Therapeutics J Med Genet: first published as 10.1136/jmedgenet-2015-102986 on 17 June 2015. Downloaded from SHORT REPORT Rescue of primary ubiquinone deficiency due to a novel COQ7 defect using 2,4–dihydroxybensoic acid Christoph Freyer,1,2 Henrik Stranneheim,1,3 Karin Naess,1,4 Arnaud Mourier,5 Andrea Felser,4 Camilla Maffezzini,2 Nicole Lesko,1,4 Helene Bruhn,1,4 Martin Engvall,1,6 Rolf Wibom,1,4 Michela Barbaro,1,6 Yvonne Hinze,5 Måns Magnusson,3 Robin Andeer,3 Rolf H Zetterström,1,6 Ulrika von Döbeln,1,4 Anna Wredenberg,1,2 Anna Wedell1,2,3 ▸ Additional material is ABSTRACT other mitochondrial dehydrogenases, and functions in published online only. To view Background Coenzyme Q is an essential mitochondrial lysosomes, endoplasmic reticulum and plasma mem- please visit the journal online electron carrier, redox cofactor and a potent antioxidant branes. Furthermore, CoQ is the only lipid-soluble (http://dx.doi.org/10.1136/ 2 jmedgenet-2015-102986). in the majority of cellular membranes. Coenzyme Q antioxidant synthesised entirely by animal cells. deficiency has been associated with a range of CoQ levels naturally diminish with age,2 but For numbered affiliations see 10 end of article. metabolic diseases, as well as with some drug can also be decreased due to treatments with, for treatments and ageing. example, statins due to a shared biosynthesis 3 Correspondence to Methods We used whole exome sequencing (WES) to pathway with cholesterol. Additionally, CoQ10 Dr Anna Wredenberg, Division investigate patients with inherited metabolic diseases deficiencies (MIM 607426, 614651, 614652, for Metabolic Diseases, and applied a novel ultra-pressure liquid chromatography 612016, 614654, 614650) have been reported in a Department of Laboratory — Medicine, Karolinska Institutet, mass spectrometry approach to measure coenzyme Q range of patients with inborn errors of metabol- 4 Stockholm, Sweden; in patient samples. ism, often identified by severe mitochondrial dys- [email protected], and Results We identified a homozygous missense function with combined deficiencies of the Prof. Anna Wedell, mutation in the COQ7 gene in a patient with complex respiratory chain complexes. They can be caused Department of Molecular fi Medicine and Surgery, mitochondrial de ciency, resulting in severely reduced by either a primary defect in the CoQ10 biosyn- Karolinska Institutet, coenzyme Q levels We demonstrate that the coenzyme Q thetic pathway or as a secondary phenomenon in a Stockholm, Sweden; analogue 2,4-dihydroxybensoic acid (2,4DHB) was able range of dysfunctions for yet unclear reasons. [email protected] to specifically bypass the COQ7 deficiency, increase Despite its central role in aerobic respiration, the cellular coenzyme Q levels and rescue the biochemical biosynthesis of CoQ is not fully understood. Based CF, HS and KN contributed fi equally to this work. defect in patient broblasts. on studies in yeast, the products of at least nine Conclusion We report the first patient with primary genes, Coq1–Coq9, are known to be involved in Received 15 January 2015 coenzyme Q deficiency due to a homozygous COQ7 CoQ synthesis, which is initiated by the condensa- http://jmg.bmj.com/ Revised 28 April 2015 mutation and a potentially beneficial treatment using tion of the isoprenoid tail with the benzoquinone Accepted 26 May 2015 5 Published Online First 2,4DHB. ring precursor. A series of benzoquinone ring 17 June 2015 modifications, including C-hydroxylations, decar- boxylations, O-methylations and C-methylations, INTRODUCTION subsequently result in CoQ10 formation. For some Coenzyme Q (CoQ), also known as ubiquinone, is a of the corresponding proteins (COQ1, COQ2, small fat-soluble redox cofactor that has been exten- COQ6, COQ3, COQ5, COQ7), the biochemical on September 30, 2021 by guest. Protected copyright. sively studied for its essential role as a mitochondrial functions are understood, whereas for others, the respiratory chain electron carrier. It consists of a mechanisms behind their involvement in the redox-active benzoquinone ring and a polyisoprene pathway remain unclear (COQ4, COQ8, COQ9). tail. The tail is believed to be important for diffusion Mutations in PDSS1, PDSS2, COQ2, COQ4, in the lipid bilayer, as well as for interactions with ubi- COQ6, COQ9, ADCK3 and ADCK4 are known quinone redox enzymes,1 and comprises 6 units in causes of primary CoQ deficiencies in humans.3 Open Access Scan to access more yeast (CoQ6), 9 units in rodents (CoQ9) and 10 in PDSS1 and PDSS2 form a heterotetramer, perform- free content humans (CoQ10). Aerobic respiration requires that ing the analogous function to Coq1 in yeast, electrons from energy-rich intermediates such as whereas ADCK3 is the mammalian Coq8 counter- NADH or FADH2 be passed to molecular oxygen, via part. ADCK4 is a paralog of ADCK3 and has been the respiratory chain in the inner mitochondrial mem- shown to interact with members of the CoQ bio- brane. CoQ is essential in this process, shuttling elec- synthesis pathway, including COQ5,6 COQ6 and trons from NADH dehydrogenase (complex I) or COQ7.7 COQ7 has further been