ADCK2 Haploinsufficiency Reduces Mitochondrial Lipid Oxidation and Causes Myopathy Associated with CoQ Deficiency

Fatty acids and glucose are the main bioenergetic substrates in mammals. Impairment of mitochondrial fatty acid oxidation causes mitochondrial myopathy leading to decreased physical performance. Here, we report that haploinsufficiency of ADCK2, a member of the aarF domain-containing mitochondrial protein kinase family, in human is associated with liver dysfunction and severe mitochondrial myopathy with lipid droplets in skeletal muscle. In order to better understand the etiology of this rare disorder, we generated a heterozygous Adck2 knockout mouse model to perform in vivo and cellular studies using integrated analysis of physiological and omics data (transcriptomics–metabolomics). The data showed that Adck2+/− mice exhibited impaired fatty acid oxidation, liver dysfunction, and mitochondrial myopathy in skeletal muscle resulting in lower physical performance. Significant decrease in Coenzyme Q (CoQ) biosynthesis was observed and supplementation with CoQ partially rescued the phenotype both in the human subject and mouse model. These results indicate that ADCK2 is involved in organismal fatty acid metabolism and in CoQ biosynthesis in skeletal muscle. We propose that patients with isolated myopathies and myopathies involving lipid accumulation be tested for possible ADCK2 defect as they are likely to be responsive to CoQ supplementation.


