Early morphological features in dominantly inherited demyelinating motor and sensory neuropathy (HMSN type I)

Journal of the Neurological Sciences, 107 (1992) 145-154

145

© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00 JNS 03681

Early morphological features in dominantly inherited demyelinating motor and sensory neuropathy (HMSN type I) A.A.W.M. GabreEls-Festen 1, E.M.G. Joosten 1, F.J.M. GabreEls 1, F.G.I. Jennekens 2 and T.W. Janssen-van Kempen 1 1Institute of Neurology, Unit,ersityHospital Nijmegen, Nijmegen (The Netherlands) and : Department of Neurology, University Hospital Utrecht, Utrecht (The Netherlands)

(Received 30 April, 1991) (Revised, received 13 August, 1991) (Accepted 16 August, 1991) Key words: Hereditary motor and sensory neuropathy; Demyelinating neuropathy; HMSN type I; Pathogenesis; Sural nerve

biopsy

Summary Seventeen cases of dominantly inherited demyelinating motor and sensory neuropathy (HMSN type I) with infantile onset were studied. Not only clinical and electrophysiological data, but also the g ratio (axon diameter to fibre diameter), considered to be a distinguishing feature between HMSN type I and HMSN type III, showed overlap. Morphological and morphometrical investigations already revealed a lack of small and large diameter myelinated axons at an early stage, and a demyelinating process most active in early childhood followed later by axonal loss. It was concluded that the histopathology of HMSN type I cannot be sufficiently explained by axonal atrophy with secondary demyelination.

Introduction Hereditary motor and sensory neuropathy type I ( H M S N type I) is a dominantly inherited demyelinating neuropathy with marked slowing of motor nerve conduction velocity (Dyck 1975). Nerve conduction slowing evolves during the first years of life ( G u t m a n et al. 1983), but it shows no (Roy et al. 1989; Nicholson 1991) or minor (Dyck et al. 1989) changes after the first decade. C o m p o u n d muscle action potential, however, decreases significantly over the years, probably reflecting progressive axonal loss (Roy et al. 1989). The pathogenesis is unknown. Although extensive demyelination favours a primary Schwann cell disorder, studies by Dyck and colleagues (1974) in two young adults suggest a primary neuronal or axonal involvement. To date, there is no systematic investigation of the morphological evolution starting early on in life.

Correspondence to." A.A.W.M. Gabre~ls-Festen, Institute of Neurology, University Hospital Nijmegen, Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: (33) 080-615291; Fax: (33) 080-540576.

We describe the morphological and morphometrical features of 17 patients with infantile onset of dominant H M S N type I. Within this group in whom time of onset was more or less the same, patients were biopsied at different ages, ranging from 2.5 years to 33 years. This enabled us to trace the evolution of the pathological processes in the peripheral nerve.

Materials and methods Patients

From 1970 to the end of 1990, a peripheral nerve biopsy was performed in 676 patients with neuropathy, of which 179 were children under 16 years. H M S N was diagnosed in 109 cases on the basis of the following criteria: chronic progressive symmetrical motor and sensory neuropathy confirmed by electrophysiological investigation; no involvement of the central nervous system, exclusion of inherited or acquired metabolic disorders, endocrinological, immunological or chronic infectious diseases, deficiencies, malignancies and disorders caused by toxic agents; no inflammatory signs in nerve biopsy.

146 TABLE 1

Histological techniques

HEREDITARY MOTOR AND SENSORY NEUROPATHY

A whole sural nerve biopsy was performed at the mid-calf level and prepared for light and electron microscopical examination, including teased fibre studies. using standard techniques (Vos et al. 1983). The total transverse fascicular area (TTFA) was assessed with the aid of a planimeter on photographic enlargements of semithin sections of the nerve. Electron microscopical photographs ( × 2200) of ultrathin sections covering approximately 10% of the T T F A were used for morphometrical analysis. Density and diameter distribution of myelinated fibres and myelinated axons were determined using a Zeiss T G Z 3 particle size analyzer. On the same prints external and axonal diameter were measured and the g ratio (axon diameter to fibre diameter) was determined. "Myelinated axons" were those presumed to belong to the myelinated fibre group, namely axons with myelin and those of larger diameters ( > 1.7 p~m) without myelin but surrounded by one or more extra Schwann cell processes (non- or demyelinated fibres) (Guzzetta et al. 1982). The density of demyelinated axons was determined and scored as a percentage of the density of myelinated fibres per case. Florid demyelination identified as a demyelinated axon with myelin debris in the Schwann cell or nearby macrophages was counted on the same prints and its density was also scored as a percentage of the density of myelinated fibres. About 50 myelinated fibres from each nerve were teased and classified according to Dyck (1975) for paranodal or segmental de- and remyelination and linear myelin ovoids (axonal degeneration).

