Our study has demonstrated that 2 mm punch biopsies are capable of identifying specific changes of molecular architecture and axonal loss in dermal myelinated nerve fibres from patients with CMT1A. These alterations provide pathological signatures that distinguish the inherited demyelinating neuropathy, CMT1A, from acquired demyelinating neuropathies such as CIDP. Moreover, the skin biopsy technique also demonstrated accumulation of intra-axonal mitochondria, which may provide new insights into the pathogenesis of axonal degeneration in CMT1A. We have found myelinated dermal nerves to be increasingly useful in investigating pathogenic mechanisms of inherited neuropathies since our initial report of the technique in 2005 (Li et al.
). For example, we have used skin biopsies to demonstrate abnormal RNA splicing of myelin protein zero in a patient with CMT1B (Sabet et al.
), to show abnormal trafficking of intracellular organelles in CMT4J (Zhang et al.
), and to quantify and identify variable PMP22 levels in compact myelin of patients with CMT1A (Katona et al.
). The present study has further expanded the utilities of this technique, and shown its potential to be used even in acquired demyelinating neuropathies. Taken together, our results have demonstrated that skin biopsy can be used to investigate pathophysiologic mechanisms in inherited, as well as acquired demyelinating neuropathies. Comparisons between findings from skin biopsies and sural nerve biopsies may further enhance our understanding of the pathogenesis of CMT1A. Moreover, skin biopsies also possess certain advantages, including its minimally invasive nature and potential repetitive use in the same subject for longitudinal studies.
Pathological studies of sural biopsies in different age groups of patients with CMT1A have suggested that extensive segmental demyelination occurs during the first decade of life in patients with CMT1A and are significantly less frequent in adulthood (Gabreels-Festen et al.
; Fabrizi et al.
; Gabreels-Festen and Wetering, 1999
). Although active segmental demyelination was not found in dermal myelinated fibres of CMT1A patients, an increased asymmetry in hemiparanodal length suggests that previous segmental demyelination may have occurred in these fibres. The increased asymmetry of paranodes in CMT1A could have resulted from insidiously accumulated de-/remyelination that occurred in the early life of the patients, as has been suggested by investigations of sural nerve biopsies in other patients with CMT1A (Gabreels-Festen et al.
Interestingly, distinct to what has been described in sural nerve studies (Dyck et al.
), the variability around the mean internodal length was not increased in dermal fibres in CMT1A. In developmentally abnormal myelination, or dysmyelination, internodal length may be uniformly shortened. For example, in periaxin null mice, internodal elongation is impaired resulting in uniformly shortened internodes as well as slow nerve conduction velocities (Court et al.
). We speculate that a developmental defect in internodal lengthening, limiting Schwann cell extension along axons, could explain our findings and be a contributing pathological mechanism in CMT1A. Limited internodal lengthening has been demonstrated in a cell culture study using the Tembler-J mouse model of PMP22 point mutations (Liu et al.
). The recent observations suggesting that PMP22 is a binding partner of α6β4-integrin (Amici et al.
), a Schwann cell protein that mediates interactions with the basal lamina complex could provide a molecular basis for this hypothesis, although further studies are necessary to confirm this. Longitudinal studies to document the changes of internodal length and segmental demyelination during development in CMT1A would be helpful to test this hypothesis further.
It is not yet clear why active segmental demyelination is not observed in dermal myelinated fibres in CMT1A since this has been reported in sural nerve biopsies in patients with CMT1 prior to the era of molecular diagnosis (Dyck et al.
) and in cases of CMT1A (Gabreels-Festen et al.
; Fabrizi et al.
). One possible explanation is that the shorter internodal lengths and smaller fibre diameters of myelinated fibres typically observed in this territory may allow for a more efficient remyelination early in life. This discrepancy stresses the importance of distinguishing changes observed in dermal biopsies from those of sural nerve biopsies in order to understand better the pathogenic mechanisms involved in CMT1A. If this was the case, our skin biopsy technique would be a better approach to show the pathological changes distinguishing the inherited from acquired demyelinating neuropathy.
The finding of shortened internodal length fits well with the uniform slowing of conduction velocities in CMT1A (Lewis and Sumner, 1982
), observed even in the youngest patients (Nicholson, 1991
; Garcia et al.
; Yiu et al.