Case report
The male index patient (subject II-3, Fig. S1A) presented to our clinic at 45 years of age with a 15-year history of slowly progressive muscle weakness and myalgia, which occurred at rest but worsened with exercise. Past medical history was unremarkable except for renal disease of unknown cause in childhood, which spontaneously improved. Family history was negative for neurological disease. On examination, moderate proximal symmetrical myopathy, more pronounced in the arms, was noted and the patient was unable to lift his arms above the horizontal position. The patient had a hyperlordotic, waddling gait and was only able to walk 100 meters without the aid of crutches.
Bilateral scapular winging was present, and bilateral atrophy of the biceps, triceps, and quadriceps was noted, whilst the deltoid muscles were well preserved. Calf hypertrophy was present. The Trendelenburg sign was positive, and the patient was unable to rise from squatting. Pulmonary function was mildly impaired (vital capacity was 85 % of normal). Nerve conduction studies did not reveal a significant polyneuropathy. There were no central neurological deficits, bulbar or ocular problems and no ataxia; cognitive function was normal. On EMG examination, a myopathic pattern was found, with copious spontaneous activity (fibrillation, myotonic discharges and complexrepetitive discharges). Echocardiogram was normal. Abdominal ultrasound examination revealed liver steatosis. MR spectroscopic imaging of the brain showed multifocal elevation of lactate, alanine, lipids and free macromolecules. A muscle biopsy performed 5 years earlier showed moderate myopathic changes with marked lipid accumulation, consistent with a lipid storage myopathy. No ragged red fibers or inflammatory changes were identified. Total carnitine was mildly reduced in muscle and a diagnosis of carnitine deficiency was made. The patient had received carnitine replacement therapy (4 g of carnitine per day) since this time without any improvement in his condition, which had continued to worsen over this time at increased speed.
Riboflavin therapy (200 mg per day) was initiated immediately based upon these findings, and two months later a further muscle biopsy was performed. Histological examination revealed some fiber atrophy and small vacuoles in many fibers (Fig. 1A), with lipid droplets apparent in some fibres with the oil-red-O stain (Fig. 1A). Only occasional ragged-red fibres were noted with the Gomori trichrome stain (not shown).
On riboflavin therapy, the plasma acyl-carnitine profile began to normalize. After 2 months, the levels of hexanoylcarnitine were within the 99 percentile limit of the normal range, while the C4, C8 and C10 species where still mildly elevated (Table S1). The plasma acyl-carnitine profile was further improved after 6 months (timepoint 4, Table S1), and this was accompanied by a moderate reduction in the levels of plasma lactate, CK, myoglobin, and total LDH (Fig. S1E). After 6 months of combined carnitine/riboflavin therapy, there was no improvement in the patient's symptoms, and he now required opiates for severe myalgia, which had worsened during this time. There was no change in the EMG, but MRS showed an improvement in cellular lactate and lipid levels in the brain, indicating a decrease in membrane destruction. As riboflavin therapy did not appear to be having an effect on disease progression, supplementation with CoQ10 in the form of nanoparticles in liquid suspension was recommended (initial dose, 75 mg CoQ per day; Fig. S1E). His clinical condition was unchanged after 4 months of combined carnitine/riboflavin/CoQ therapy, but there was a slight improvement in the plasma lactate and myoglobin levels (Fig. S1E). One month later, the patient stopped taking CoQ10, but after 3 weeks, his pain worsened, his muscle strength decreased, and he was unable to lift his arms, indicating that the symptoms were partially dependent on CoQ10. After 4 months, the patient resumed CoQ10 at a reduced dose (35 mg per day), but his muscle strength continued to diminish and the EMG results were unchanged, so the dose of CoQ10 was increased (Fig. S1E). As the C4, C6, C8 and C10 acyl-carnitines were still elevated (Table S1, time point 5), and riboflavin and carnitine supplementation were without effect on the clinical course, both riboflavin and carnitine were stopped after ~18 months.
The patient's clinical symptoms progressively worsened, so that 3 years after he first presented to us he could only walk 50 m with the aid of crutches or a walker and within 1 year this was reduced to 20 m and he could no longer rise from the sitting position. Nerve conduction studies performed at this time revealed a mild axonal polyneuropathy. One year later, walking distance was reduced to 10 m and non-invasive ventilation was initiated because of global respiratory failure. Measurement of testosterone in plasma at this time revealed low levels of both total (patient 5.44; range 9.08-55.23 nmol/L) and free (18.1; range 25.0-80.0 nmol/L) testosterone. The levels of LSH (<0.5; range 2.0-12.0 units/L), FSH (<0.4; range 1.0-8.0 units/L), and cortisol (91; normal range 125-667nmol/L) were also reduced whilst that of DHEAS was increased (11.9; range 1.9-8.41 units/L). A diagnosis of primary partial pituitary failure, involving the gonadotropic, corticotropic, and somatotropic axes was made.
The patient reported an improvement in his feeling of well-being. Despite these interventions, eight years after presentation, the patient could not stand freely, required a wheel chair, and help with all activities of daily living. A muscle MRI performed 2 years later revealed severe fatty degeneration of the shoulder girdle, deltoid, biceps, hamstring and calf muscles, whilst the triceps were well preserved ( Fig. S1B). A biopsy of the triceps revealed some cox-negative and ragged red-fibres, but no lipid storage. Paracrystalline inclusions were observed in approximately one quarter of the mitochondria (not shown). Treatment with ubiquinol, 150 mg/per day (GeriMed, GmBH), was initiated 10 years after presentation, and was associated with stabilization of the plasma levels of lactate, CK, myoglobin, and LDH, which returned to normal or near normal values. The levels of C8 and C10 acyl-carnitines in the plasma remained moderately elevated. Twelve years after he presented, the patient died of bolus aspiration at the age of 57 due to newly developed dysphagia.
As the muscle biopsy findings were compatible with a mitochondrial disorder, we measured the activity of the mitochondrial respiratory chain complexes in muscle homogenate from the patient.
The activities of complexes I, III, and IV compared to the activity of citrate synthase, a mitochondrial marker, were within the control range, whilst the combined activities of complexes I and III, which requires the participation of endogenous CoQ, was reduced ( Fig. 1H), indicative of a CoQ deficiency. The combined activities of complex II+III were also reduced in cultured fibroblasts from both the index patient (ADCK2-II-3 in Fig. 1I) and his asymptomatic sister, II-2 ( Fig. 1I).
Accordingly, CoQ levels were significantly reduced in cultured fibroblasts from the index patient and subject II-2 compared to control fibroblast cultures ( Fig 1E). Sequencing of the entire mitochondrial genome in the patient did not reveal any pathogenic mutations (not shown).
Plasma lactate levels were elevated in the index patient, but normal in subject II-2. We next investigated lactate production in cultured fibroblasts from both individuals. Fibroblasts (25 x 10 3 cells/mL) from controls and subjects II-3 and II-2 were seeded and grown for 6 days in glucose-rich medium, and then the lactate concentration was measured in the culture medium. Lactate production was elevated in cultured fibroblast from both the index patient and his sister compared with that in control fibroblasts (control: 0.050±0.003 nmol/cell; II-2: 0.350±0.005 nmol/cell; II-3: 0.290±0.009 nmol/cell; p<0.002; n=4).