No. of biopsies <16y

>16y

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14

32

46

11 2 5 3

4 8 2

15 10 5 5

1 12

16 6

17 18

48

68

116

Demyelinating forms H M S N type I dominant sporadic/recessive parents examined parents not examined H M S N type III Demyelinating H M S N with tomacula (GabreEls-Festen et al. 1990)

Neuronal forms H M S N type II dominant recessive with early childhood onset (Ouvrier et al. 1981; GabreEls-Festen et al. 1991) Total HMSN

Uncomplicated chronic progressive axonal polyneuropathies with onset in the second or later decades and without familial occurrence were not included as sporadic HMSN type II, because this diagnosis can often only be made by exclusion. Seven selected biopsies (3 sporadic HMSN type I and 4 early onset HMSN type II) were provided by the Department for Neuromuscular Diseases of the University of Utrecht. Table 1 summarizes the definitive HMSN cases. All patients were examined by (child) neurologists and paediatricians experienced in neuromuscular diseases. In 46 patients dominant HMSN type I was diagnosed. Careful inquiry after the first signs was made. In 16 cases, parents had already noted motor dysfunctions within the first two years of life. The disease onset of one patient was probably early, but the exact time is not known. Fifteen patients had an unequivocally affected parent confirmed by clinical examination. In several cases electrophysiological examination of parents was also performed. The parents of 2 patients could not be investigated, but both patients had an affected child as confirmed clinically and electrophysiologically.

Electrophysiological techniques Nerve conduction velocities were examined according to a standardized protocol (Spaans 1984). Motor nerve conduction velocities (MNCV) of the median (elbow-wrist) and peroneal (head of fibula-ankle) nerves were recorded using surface electrodes. Sensory action potentials were recorded from the median nerve at the wrist on stimulating the index finger with ring electrodes and from the sural nerve orthodromically. A general E M G investigation was performed with concentric needle electrodes.

Results

Clinical features Age and sex of the 17 patients and time of disease onset are listed in Table 2. In one patient hypotonia was noticed at birth and was confirmed by paediatric examination at one month of age. Eight patients walked before the 18 th month, but from the beginning gait was abnormal with frequent falling, walking o n tiptoe and poor running. The precise time of disease onset or age of walking of patient 15 (father of case 8) is not known. At the age of 6 years, he consulted a physician because of walking difficulties. Pes cavus was evident in most patients at first examination. Scoliosis was often present from late childhood onwards. Distal weakness was present in all patients; impairment of intrinsic hand muscles was not evident in the two youngest patients. Proximal leg muscles were slightly affected in patients 6 and 15. Patient 13 showed shoulder amyotrophy. Total areflexia was the rule, in 5 patients proximal arm reflexes were still slightly elicitable. Sensory involvement could be demonstrated in about three-quarters of

147 the patients. Slight sensory ataxia was noted in 5 patients, 3 of them belonged to the same kinship. Cranial nerves did not exhibit abnormalities, except in patient 13, who showed fine nystagmus and slight pupillary inequality. Clinically enlarged nerves were palpable in patients 13, 15 and 16. Cerebrospinal fluid (CSF) protein content was elevated in 3 out of 10 patients in whom it was examined (Table 2). Three out of 12 patients had a creatine kinase (CK) elevation to maximally 3 times the normal value. All patients showed a severe slowing of MNCV, ranging in 16 from 7 to 19 m / s in the median nerve. Only one had a less severe slowing of 28 m / s . Sensory action potentials were absent in all except 3 patients in whom a small sensory action potential could be recorded (Table 2).

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Family investigation None of the parents in any of the families was aware of any consanguinity. The mother was the affected parent in 5 cases, the father in 12 cases. Recorded median MNCV of the affected parents was 20.6 _+ 4.8 m / s . In several instances siblings or offspring were also examined. Patients 8, 12 and 15 belong to one kinship (Fig. 1). In this family persons allied by marriage were also examined.

