Myelin protein zero mutation-related hereditary neuropathies: Neuropathological insight from a new nerve biopsy cohort

Abstract

Myelin protein zero (MPZ/P0) is a major structural protein of peripheral nerve myelin. Disease-associated variants in the MPZ gene cause a wide phenotypic spectrum of inherited peripheral neuropathies. Previous nerve biopsy studies showed evidence for subtype-specific morphological features. Here, we aimed at enhancing the understanding of these subtype-specific features and pathophysiological aspects of MPZ neuropathies. We examined archival material from two Central European centers and systematically determined genetic, clinical, and neuropathological features of 21 patients with MPZ mutations compared to 16 controls. Cases were grouped based on nerve conduction data into congenital hypomyelinating neuropathy (CHN; n = 2), demyelinating Charcot-Marie-Tooth (CMT type 1; n = 11), intermediate (CMTi; n = 3), and axonal CMT (type 2; n = 5). Six cases had combined muscle and nerve biopsies and one underwent autopsy. We detected four MPZ gene variants not previously described in patients with neuropathy. Light and electron microscopy of nerve biopsies confirmed fewer myelinated fibers, more onion bulbs and reduced regeneration in demyelinating CMT1 compared to CMT2/CMTi. In addition, we observed significantly more denervated Schwann cells, more collagen pockets, fewer unmyelinated axons per Schwann cell unit and a higher density of Schwann cell nuclei in CMT1 compared to CMT2/CMTi. CHN was characterized by basal lamina onion bulb formation, a further increase in Schwann cell density and hypomyelination. Most late onset axonal neuropathy patients showed microangiopathy. In the autopsy case, we observed prominent neuromatous hyperinnervation of the spinal meninges. In four of the six muscle biopsies, we found marked structural mitochondrial abnormalities. These results show that MPZ alterations not only affect myelinated nerve fibers, leading to either primarily demyelinating or axonal changes, but also affect non-myelinated nerve fibers. The autopsy case offers insight into spinal nerve root pathology in MPZ neuropathy. Finally, our data suggest a peculiar association of MPZ mutations with mitochondrial alterations in muscle.

Keywords: Charcot-Marie-tooth; MPZ; congenital hypomyelinating neuropathy; inherited neuropathy; myelin protein zero.

FIGURE 1

CMT‐type dependent differences in ultrastructural alterations of density of axons and Schwann cells as well as myelin thickness. (A, B) Electron microscopy images of cross sections through the sciatic nerve. Nerve of a control patient (A), showing normally myelinated axons (arrows) and several non‐myelinated axons (black asterisks) in one nucleated Schwann cells unit (white asterisk marking the nucleus). MPZ‐mutation‐induced neuropathy in CMT1‐patient #11 (B), showing two denervated nucleated Schwann cells (white pound key, #), one non‐myelinated axon (black asterisk) in a nucleated Schwann cell unit (white asterisk marking the nucleus) and collagen pockets (black arrowheads). (C–K) Graphs showing quantification of ultrastructural findings, sorted by CMT‐type: Densities of myelinated fibers (C) and unmyelinated axons (D), unmyelinated axons per nucleated Schwann cells units (E), density of all Schwann cell nuclei (F), myelinating Schwann cell nuclei (G), non‐myelinated Schwann cell nuclei (H), density of denervated Schwann cells (i.e., nucleated Schwann cell units without any axon; (I), density of collagen pockets (J), mean g‐ratio per case (K). One‐way ANOVA with Tukey's multiple comparison test was used as statistical test. Groups for analyses were: controls, CHN, CMT1, CMTi combined with CMT2. For CHN case #1, two nerve biopsies were available, taken 6 years apart from each other, at 7 months and 6 years of age, labeled with 1 and 2, respectively. Data points of clinically defined CMT1 case #3 are shown in brackets. Limited image quality did not allow to determine unmyelinated axon density and density of collagen pockets in CHN case #2. Numbers of analyzed biopsies are displayed below the graph.