A truncating mutation in ADCK2 identified in the patient and subject II-2.
As the clinical findings were strongly indicative of MADD, we sequenced ETFDH, ETFA, and EDFB in DNA from the index patient, II-3. No mutations were identified in any of these genes.
Next, targeted sequencing of the genes known or predicted to be involved in CoQ biosynthesis was other family members revealed that the patient's clinically unaffected mother, subject I-1, and sister, subject II-2, were heterozygous for this sequence change, whilst the other sibling, subject II-1, does not harbor this change (Figs. S1C and S1D). These results were confirmed by mitochondrial panel sequencing and no other sequence changes were detected upon sequencing of the cDNA obtained from the index patient.
The c.997C>T mutation resides 83 nucleotides upstream of an exon junction in exon 2 of the 8 exon gene, and thus would be predicted to lead to the rapid decay of the affected mRNA. Extraction of RNA from blood and cultured fibroblasts from the index patient and individual II-2, followed by reverse transcription and subsequent sequencing of the cDNA, revealed decreased abundance of mRNA harboring the premature translation-termination codon arising from the c.997C>T mutation, confirming that this mRNA species undergoes non-sense mediated decay in these tissues (Fig. S1C).
Further evidence for this is provided by the finding that the amount of ADCK2 mRNA (Fig. 1B) and protein (Figs. 1C and 1D) is significantly decreased in cultured fibroblasts from the index patient and individual II-2. To understand the role of this mutation in the level of CoQ in patient' fibroblasts, we transformed patient's cells with the plasmid pRRL harboring the WT allele of ADCK2. Figures 1F and 1G shows that these cells increased both mRNA and CoQ content.

Supplementary Table 1. Acyl-carnitine analysis in plasma from the index patient II-3.
The values shown are the fold increase of the measured value over the 99-percentile value for adults. The numbers shown in the upper column (4-17) refer to the time points indicated in panel C of Figure S1E. Bold type represents significant differences.   Quantitative real-time PCR (qPCR) of selected genes whose expression was affected by the Adck2 haploinsufficiency. Values represent the fold change in gene expression of both liver and muscle genes in Adck2+/-mice relative to wild type animals. Not detected (n.d.)

Supplementary figures
Supplementary Figure 1related to Fig.1. Laboratory findings in the index patient. A. Pedigree of the index patient, subject II-3. B. ADCK2 deficiency is associated with selective muscular fatty degeneration in the index patient. TI-weighted skeletal muscle MRI of the right arm showing pronounced fatty degeneration of the biceps, but not the triceps; the right deltoid showing peripheral sparing and central involvement; the thighs, showing preferential degeneration of the hamstring muscles, the lower legs, showing preferential degeneration of the soleus and the medial head of the gastrocnemius. C. Sequencing chromatograms showing the heterozygous c.997C>T sequence change in i) blood from the index patient II-3, ii) his mother I-1, iv) his sister, II-2, and iii) wild type sequence in blood from his sister II-1. D. Sequence chromatograms obtained following reverse transcription of RNA isolated from blood (i-iii) and fibroblasts (iv-vi) of family members and subsequent sequencing of the cDNA. Note that only a small peak of c.997C>T is apparent in mRNA isolated from blood and fibroblasts of II-3 (ii and iv) and II-2 (iii and vi) indicating that the allele harboring this mutation has undergone nonsense-mediated decay. Only wild type sequence was Figure S1 A B C D E visible in cDNA from control blood (i) and fibroblasts of   Figure 1C.  . 3. ADCK2 protein is located inside mitochondria. A. Coimmunolocalization of cytochrome c and endogenous ADCK2 protein in HEK293 cells by confocal microscopy. B. Subcellular fractions from HEK293 cells were immunoblotted for ADCK2, lactate dehydrogenase (LDH), calnexin, TOM20, and mitofusin-2. Cyt, cytosol; ER, endoplasmic reticulum; MAM, mitochondria-associated membranes; Mito, mitochondria. Note the enrichment of ADCK2 in MAM and mitochondrial fractions. C. Mitochondrial-enriched fractions from HEK293 cells were treated with proteinase K combined with swelling and Triton X-100. Immunoblotting was then performed with the indicated primary antibodies. TOM20, outer membrane marker; calnexin and OPA1, intermembrane space markers; ornithine aminotransferase (OAT), matrix marker.  Table 1).

Figure S6
Repressed Ac vated A B C