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The duration of the follow-up was 0 to 21 years (mean 11.4 years). The age at last investigation ranged from 9 to 51 years (mean 25.6 years). Progression was slow in all patients. From adolescence onwards, the patients hardly experienced progression, although throughout the years a slight increase of signs was usually manifest. All but 2 patients used orthopaedic footwear. Maximum walking distance ranged from 2 to 10 km in adult life. However, patients 8, 12 and 15, all of one kinship, were more severely handicapped. They also needed braces, which enabled them to walk some hundred metres. In adult life, sensory dysfunction of distal lower limbs was always present, even if only slightly. Many patients complained of cold extremities and discoloured skin. Most adult patients had developed a slight scoliosis. All patients were of normal stature compared with their family.

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Density of myelinated fibres was markedly reduced, partly due to an increase in total transverse fascicular area. Demyelinated fibres and florid demyelination were seen in the young patients (Fig. 2a). Demyelinated axons often exhibited a high density of neurofilaments (Fig. 3a). Even in the youngest patients thick myelin sheaths were observed around some axons, which showed normal density of neurofilaments and neurotubules (Fig. 3b). In longitudinal sections small focal myelin thickenings (tomacula) were occasionally

148 observed. Infrequent and small onion bulbs were observed in the 2 patients aged 2.5 years (Fig. 2a). Well developed onion bulb formations of about 2 - 4 lamellae were present around many myelinated fibres in the older patients (Fig. 2b). Some short fragments of double-layered basement m e m b r a n e s were often intermingled with Schwann cell lamellae. Active axonal degeneration was not seen. After the age of 6, a varying number of denervated onion bulbs was observed, and bands of Biingner or complex Schwann cell bands with several small nonmyelinated axons were present (Fig. 2c). The degree of axonal loss showed a marked individual variation. Rarely, a cluster of small myelinated fibres was observed inside an onion bulb. Unmyelinated fibres in between the onion bulb structures were without abnormalities. Patients 8, 12 and 15, all belonging to one kinship, showed only small and thinly myelinated fibres (Fig. 4a). A moderate to massive increase in endoneural collagen fibres was observed in all cases. Lymphocytic infiltration was not present in any case. Teased fibre examination showed extensive abnormalities in all cases. Nearly all fibres showed a marked variation in internodal length and diameter. Light microscopically several segments or paranodal areas seemed demyelinated. Linear myelin ovoids as seen in axonal degeneration were not observed.

Morphometry (Table 3) Myelinated fibre (MF) density was reduced in all cases, ranging from 7 to 52% (mean 26%) of age-related values. Density reduction could be ascribed partly to markedly increased total transverse fascicular area (T-FFA). The total number of fibres (MF density x T T F A ) showed a smaller reduction (mean 60% of age-related values, range 9-92%). There was no clear correlation between clinically enlarged nerves (cases 13, 15 and 16) and the most increased T T F A s of the sural nerve (Table 3). The density of demyelinated

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fibres expressed as percentage of myelinated fibre density was pronounced in biopsies taken in childhood. After the age of 10, demyelinated fibres were rare in our series, even in the severest cases belonging to one kinship. In addition; florid demyelination was seen but only up to about 6 years of age. The myelinated fibre size histogram showed a rather flat configuration (Fig. 5). The first peak was often displaced to larger diameters. In some cases a second dilute peak was present at about 7 ~m. Comparison with normal age-related histograms showed that fibre loss of both small and large diameters had occurred. Fibres with a diameter > 8 # m were few or absent. A percentual loss of fibres < 3 / x m was most pronounced in childhood. Axon histograms showed the same pattern; axons < 2 /xm being diminished percentually in early childhood. A varying loss of larger diameter axons occurred in all cases. The myelinated fibre size histogram of the 3 kinship cases differed, showing a narrow unimodal peak with the top between 2 and 3 /zm. Fibres > 8 ~zm were absent and 4(1 to 65% of the fibres had a diameter of less than 3 / z m . In these cases the g ratio came to 0.88, 0.72 and 0.81 respectively. Regression lines for the relationship of the axon diameter to the total fibre diameter of cases 8, 12 and 15 and of age related controls are shown in Fig. 4b. The regression lines of the 3 cases approach the regression line of which axon diameter = total fibre diameter. In the other cases, the g ratio ranged from 0.52 to 0.66, which is slightly below the normal range.