FIGURE 2

CMT‐type dependent differences in ultrastructural alterations of myelin structure, onion bulb formation and axon regeneration. (A–F) Electron microscopy images of cross sections through peripheral nerves of patients with MPZ‐neuropathy: Onion bulb formation around a large caliber axon in CMT1 patient #4 with lamellae containing Schwann cell cytoplasm in addition to basement membranes (A). Regenerative cluster in CMTi patient #16 (B). Basal lamina onion bulb (BLOB) formation around a severely hypomyelinated larger caliber axon in CHN case #1. In contrast to regular onion bulbs, lamellae of BLOB contain Schwann cell basal laminae without cytoplasm (C). Uncompacted myelin lamellae in CMT1 patient #3. Uncompacted myelin is caused by separation of myelin lamellae at the major dense line [32]. In the four MPZ patients reported here, the lack of compaction could affect either the central or peripheral parts or the entirety of the myelin sheath. While it was often the only anomaly observed at this specific location of a fiber, it sometimes occurred in the context of excessive myelin folding (D). Tomaculum (E) and focally folded myelin (F) in CMT1 patient #9. Scale bars in A and B are 5 μm, and 2 μm in C–F. (G–M) Graphs showing quantification of ultrastructural findings, sorted by CMT‐type: Density of onion bulbs (G) and macrophages (H). Stacked bar diagrams displaying presence or absence of regenerative clusters (I), tomacula (J), uncompacted myelin (K), focally folded myelin (L), and basal lamina onion bulbs (M). One‐way ANOVA with Tukey's multiple comparison test was used as statistical test, also for numerical data on the density of regenerative clusters, tomacula, focally folded myelin BLOB, shown in graph I, J and L, M. For K and M, Fisher exact test was used. Groups for analyses were: controls, CHN, CMT1, CMTi combined with CMT2. For CHN case #1, two nerve biopsies were available, taken 6 years apart from each other, at 1 and 6 years of age, labeled with 1 and 2, respectively. Data points of clinically defined CMT1 case #3 are shown in brackets. Numbers of analyzed biopsies are displayed below the graph or in the bars.

FIGURE 3

Biopsy and autopsy findings in a CMT1 patient with p.R98C mutation. (A) X‐ray (a.p.) of the chest: severe scoliosis of the thoracic vertebral column. Marked chest deformation. (B) Sural nerve biopsy in 1998 revealed severe demyelinating neuropathy and angiopathy of endoneurial capillaries; scale bar = 100 μm. (Semithin section: toluidine blue staining) (C) Sciatic nerve, taken in 2006 during autopsy, showing advanced stage of severe neuropathy with only few remaining thinly myelinated axons (semithin section: toluidine blue staining). (D, E) Post mortem investigation of median nerve: HE (D) and EMA immunohistochemistry (E) show marked concentric thickening of the perineurium of a nerve fascicle (between arrowheads). Renaut body in the center of the nerve fascicle (arrows). Scale bars = 100 μm. (F) Leptomeningeal neuromatous sprouting observed in autopsy material: Neurofilament‐positive axons (F) surrounded by S100‐positive Schwann cells (insert in F; 2.5‐fold zoomed) forming minifascicles. Nerve root showing surplus perineurial septae (G–J): neurofilament (G), S100 (H), GLUT1 (I), and EMA (J). Scale bars = 100 μm.

FIGURE 4

Skeletal muscle pathology in patients with MPZ neuropathies. Neurogenic atrophy with groups of atrophic fibers, angular atrophic fibers, and targetoid fibers in CMT1 patient #4 in trichrome stained cryo cross sections (A) and mild Z‐band streaming in CMT1 patient #13 (B). CMTi patient #15 showed massive accumulation of altered autophagic material. This material showed the predominantly membranous, myelin‐like morphology and was surrounded by partially membrane‐bound vacuolar structures together with degraded organelles including mitochondria, which is typical for autophagic vacuoles (C). Paracrystalline inclusions, so called “parking lots” in mitochondria of CMT1 patients #6 (D) and #5 (E) seen in electron microscopy (EM) images. CMTi patient #15 also showed paracrystalline inclusions in mitochondria (F). EM image of a cross section of skeletal muscle of CMT patient (#20) showing accumulation of structurally altered mitochondria with concentric christae formation (G). Paraphenylendiamine‐stained semithin longitudinal section of skeletal muscle of the same patient #20 showing accumulation of numerous large diameter lipid droplets within muscle fibers (H). Scale bars are: 100 μm in A, 2 μm in B–G, and 20 μm in H.