Biallelic COX10 Mutations and PMP22 Deletion in a Family With Leigh Syndrome and Hereditary Neuropathy With Liability to Pressure Palsy

Clinical Characteristics

Table 1 summarizes the family's clinical characteristics. Patients 1 (III-1, eFigure 1, links.lww.com/NXG/A545) and 3 (III-3) presented with spastic paraplegia, intellectual disability, cerebellar ataxia, and respiratory failure. Brain MRI for patient 1 revealed diffuse high-intensity lesions with coarsening and vacuolization in the white matter (eFigure 2, A–F) as well as multiple lesions in the brainstem and cerebellum (eFigure 2, G–I). Patients 1 and 3 had marked peripheral neuropathy. Patient 4 (II-5) developed compression neuropathy after surgery (eTable 1).

Histology and Respiratory Chain Enzyme Activity of Biopsied Muscle

A biopsy specimen from patient 1 revealed variation in muscle fiber diameter (eFigure 3A, links.lww.com/NXG/A545). A moderate increase in lipid was found in muscle fibers (eFigure 3B). Modified Gomori trichrome staining revealed no ragged-red fibers (eFigure 3C). COX activity was found to be reduced in a widespread and severe manner (eFigure 3, D and E). The uniform staining pattern of type 2 fibers was revealed by ATPase histochemistry (eFigure 3F).

Measurement of respiratory chain complex enzyme activity in biopsied muscle revealed that the levels of all enzymes were lower than those in normal tissues (eTable 2, links.lww.com/NXG/A545).⁵ The enzyme activity in fibroblast revealed a selective decrease in complex IV (eTable 2). The results of the enzymes were consistent with complex IV deficiency.

Autopsy Findings

An autopsy was performed on patient 3 (III-3) at age 9 years. The brain showed diffuse and severe white matter degeneration (Figure 1, A and B). The loss of myelin and axons appeared to be similar in extent in the degenerated white matter (Figure 1, C and D), and vacuolar changes of the neuropil accompanied by gliosis were observed (Figure 1E). Atrophy and myelin pallor was also evident in the cerebellar white matter, middle cerebellar peduncles, and pontine base (Figure 1F). Spotty demyelinating lesions were found in the left medial lemniscus (Figure 1F) and the reticular formation of the medulla oblongata (Figure 1, G and H). Axons were relatively preserved in both lesions, as well as the ventral part of the medulla oblongata, despite the significant loss of myelin (Figure 1, I and J). Olivary hypertrophy with severe gliosis (Figure 1K), Purkinje cell loss in the cerebellar cortex, and gliosis in the dentate nucleus (Figure 1L) were evident.

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Figure 1 Neuropathologic Findings of the Autopsied Brain (Patient 3)

(A) The brain weight was 1,100 g. A coronal section of the left cerebral hemisphere showed marked volume loss and grayish discoloration of the white matter. Severely softened deep white matter with cavity-like changes (arrow). (B) Diffuse myelin pallor in the affected white matter. U-fibers are mostly spared. (C and D) Myelin (C) and axons (D) are depleted to similar extents accompanied by some swelling axons in the moderately affected parietal white matter. (E) Neuropil vacuolar changes with gliosis and macrophage infiltration (inset) in relatively preserved white matter of the frontal lobe. (F) Atrophy and myelin pallor of the cerebellar white matter, middle cerebellar peduncles, and pontine base. A spotty demyelinating lesion in the left medial lemniscus (arrowhead). (G) Diffuse myelin pallor of the ventral part of medulla oblongata with bilateral olivary hypertrophy. A spotty demyelinating lesion crossing the midline raphe (arrow). (H) A high-magnification image was taken from the demyelinating lesion indicated by an arrow in (G). Tissue rarefaction and neuropil vacuolation are visible, and capillary prominence, gliosis, and macrophage infiltration are evident. Some neurons are relatively preserved in the lesion (triangles). (I and J) Images taken from the same lesion pointed with an arrow in (G). In the myelin basic protein (MBP) severely depleted lesion (I arrow), SMI-31-labeled axons are relatively preserved (J arrow). (K) Neuronal loss with marked gliosis in the inferior olivary nucleus. (L) Mild neuropil vacuolar changes accompanied by capillary prominence and gliosis in the dentate nucleus. (B, C, F, and G) Klüver-Barrera, and (E, H, K, and L) hematoxylin and eosin staining, (D and J) SMI-31, and (I) MBP immunohistochemistry. Bar = 145 μm for C and D, 50 μm for E, K, and L, 16 μm for inset in E, 43 μm for H, and 400 μm for I and J.

Genetic Analysis

Whole-exome sequence analysis showed COX10 variants of p.Arg159Gln and novel p.Pro295Leu in patients 1 and 3 (Figure 2A). An in silico analysis suggested that the p.Pro295Leu variant was pathogenic, whereas the p.Arg159Gln was predicted to be benign (eTable 3, links.lww.com/NXG/A545). CNV analysis of COX10 revealed a decreased gene dosage, indicating the presence of a deletion in patients 1, 3, and 4 (Figure 2B). Furthermore, CNV analysis revealed a decreased gene dosage in PMP22 (Figure 2C). A DNA microarray analysis revealed an approximately 1.4-Mb deletion spanning from intron 6 of COX10 to PMP22 (Figure 2D).

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Figure 2 Genetic Analysis

(A) Electropherograms of the p.Pro295Leu missense mutation in exon 6 of COX10 in patients 1 and 3 and their mother are shown. There were no pathogenic mitochondrial mutations in this family. (B) A CNV analysis of COX10 revealed a decreased gene dosage in intron 6 and exon 7 of COX10 in patients 1 (III-1), 3 (III-3), and 4 (II-5). The results are presented as the mean ± SEM (n = 3). (C) Decreases in CNV in PMP22 in patients 1 (III-1), 3 (III-3), and 4 (II-5). The results are presented as the mean ± SEM (n = 3). (D) Microarray analysis of patient 1 (III-1) revealed an approximately 1.4-Mb deletion extending from COX10 intron 5 to PMP22 (hg19 chr17: 14,087,933–15,484,859; 1,397 kbp). The deletion is predicted to result in a truncated COX10 lacking 130 amino acids at its C-terminus. CNV = copy number variant.

Protein Analysis

Proteins were extracted from the frozen tissue (cerebral cortex and white matter) in patient 3. In both mitochondrial and total lysate fractions, Western blot analysis revealed decreased expression levels of the COX1/MT-CO1, COX2/MT-CO2, and COX4 complex IV subunits (eFigure 4, A and B, links.lww.com/NXG/A545).