281±

23. Delague V, Bareil C, Tuffery S, Bouvagnet P, Chouery E, Koussa S, Maisonobe T,290 Loiselet J, Megarbane A, Claustres M (2000) Mapping of a new locus for autosomal recessive demyelinating Charcot-Marie-Tooth disease to 19q13.1±13.3 in a large consanguineous Lebanese family: exclusion of MAG as a candidate gene. Am J Hum Genet 67:236±243

24. Denny-Brown D (1951) Hereditary sensory radicular neuropathy. J Neurol Neuro- surg Psychiatry 14:237±247

25. Dyck PJ, Lambert EH, Nichols PC (1971) Quantitative measurement of sensation related to compound axon potential and number and sizes of myelinated and unmyelinated fibers of sural nerve in health, Friedreich's ataxia, hereditary sensory neuropathy, and tabes dorsalis. In: Cobb VA (ed) Handbook of electroencephalog- raphy and clinical neurophysiology, vol 9. Elsevier, Amsterdam, pp 81±93

26. Dyck PJ, Giannini C, Lais A (1993) Pathologic alterations of nerves. In: Dyck PJ, Thomas PK, Griffin JW, Low PA, Poduslo JF (eds) Peripheral neuropathy, 3^rdedn.

Saunders, Philadelphia, pp 514±595

27. Dyck PJ (1993) Neuronal atrophy and degeneration predominantly affecting peripheral sensory and autonomic neurons. In: Dyck PJ, Thomas PK, Griffin JW, Low PA, Poduslo JF (eds) Peripheral neuropathy, 3^rdedn. Saunders, Philadelphia, pp 1065±1093

28. Dyck PJ, Chance P, Lebo R, Carney JA (1993) Hereditary motor and sensory neuropathies. In: Dyck PJ, Thomas PK, Griffin JW, Low PA, Poduslo JF (eds) Periph- eral neuropathy, 3^rdedn. Saunders, Philadelphia, pp 1094±1136

29. Einarsdottir E, Carlsson A, Minde J, Toolanen G, Svensson O, Solders G, Holm- gren G, Holmberg D, Holmberg M (2004) A mutation in the nerve growth factor beta gene (NGFB) causes loss of pain perception. Hum Mol Genet 13:799±805 30. Evgrafov OV, Mersiyanova I, Irobi J, Van Den Bosch L, Dierick I, Leung CL, Scha-

gina O, Verpoorten N, Van Impe K, Fedotov V, Dadali E, Auer-Grumbach M, Windpassinger C, Wagner K, Mitrovic Z, Hilton-Jones D, Talbot K, Martin JJ, Vas- serman N, Tverskaya S, Polyakov A, Liem RK, Gettemans J, Robberecht W, De Jonghe P, Timmerman V (2004) Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. Nat Genet 36:602±606

31. Fabrizi GM, Taioli F, Cavallaro T, Rigatelli F, Simonati A, Mariani G, Perrone P, Rizzuto N (2000) Focally folded myelin in Charcot-Marie-Tooth neuropathy type 1B with Ser49Leu in the myelin protein zero. Acta Neuropathol (Berl) 100:299±304 32. Fabrizi GM, Simonati A, Taioli F, Cavallaro T, Ferrarini M, Rigatelli F, Pini A, Mostacciuolo ML, Rizzuto N (2001) PMP22 related congenital hypomyelination neuropathy. J Neurol Neurosurg Psychiatry 70:123±126

33. Fabrizi GM, Cavallaro T, Angiari C, Bertolasi L, Cabrini I, Ferrarini M, Rizzuto N (2004) Giant axons and neurofilament accumulation in Charcot-Marie-Tooth disease type 2E. Neurology 62:1429±1431

(26)

34. Flachenecker P, Janka M, Goldbrunner R, Toyka KV (1999) Clinical outcome of sural nerve biopsy: a retrospective study. J Neurol 246:93±96

35. Gabreels-Festen A, van Beersum S, Eshuis L, LeGuern E, Gabreels F, van Engelen B, Mariman E (1999) Study on the gene and phenotypic characterisation of autosomal recessive demyelinating motor and sensory neuropathy (Charcot-Marie- Tooth disease) with a gene locus on chromosome 5q23-q33. J Neurol Neurosurg Psychiatry 66:569±574