Discussion The clinical features of H M S N type I with an infantile onset described in this report are essentially the same as those described in unselected cases of dominant H M S N type I (Dyck 1975; Buchthal and Behse

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149 1977; Brust et al. 1978; Davis et al. 1978; Harding and Thomas 1980; Bouch6 et al. 1983). The relatively large number of cases with early signs might give the impression of a skewed age of onset in dominant HMSN type I. However, early signs will be anticipated by the parents' knowledge of a dominant inherited disease (Harding and Thomas 1980) and in our setting with experienced (child) neurologists, attention was paid to early complaints of motor dysfunction. Subsequent clinical, electrophysiological and morphological examination will permit early detection before the admission of definite symptoms (Berciano et al. 1989). Yet, an over-representation of severe cases in our series is likely, as patients with severe clinical features tend to have an early onset. All except one patient showed a severe slowing of median MNCV (mean 15.7 + 5.6 m / s ) . This was strikingly less than 21.7 + 7.0 m / s registered by Harding and Thomas (1980) in dominant HMSN type I cases not selected for early age of onset. The mean median MNCV of the affected parents of our series was 20.6 + 4.8 m / s . Clinical, electrophysiological and morphological features of the 3 patients of one kinship mostly fell within the diagnostic criteria of HMSN type III (Dyck 1975; Ouvrier et al. 1987). Although high g ratios ( > 0.81) are considered to be a distinguishing morphological hallmark of HMSN type III (Dyck et al. 1971; Ouvrier et al. 1987), we determined g ratios of 0.72, 0.81 and 0.88 in this kinship. Autosomal dominant inheritance in this family is beyond doubt (Fig. 1) and excludes HMSN type III or X-linked inheritance (Rozear et al. 1987). Presumed homozygosity for the dominant HMSN type I gene as described by Killian and Kloepfer (1979) and Hagberg and Westerberg (1983) can produce clinical features of HMSN type III, although morphologically these cases are consistent with HMSN type I (Killian and Kloepfer 1979). Homozygosity can be excluded in at least two cases, because it was documented that cases 8 and 12 had only one affected parent. Another exceptional case of dominantly inherited motor and sensory neuropathy was reported by Kasman et al. (1976) in which a father and son showed a nearly complete lack of peripheral myelin. Possibly, these more severe cases of dominant HMSN type I have to be attributed to a distinct genotype. Our investigation has enabled us to obtain an impression of the early pathological processes. It has to

Fig. 2. Low power electronmicrographs, a: case 1 (aged 2.5 years). Several demyelinated fibres (T), demyelinated fibre with myelin debris in nearby macrophage (1" I"), few and small onion bulbs, b: case 7 (aged 7 years). Many medium sized onion bulbs, c: case 12 (aged 23 years). Large, frequently denervated onion bulbs. Bar = I0 tzm.

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Fig. 3. Case 1. a: high density of neurofilaments and neurotubuli in demyelinated fibre, b: thick myelin sheath in comparisonwith ax~vndiamctu:r. Normal density of neurofilaments. Bar = 1 gin.

be noted that the clinical and electrophysiological severity in the early biopsied cases does not differ from that of the cases biopsied later in life (Table 2). Florid demyelination and the occurrence of demyelinated fibres is most pronounced in biopsies taken in the first years of life. During this period, MNCV slowing evolves as is apparent from electrophysiological studies (Gutman et al. 1983). Initially, onion bulb formation is scanty, but from age 6 onwards well-developed onion bulbs can be observed. This confirms earlier observations of onion bulb development in children (Meier et al. 1976; Ouvrier et al. 1987). After the first decade demyelinated fibres are rare. Obviously the processes of de- and remyelination are kept in balance. According to some authors (Roy et al. 1989; Nicholson 1991) MNCV slowing, the counterpart of demyelination, also does not change with time or age from late childhood onwards. However, Dyck et al. (1989) noted an increase in ulnar MNCV by a few meters per second in patients who were 5 to 14 or 15 to 39 years old at first examination, and a decrease in patients who were older. They gave no explanation for this observation. In our series, denervated onion bulbs and bands of Biingner occur in varying degrees after the age of 6, also resulting in a variable loss of large diameter fibres. At later stages, slight cluster formation is noted. Axonal loss is likely to be correlated with clinical deterioration and a decrease in compound muscle action potential, all developing over the years (Dyck et al. 1989; Roy et al. 1989).