). Uniform slowing in CMT1A would not be adequately explained by active segmental de-/remyelination, since non-uniform slowing, temporal dispersion and conduction block are usually seen in acquired demyelinating neuropathies, where active segmental demyelination is the main pathological feature (Hahn, 2005
). Shortened internodal length without segmental demyelination is sufficient to reduce nerve conduction velocities in periaxin-null mice (Court et al.
). Thus, uniformly shortened internodal length in patients with CMT1A may serve, at least partially, as a pathogenic mechanism for the uniform slowing of conduction velocity in CMT1A.
In the present study, we were able to quantify the axonal loss of myelinated nerve fibres by measuring the density of Meissner corpuscles. Attempts to quantify axonal loss by counting axons within fascicles produced variable results between fascicles, such that axonal loss could not be ascertained with confidence. Moreover, no significant differences between controls and patients with CMT1A could be determined by this technique. It is possible that the length of nerves innervating the hands may be insufficient to document axonal loss in mild forms of length-dependent hereditary neuropathies. However, areas in lower extremities typically have more severe axonal loss with few if any myelinated nerve fibres making it impossible to measure internodes and segmental demyelination. Thus, for the purpose of our study, skin biopsies from hands were the more appropriate area to perform these measurements. Alternatively, the density of Meissner corpuscles in CMT1A patients was significantly reduced compared with controls. Whether sensory nerves going to Meissner corpuscles are preferentially affected in CMT1A compared with other myelinated sensory axons in skin is unknown. Whatever the underlying reason for this difference is, Meissner corpuscle density appears to be a more sensitive index for evaluating axonal loss in CMT1A, which is consistent with the observation of a previous study (Dyck et al.
Since axonal loss is closely related to the neurological impairment in patients with CMT1A (Dyck et al.
; Krajewski et al.
), mechanisms underlying the axonal degeneration are necessary to understand the pathogenesis of the neuropathy. PMP22 is expressed in myelinating Schwann cells, but not in the neurons they ensheath (Welcher et al.
). Thus, axonal loss must result from abnormal interactions between mutant Schwann cells, which overexpress PMP22, and the underlying axons. Our study identified an increase of mitochondrial density in myelinated axons of CMT1A, suggestive of an impairment of mitochondrial transport along the axons. Abnormal mitochondrial trafficking resulting from mutations of nuclear encoded mitochondrial genes, causes severe axonal loss and impaired transport of mitochondria in the most common dominant (CMT2A) (Zuchner et al.
) and recessive (CMT4A) (Baxter et al.
, Cuesta et al.
) axonal forms of CMT. Thus, the mitochondrial abnormalities we have identified in axons of patients with CMT1A may provide important insights into potential mechanisms for axonal degeneration in this demyelinating disorder.
The number of mitochondria is known to increase in the nodal and paranodal regions of axons (Berthold et al.
). We recognized that one potential explanation for the observed increased mitochondrial density in CMT1A axons could be the increased mitochondrial number in the nodes/paranodes of dermal myelinated nerves, consequent to the internodal length reduction in CMT1A. However, this is an unlikely explanation for several reasons. First, there were no nodal regions in any of the sections we analysed for mitochondrial density. Second, in several axonal sections that were not in nodal or paranodal regions, there was clearly an increased mitochondrial density in patients with CMT1A. Consistent with this notion, an illustrative case in shows a remarkable accumulation of intra-axonal mitochondria, but no morphological features of node or paranode. Therefore, this increase of mitochondrial number is independent of any regional effect of nodes/paranodes.
In summary, this study identifies several important features of CMT1A, including differences between CMT1A and CIDP. First, internodal length in CMT1A is shortened in the absence of active segmental demyelination which may, at least partially, explain the uniform slowing of conduction velocity in this disease. This finding also suggests a potential developmental defect in CMT1A during internodal lengthening. While sural biopsies from previous studies showed segmental demyelination in CMT1A, this difference between the sural and dermal myelinated nerve fibres stresses the importance of distinguishing changes observed in dermal and sural biopsies in order to understand better the biological property of dermal nerve Schwann cells or axons that protects them from demyelination. Second, the density of Meissner corpuscles proved to be a useful index of axonal loss in CMT1A. It remains to be determined whether this can be used as a surrogate marker for the progression of axonal degeneration in clinical trials. Finally, we found an abnormal accumulation of intra-axonal mitochondria in dermal myelinated axons of CMT1A, which may shed light on the pathogenesis of axonal degeneration in this disease.