36. Gabreels-Festen AA, Hoogendijk JE, Meijerink PH, Gabreels FJ, Bolhuis PA, van Beersum S, Kulkens T, Nelis E, Jennekens FG, de Visser M, van Engelen BG, Van Broeckhoven C, Mariman EC (1996) Two divergent types of nerve pathology in patients with different P0 mutations in Charcot-Marie-Tooth disease. Neurology 47:761±765

37. Gillespie CS, Sherman DL, Fleetwood-Walker SM, Cottrell DF, Tait S, Garry EM, Wallace VC, Ure J, Griffiths IR, Smith A, Brophy PJ (2000) Peripheral demyelination and neuropathic pain behavior in periaxin-deficient mice. Neuron 26:523±

38. Ginsberg L, Malik O, Kenton AR, Sharp D, Muddle JR, Davis MB, Winer JB, Orrell531 RW, King RH (2004) Coexistent hereditary and inflammatory neuropathy. Brain 127:193±202

39. Goebel HH, Veit S, Dyck PJ (1980) Confirmation of virtual unmyelinated fiber absence in hereditary sensory neuropathy type IV. J Neuropathol Exp Neurol 39:

670±675

40. Guilbot A, Williams A, Ravise N, Verny C, Brice A, Sherman DL, Brophy PJ, Le- Guern E, Delague V, Bareil C, Megarbane A, Claustres M (2001) A mutation in periaxin is responsible for CMT4F, an autosomal recessive form of Charcot-Marie- Tooth disease. Hum Mol Genet 15:415±421

41. Hahn AF, Ainsworth PJ, Bolton CF, Bilbao JM, Vallat JM (2001) Pathological findings in the x-linked form of Charcot-Marie-Tooth disease: a morphometric and ultrastructural analysis. Acta Neuropathol 101:129±139

42. Harding AE, Thomas PK (1980) The clinical features of hereditary motor and sensory neuropathy types I and II. Brain 103:259±280

43. Harding AE (1993) Inherited neuronal atrophy and degeneration predominantly of lower motor neurons. In: Dyck PJ, Thomas PK, Griffin JW, Low PA, Poduslo JF (eds) Peripheral neuropathy, 3^rdedn. Saunders, Philadelphia, pp 1051±1064 44. Hattori N, Yamamoto M, Yoshihara T, Koike H, Nakagawa M, Yoshikawa H, Oh-

nishi A, Hayasaka K, Onodera O, Baba M, Yasuda H, Saito T, Nakashima K, Kira J, Kaji R, Oka N, Sobue G (2003) Study Group for Hereditary Neuropathy in Ja- pan. Demyelinating and axonal features of Charcot-Marie-Tooth disease with mutations of myelin-related proteins (PMP22, MPZ and Cx32): a clinicopathological study of 205Japanese patients. Brain 126:134±151

45. Hayasaka K, Himoro M, Sawaishi Y, Nanao K, Takahashi T, Takada G, Nicholson GA, Ouvrier RA, Tachi N (1993) De novo mutation of the myelin P0 gene in De- jerine-Sottas disease (hereditary motor and sensory neuropathy type III). Nat Genet 5:266±268

46. Herget T, Esdar C, Oehrlein SA, Heinrich M, Schutze S, Maelicke A, van Echten- Deckert G (2000) Production of ceramides causes apoptosis during early neural differentiation in vitro. J Biol Chem 275:30344±30354

47. Herrman DN, Griffin J (2002) Intermediate filaments A common thread in neuro- muscular disorders. Neurology 58:1141±1143

48. Houlden H, King RH, Hashemi-Nejad A, Wood NW, Mathias CJ, Reilly M, Tho- mas PK (2001) A novel TRK A (NTRK1) mutation associated with hereditary sensory and autonomic neuropathy type V. Ann Neurol 49:521±525

(27)

49. Indo Y, Tsuruta M, Hayashida Y, Karim MA, Ohta K, Kawano T, Mitsubuchi H, Tonoki H, Awaya Y, Matsuda I (1996) Mutations in the TRKA/NGF receptor gene in patients with congenital insensitivity to pain with anhidrosis. Nat Genet 13:485±488

