Characterization of Coq10 Biosynthesis in Fibroblasts of Patients with Primary and Secondary Coq10 Deficiency

J Inherit Metab Dis (2014) 37:53–62 DOI 10.1007/s10545-013-9620-4

ORIGINAL ARTICLE

Characterization of CoQ10 biosynthesis in fibroblasts of patients with primary and secondary CoQ10 deficiency

Nuria Buján & Angela Arias & Raquel Montero & Judit García-Villoria & Willy Lissens & Sara Seneca & Carmen Espinós & Plácido Navas & Linda De Meirleir & Rafael Artuch & Paz Briones & Antonia Ribes

Received: 2 October 2012 /Revised: 7 May 2013 /Accepted: 13 May 2013 /Published online: 18 June 2013 # SSIEM and Springer Science+Business Media Dordrecht 2013

Abstract Primary coenzyme Q10 (CoQ10) deficiencies are labelled CoQ10 were analysed by high performance liquid associated with mutations in genes encoding enzymes im- chromatography-tandem mass spectrometry. The mean and 13 2 portant for its biosynthesis and patients are responsive to the reference range for C6-CoQ10 and H3-CoQ10 biosyn- CoQ10 supplementation. Early treatment allows better prog- thesis were 0.97 (0.83–1.1) and 0.13 (0.09–0.17) nmol/Unit of nosis of the disease and therefore, early diagnosis is desir- citrate synthase, respectively. We validated the methodology able. The complex phenotype and genotype and the frequent through the study of one patient with COQ2 mutations and six secondary CoQ10 deficiencies make it difficult to achieve a patients with CoQ10 deficiency secondary to other inborn definitive diagnosis by direct quantification of CoQ10.We errors of metabolism. Afterwards we investigated 16 patients’ developed a non-radioactive methodology for the quantifica- fibroblasts and nine showed decreased CoQ10 biosynthesis. tion of CoQ10 biosynthesis in fibroblasts that allows the iden- Therefore, the next step is to study the COQ genes in order to tification of primary deficiencies. Fibroblasts were incubated reach a definitive diagnosis in these nine patients. In the 2 13 72 h with 28 μmol/L H3-mevalonate or 1.65 mmol/L C6-p- patients with normal rates the deficiency is probably second- 2 13 hydroxybenzoate. The newly synthesized H3-and C6- ary. In conclusion, we have developed a non-invasive non- radioactive method suitable for the detection of defects in Communicated by: Piero Rinaldo CoQ10 biosynthesis, which offers a good tool for the stratification of patients with these treatable mitochondrial diseases. Nuria Buján and Angela Arias contributed equally to this work. N. Buján : A. Arias : J. García-Villoria : A. Ribes (*) ’ Secció d Errors Congènits del Metabolisme-IBC, Servei de Introduction Bioquímica i Genètica Molecular, Hospital Clínic, CIBERER, Edifici Helios III, planta baixa, C/Mejía Lequerica s/n, 08028 Barcelona, Spain Coenzyme Q10 (CoQ10) is a lipophilic molecule critical for e-mail: [email protected] the transport of electrons from complex I and complex II (and also from the β-oxidation pathway via the electron R. Montero : R. Artuch Servei de Bioquímica, Hospital Sant Joan de Déu, CIBERER, transfer flavoprotein, ETF) to complex III in the mitochon- Barcelona, Spain drial respiratory chain (RC) (Festenstein et al 1955; Crane et : : al 1957; Frerman 1987). It also participates in extra- W. Lissens S. Seneca L. De Meirleir mitochondrial electron transport and functions as an antiox- UZ Brussel, Vrije Universiteit, Brussel, Belgium idant in cell membranes preventing lipid, protein and DNA

C. Espinós oxidation. Moreover, CoQ10 is involved in the regulation of Instituto de Biomedicina de Valencia, CSIC, CIBERER, mitochondrial uncoupling proteins and mitochondrial per- Valencia, Spain meability transition pore; it is also required for pyrimidine P. Navas nucleoside biosynthesis and may modulate apoptosis Universidad Pablo de Olavide-CSIC, CIBERER, Sevilla, Spain (Turunen et al 2004).