shown to interact succinate dehydrogenase (complex II) to the cyto- with COQ9,8 suggesting the presence of a large chrome bc1 complex (complex III). Although pre- CoQ10 biosynthesis complex. dominantly located in the inner mitochondrial To cite: Freyer C, membrane, CoQ is present in almost all cellular mem- MATERIALS AND METHODS Stranneheim H, Naess K, branes in aerobic organisms.2 In addition to its role as Biochemistry et al. J Med Genet an electron shuttle during aerobic respiration, CoQ is Mitochondrial ATP production rates (MAPRs) and – 2015;52:779 783. also a cofactor of various uncoupling proteins and respiratory chain enzyme activities in skeletal Freyer C, et al. J Med Genet 2015;52:779–783. doi:10.1136/jmedgenet-2015-102986 779 Therapeutics J Med Genet: first published as 10.1136/jmedgenet-2015-102986 on 17 June 2015. Downloaded from muscle and mitochondrial oxygen consumption of fibroblasts albumin to 652 mg/L (ref <225). Urine analysis revealed moder- were determined as described in online supplementary ately increased excretion of fumarate and malate. A muscle information. biopsy was performed at 2 years 3 months to measure MAPR and respiratory chain enzyme activities (see online Next generation sequencing, MIP analysis and Sanger supplementary material and methods), revealing a combined sequencing complex I+III and IV deficiency (figure 1A, B). Histological Whole exome sequencing was performed on DNA from the examination of the biopsy showed small fibre size indicating patient and parents’ fibroblasts, using Illumina technology. neurogenic damage but no obvious myopathic changes. Electron Following mutation identification pipeline (MIP) analysis, among microscopy revealed no obvious abnormalities of mitochondria the top 20 variants only the c.422T>A transition in COQ7 or other structures. Sanger sequencing of the complete mtDNA assumed homozygosity and a mitochondrial association. Sanger genome and the POLG gene revealed no pathogenic mutations. sequencing was performed on genomic DNA extracted from We performed whole exome sequencing on genomic DNA blood from the patient, unaffected sibling and both parents (for samples from the patient and his parents, followed by in-house details see ref. 9 and online supplementary information). computational analysis, using the MIP910(see online supple- mentary information). After MIP analysis, only three candidate Measuring absolute ubiquinone levels genes assumed an autosomal recessive inheritance of potentially Ubiquinone quantification by ultra-pressure liquid chromatog- disease-causing variants and only a single candidate gene was raphy (UPLC)—tandem quadrupole mass spectrometry analysis associated with mitochondria. Other candidates were dismissed was performed as described in online supplementary information. for failure to show an appropriate inheritance model. Sanger sequencing confirmed (see online supplementary material and RESULTS methods) homozygosity for a thymidine to adenosine transver- In this study, we report the first case of COQ7 deficiency. The sion at nucleotide 422 of COQ7 (MIM 601683; NM_016138: affected boy was born in 2005 as the second child of consan- exon4:c.422T>A:p.Val141Glu) in the patient and heterozygos- guineous Syrian parents, with an older healthy sister born in ity in both parents and the unaffected sibling (figure 1C). The 2002. Pregnancy was complicated by oligohydramniosis, fetal variant was not present in any of the public databases or in our lung hypoplasia and growth retardation. The boy was born full in-house database containing data from 156 individuals. term but small for gestational age (birth weight 2070 g, length COQ7 is a di-iron oxidase responsible for the penultimate 46 cm, head circumference 30.5 cm). At birth, he had muscular step of CoQ synthesis, hydroxylating 5-demethoxyubiquinol hypotonia, contractures of the extremities and respiratory dis- (DMQH2) in the presence of NADH.11 12 The highly con- tress with persistent pulmonary hypertension of the newborn. served glutamic acid p.Glu142 residue is predicted to be part of His Apgar score was 1,5,7. Lung hypoplasia was confirmed and the di-iron motif,13 and with p.Val141 being conserved in renal dysfunction was diagnosed, with plasma creatinine ele- eukaryotes (figure 1D), it is highly likely that the p.Val141Glu vated up to 196 mmol/L (ref <100) on day 2. Ultrasound mutation affects COQ7 function and impairs iron binding. revealed small dysplastic kidneys with impaired cortical differen- To identify whether CoQ levels are affected in the patient, we tiation. Secondary, there was systemic hypertension and left ven- developed a novel method to analyse CoQ10 levels in isolated tricular cardiac hypertrophy. Plasma creatinine normalised mitochondria, using UPLC—tandem quadrupole mass spectrom- during the first week. Blood pressure normalised after etry analysis (see online supplementary material and methods). In http://jmg.bmj.com/ 3 months, the cardiac hypertrophy regressed and pulmonary our analysis, this method was highly sensitive to even low levels of function normalised within the first 8 months.