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. 2011 Jun 10;88(6):845-851.
doi: 10.1016/j.ajhg.2011.05.010.

A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis

Satomi Mitsuhashi  1 , Aya Ohkuma  1 , Beril Talim  2 , Minako Karahashi  3 , Tomoko Koumura  3 , Chieko Aoyama  4 , Mana Kurihara  5 , Ros Quinlivan  6 , Caroline Sewry  7 , Hiroaki Mitsuhashi  1 , Kanako Goto  1 , Burcu Koksal  2 , Gulsev Kale  2 , Kazutaka Ikeda  8 , Ryo Taguchi  8 , Satoru Noguchi  1 , Yukiko K Hayashi  1 , Ikuya Nonaka  1 , Roger B Sher  9 , Hiroyuki Sugimoto  4 , Yasuhito Nakagawa  3 , Gregory A Cox  9 , Haluk Topaloglu  10 , Ichizo Nishino  11
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A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis

Satomi Mitsuhashi et al. Am J Hum Genet. .

Abstract

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.

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Figures

Figure 1
Figure 1
Muscle Pathology of the Affected Individuals Cross-sections of muscle fiber from a human control and individual 4. (A) On H&E staining, nonspecific dystrophic features with necrotic and regenerating fibers, internalized nuclei, and endomysial fibrosis are seen. The scale bar represents 25 μm. (B) On cytochrome c oxidase staining, enlarged mitochondria at the periphery and central areas devoid of mitochondria were seen. The scale bar represents 20 μm. (C) On NADH-TR staining, the intermyofibrillar network was preserved even in the central areas that are devoid of mitochondria, suggesting the presence of myofibrils and only absence of mitochondria. The scale bar represents 20 μm. (D) Electron microscopy confirmed enlarged mitochondria. The scale bar represents 1 μm.
Figure 2
Figure 2
Choline Kinase Activity and Phospholipid Analyses (A) In muscle tissue from individuals 2, 3, and 4, CHK activity cannot be detected (n = 3). Data represent the mean of three individuals. (B) PC and PE content in frozen biopsied muscle tissues from individuals 2, 3, and 4 and hindlimb muscles from 8-week-old rmd mice (n = 4) and control littermates (n = 5) were analyzed by thin-layer chromatography followed by phosphorus analysis. PC and the PC/PE ratio are significantly decreased in affected individuals and rmd mice (n = 3 for humans, n = 4 for rmd mice, n = 5 for littermates). (C) Fatty acid composition of PC molecular species in muscles and isolated mitochondria from hindlimb muscles of rmd mice are determined by electrospray ionization mass spectrometry (ESI-MS). We observed that 34:1-PC (16:0-18:1), 36:4-PC (16:0-20:4), and 38:6-PC (16:0-22:6) species are significantly decreased, whereas 36:2-PC (18:0-18:2) is increased in rmd muscle. Similarly, in isolated mitochondria from hindlimb muscle, 36:4-PC (16:0-20:4) and 38:6-PC (16:0-22:6) species are decreased, whereas 36:2-PC (18:0-18:2) is increased. In muscle and isolated mitochondria, the 38:6-PC molecular species is profoundly decreased (n = 6 for muscle, n = 5 for isolated mitochondria). Mitochondria from skeletal muscles of whole hindlimbs of rmd mice were isolated by the differential centrifugation method. Fresh muscle was minced and homogenized with a motor-driven Teflon pestle homogenizer with ice-cold mitochondrial isolation buffer (10 mM Tris-HCl [pH 7.2], 320 mM sucrose, 1mM EDTA, 1mM DTT, 1 mM PMSF, 1 mg/ml BSA, and protease inhibitor cocktail [Roche]) and centrifuged at 1,500 × g for 5 min. The supernatant fraction was centrifuged at 15,000 × g for 20 min, the pellet was resuspended in mitochondrial isolation buffer, and the centrifugation/resuspension was repeated twice more. All data are presented as means ± standard deviation (SD). Means were compared by analysis with a two-tailed t test via R software version 2.11.0.
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