50. Jordanova A, De Jonghe P, Boerkoel CF, Takashima H, De Vriendt E, Ceuterick C, Martin JJ, Butler IJ, Mancias P, Papasozomenos SCh, Terespolsky D, Potocki L, Brown CW, Shy M, Rita DA, Tournev I, Kremensky I, Lupski JR, Timmerman V (2003) Mutations in the neurofilament light chain gene (NEFL) cause early onset severe Charcot-Marie-Tooth disease. Brain 126:590±597

51. Junghenn H, Krçcke W, Wadulla H (1949) Zur Frage der familiåren Syringomye- lie: klinisch-anatomische Untersuchungen çber familiåre neuro-vasculåre Dystro- phie der Extremitåten. Arch Psychiatr Nervenkr 182:153±160

52. Kalaydjieva L, Nikolova A, Turnev I, Petrova J, Hristova A, Ishpekova B, Petkova I, Shmarov A, Stancheva S, Middleton L, Merlini L, Trogu A, Muddle JR, King RH, Thomas PK (1998) Hereditary motor and sensory neuropathy-Lom, a novel demyelinating neuropathy associated with deafness in gypsies. Clinical, electrophysiological and nerve biopsy findings. Brain 121:399±408

53. Kessali M, Zemmouri R, Guilbot A, Maisonobe T, Brice A, LeGuern E, Grid D (1997) A clinical, electrophysiologic, neuropathologic, and genetic study of two large Algerian families with an autosomal recessive demyelinating form of Char- cot-Marie-Tooth disease. Neurology 48:867±873

54. King RH, Tournev I, Colomer J, Merlini L, Kalaydjieva L, Thomas PK (1999) Ul- trastructural changes in peripheral nerve in hereditary motor and sensory neuropathy-Lom. Neuropathol Appl Neurobiol 25:306±312

55. Kuhlenbåumer G, Young P, Hunermund G, Ringelstein B, Stogbauer F (2002) Clin- ical features and molecular genetics of hereditary peripheral neuropathies. J Neu- rol 249:1629±1650

56. Lafreniere RG, MacDonald ML, Dube MP, MacFarlane J, O'Driscoll M, Brais B, Meilleur S, Brinkman RR, Dadivas O, Pape T, Platon C, Radomski C, Risler J, Thompson J, Guerra-Escobio AM, Davar G, Breakefield XO, Pimstone SN, Green R, Pryse-Phillips W, Goldberg YP, Younghusband HB, Hayden MR, Sherrington R, Rouleau GA, Samuels ME; Study of Canadian Genetic Isolates (2004) Identifica- tion of a novel gene (HSN2) causing hereditary sensory and autonomic neuropathy type II through the Study of Canadian Genetic Isolates. Am J Hum Genet 74:1064±1073

57. Leyne M, Mull J, Gill SP, Cuajungco MP, Oddoux C, Blumenfeld A, Maayan C, Gu- sella JF, Axelrod FB, Slaugenhaupt SA (2003) Identification of the first non-Jewish mutation in familial dysautonomia. Am J Med Genet 118:305±308

58. Low PA, Burke WJ, McLeod JG (1978) Congenital sensory neuropathy with selec- tive loss of small myelinated fibers. Ann Neurol 3:179±182

59. Lupski JR, de Oca-Luna RM, Slaugenhaupt S, Pentao L, Guzzetta V, Trask BJ, Sau- cedo-Cardenas O, Barker DF, Killian JM, Garcia CA, et al (1991) DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell 66:219±232

60. Madrid R, Bradley WG (1975) The pathology of neuropathies with focal thickenings of the myelin sheath (tomaculous neuropathy). J Neurol Sci 25:45

61. Mancardi GL, Mandich P, Nassani S, Schenone A, James R, Deferrari R, Bellone E, Giunchedi M, Ajmar F, Abbruzzese M (1995) Progressive sensory-motor polyneuropathy with tomaculous changes is associated to 17p11.2 deletion. J Neurol Sci 13:30±34

62. Mandich P, Mancardi GL, Varese A, Soriani S, Di Maria E, Bellone E, Bado M, Gross L, Windebank AJ, Ajmar F, Schenone A (1999) Congenital hypomyelination due to myelin protein zero Q215X mutation. Ann Neurol 45:676±678