Mitochondrial DNA variants in inclusion body myositis characterized by deep sequencing

Abstract

Muscle pathology in inclusion body myositis (IBM) typically includes inflammatory cell infiltration, muscle fibers with rimmed vacuoles and cytochrome c oxidase (COX)-deficient fibers. Previous studies have revealed clonal expansion of large mitochondrial DNA (mtDNA) deletions in the COX-deficient muscle fibers. Technical limitations have prevented complete investigations of the mtDNA deletions and other mtDNA variants. Detailed characterization by deep sequencing of mtDNA in muscle samples from 21 IBM patients and 10 age-matched controls was performed after whole genome sequencing with a mean depth of mtDNA coverage of 46,000x. Multiple large mtDNA deletions and duplications were identified in all IBM and control muscle samples. In general, the IBM muscles demonstrated a larger number of deletions and duplications with a mean heteroplasmy level of 10% (range 1%-35%) compared to controls (1%, range 0.2%-3%). There was also a small increase in the number of somatic single nucleotide variants in IBM muscle. More than 200 rearrangements were recurrent in at least two or more IBM muscles while 26 were found in both IBM and control muscles. The deletions and duplications, with a high recurrence rate, were mainly observed in three mtDNA regions, m.534-4429, m.6330-13993, and m.8636-16072, where some were flanked by repetitive sequences. The mtDNA copy number in IBM muscle was reduced to 42% of controls. Immunohistochemical and western blot analyses of IBM muscle revealed combined complex I and complex IV deficiency affecting the COX-deficient fibers. In conclusion, deep sequencing and quantitation of mtDNA variants revealed that IBM muscles had markedly increased levels of large deletions and duplications, and there were also indications of increased somatic single nucleotide variants and reduced mtDNA copy numbers compared to age-matched controls. The distribution and type of variants were similar in IBM muscle and controls indicating an accelerated aging process in IBM muscle, possibly associated with chronic inflammation.

Keywords: inclusion body myositis; mitochondrial DNA; mtDNA deletions; mtDNA duplications; mtDNA point mutations; mtDNA rearrangements; muscle disease.

FIGURE 1

Enzyme‐histochemical (A and F) and immuno‐histochemical (B‐E) analysis of serial sections of an IBM muscle biopsy (sample 588). (A and F) Several muscle fibers with reduced cyotochrome c oxidase (COX) activity (marked with a red square) at two different levels. The fibers appear blue in the combined COX/succinate dehydrogenase (SDH) enzyme histochemical staining. Some of the COX‐deficient fibers show mitochondrial proliferation and several are atrophic. (B) The NDUFB8 subunit in complex I is reduced in the COX‐deficient fibers. (C) The SDHB subunit in complex II shows normal presence in relation to the distribution of mitochondria. (D) The COX1 subunit in complex IV is reduced in the COX‐deficient fibers. (E) VDAC shows the distribution of mitochondria. (A‐E) Red squares indicate COX deficient fibers

FIGURE 2

Circular map of the deletions (blue) and duplications (red) identified by the pipeline in three IBM and three control samples (all 31 samples are illustrated in Figure S1). The color intensity is related to the abundance of each rearrangement. A heteroplasmy cut‐off of 0.05% is used for visualization. The deletions are localized mainly to the major arc of mtDNA between the origins of heavy (OH) and light (OL) chain replication

FIGURE 3

Overview of structural mtDNA alterations in muscle samples from IBM patients and age‐matched controls without muscle disease. (A) Sample‐wise distribution of heteroplasmy of the identified deletions and duplications. The color intensity is related to the abundance of each rearrangement. (B) The number of deletions and duplications in each sample and their respective contribution to sum % heteroplasmy. (C) The IBM muscle samples show a higher amount of deletions and duplications in the mitochondrial genome than the controls and the number of structural alterations correlates with the level of heteroplasmy

FIGURE 4

Recurrent mtDNA rearrangements. (A) The number of recurrent deletions and duplications in IBM and control muscles. (B) The most frequently recurrent deletions and duplications among IBM samples. One specific deletion was identified in all 21 IBM muscle samples. (C) Circular map of the most frequently recurrent deletions (blue) and duplications (red) illustrated in (B). The origins of heavy (OH) and light (OL) strand replication are indicated

FIGURE 5

Age (years) of the patients and controls and estimated mtDNA (Mt) copy number relative to the nuclear DNA of each sample. The IBM patient samples show reduced mtDNA copy number on average. The mean level is indicated

FIGURE 6

Western blot analyses using antibodies to subunits of the respiratory complexes I to V. There is a consistent decrease of NDUFB8 (Complex I) protein in the muscle samples from seven IBM patients compared to controls. Complex II is increased in some IBM samples, whereas complex III and IV are variably reduced, and complex V appears similar to controls. The analysis was performed in two different gels. Results from complex I, IV, and V analyzed in one gel are indicated with *, and complex II and III analyzed in the other gel are indicated with **. The bands corresponding to myosin heavy chain in the lower part of the panel were used as loading control for the corresponding gels