The myelinated fibre size histograms showed a rather fiat configuration with a slight shift of the first peak to larger diameters and a lack of both small and large diameter fibres already in the young children (Table 3, Fig. 4) These histogram changes were also observed by Behse and Buchthal (1977), Joosten (1982) and Gherardi et al. (1983) in their series of adult HMSN type 1 cases and by Ohnishi et al. (1977) in primary demyelinating lead neuropathy, and are strikingly different from the histogram changes of the neuronal types of HMSN (Behse and Buchthal 1977; Joosten 1982; Gherardi et al. 1983; GabreEls-Festen et al. 1991). The pathogenesis of HMSN type I has still not been elucidated. Based on morphometrical studies of the sural nerves of 2 young adults, Dyck and colleagues (1974) proposed a primary neuronal disorder leading to an axonal atrophy starting distally, with secondary segmental de- and remyelination. Indeed, the absence of the largest fibres in the histogram could be explained by axonal atrophy and axonal de- and regeneration (Nukada et al. 1983), but this does not hold for the lack of the smallest fibres. Moreover, the transplantation studies of Aguayo et al. (1978) and the uniform and marked slowing of conduction velocities of all nerve segments in HMSN type I (Lewis and Sumner 1982) favour a generalized disturbance of Schwann cells or myelin sheaths. From experimental and animal models of primary segmental demyelination, it is known that demyelination causes a close packing of axonal neurofilaments

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d M e a n o f a g e - m a t c h e d c o n t r o l s : 0.73 ( 2 - 5 y, n = 2); 0.67 ( 6 - 1 0 y, n = 2); 0.67 ( 1 1 - 2 0 y, n - 3); 0.66 ( 2 1 - 5 0 y, n = 3).

~' M e a n o f a g e - m a t c h e d c o n t r o l s : 14170 ( 2 - 5 y, n = 6); 13180 (6 10 y, n = 11); 10530 ( 1 1 - 2 0 y, n = 5); 9 6 4 0 ( 2 1 - 3 0 y, n = 7); 9 7 6 0 ( 3 1 - 5 0 y, n = 5). h M e a n o f a g e - m a t c h e d c o n t r o l s : 0.61 ( 2 - 5 y, n = 4); 0.72 ( 6 - 1 0 y, n = 5), 0.87 ( 1 1 - 3 0 y, n = 5); 1.16 ( 3 1 - 5 0 y, n = 3). c M e a n o f a g e - m a t c h e d c o n t r o l s : % < 3 izm: 22 ( 2 - 5 y, n = 7); 26 ( 6 - 1 0 y, n = 11); 21 ( 1 1 - 2 0 y, n = 5); 15 ( 2 1 - 3 0 y, n = 7); 18 ( 3 1 - 5 0 y, n = 5). % > 8 Izm: 17 ( 2 - 5 y, n = 6 ) ; 17 ( 6 - 1 0 y, n = 11); 2 3 ( 1 1 2 0 y , n = 5); 25 ( 2 1 - 3 0 y, n = 7); 24 ( 3 1 - 5 0 y, n = 5 ) .

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MORPHOMETRY

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colleagues (1974) have described in their young adults the later events of HMSN type I, when the sequential processes of demyelination and axonal atrophy have become entwined. Whether the high density of neurofilaments frequently observed in demyelinated axons represents local axonal pathology, induced by segmental demyelination, requires further study.

large and intermediate diameter fibres induced by demyelination, while demyelination and subsequent attenuation of the smallest axons might result in their degeneration. Axonal atrophy occurring secondary to segmental demyelination will in turn induce a process of distally preponderant segmental de- and remyelination (Dyck et al. 1981). In our opinion, Dyck and

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153

Acknowledgements The authors wish to express their gratitude to Dr. M.W.I. Horstink, Dr. J.B. Krijgsman, Dr. W.O. Renier and Dr. R.C.A. Sengers for referring their patients, to Dr. H.M. Vingerhoets for electrophysiological examinations, to Dr. J. Hoogendijk for family investigations, and to Mr. J. Moleman for help with morphometrical analysis. This investigation is part of the research programme "Disorders of the Neuromuscular System" of the University of Nijmegen, The Netherlands.

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