In humans CoQ10 is synthesized in cells and tissues and P. Briones no uptake is usually required; 2–4 % of the dietary CoQ is Secció d’Errors Congènits del Metabolisme-IBC, 10 Servei de Bioquímica i Genètica Molecular, Hospital Clínic, recovered in the circulation, but its transfer to the organs CIBERER, CSIC, Barcelona, Spain seems very limited (Turunen et al 2004). 54 J Inherit Metab Dis (2014) 37:53–62

CoQ10 is composed of a benzoquinone ring derived from RC complexes I+III and II+III, and low levels of CoQ10 tyrosine and a decaprenyl side-chain coming from the (Rahman et al 2012; Ogasahara et al 1989; Rötig et al 2000; mevalonate (MV) pathway after successive additions of Salviati et al 2005; Horvath et al 2006; Quinzii et al 2007; isopentenyl-diphosphate (IPP) molecules to farnesyl- Rustin et al 2004) in muscle or fibroblasts. The diversity of diphosphate (FPP) catalyzed by prenyl-diphosphate synthase symptoms along with the large number of genes involved in

(COQ1) (Fig. 1) (Dallner and Sindelar 2000). Decaprenyl the synthetic pathway and the frequent secondary CoQ10 diphosphate (DPP) and p-hydroxybenzoate (PHB) are con- deficiencies make it difficult to achieve a definitive diagnosis. densed by PHB-polyprenyltransferase (COQ2), and further CoQ10 deficiencies are primary when due to mutations in modified by at least six enzymes catalyzing methylation, de- genes involved in CoQ10 biosynthesis (COQ genes), where carboxylation, and hydroxylation reactions to synthesize the even haploinsufficiency for the COQ4 gene has been de- final CoQ10 molecule. The MV pathway comprises the reac- scribed to cause CoQ deficiency (Salviati et al 2012). It can tions from acetyl-coenzyme A (acetyl-CoA) to FPP, which is also be secondary to genes not directly involved in it, such as precursor for CoQ10, cholesterol, dolichol and isoprenylated APTX (aprataxin) (Quinzii et al 2005), ETFDH (electron- proteins (Turunen et al 2004; Dallner and Sindelar 2000). transferring-flavoprotein dehydrogenase) (Gempel et al

Primary CoQ10 deficiencies are described as genetic dis- 2007; Liang et al 2009)orBRAF (Aeby et al 2007). orders with good response to supplementation with CoQ10. Secondary deficiencies have also been reported in patients Early treatment based on early diagnosis is critical to max- with mitochondrial DNA (mtDNA) mutations or deletions imize the efficacy of ubiquinone supplementation (López et (Rahman et al 2012; Sacconi et al 2010;Matsuokaet al 2010). These mitochondrial disorders are rare condi- al 1991), and some specific genetic factors may confer tions that have been reported in individuals with various susceptibility to develop secondary CoQ10 deficiency clinical phenotypes showing decreased activities of the (Sacconi et al 2010).

Acetyl-CoA

3-HMG-CoA HMG-CoA reductase

MEVALONATE

Mevalonate kinase Mevalonate-P Phosphomevalonate kinase Mevalonate-PP Tyrosine Mevalonate-5-pyrophosphate decarboxylase Isopentenyl-PP Dimethylallyl-PP IPP-isomerase GPP-synthase; COQ1 Geranyl-PP

FPP-synthase

4-OH-BENZOATE (PHB) Decaprenyl-PP Farnesyl-PP Prenyldiphosphate synthase PDSS1-PDSS2 4-OH benzoate prenyltransferase(COQ2)

Decaprenyl-4-OH-benzoate

COQ4, unknown Dolichol COQ3, methyltransferase Cholesterol COQ6, mono-oxygenase

COQ5, C- methyltransferase

COQ7, mono-oxygenase

COQ8, CABC1, ADCK3, protein kinase

COQ9, unknown

COQ10, CoQbinding protein

CoQ10

Fig. 1 Biosynthetic pathway of CoQ10. Modified from Dallner and Sindelar (Dallner and Sindelar 2000) J Inherit Metab Dis (2014) 37:53–62 55

Skeletal muscle is accepted as the tissue of choice for protein and citrate synthase (CS) activity. For CoQ10 deter- CoQ10 evaluation, but obtaining a muscle biopsy is invasive. mination, 10 μL of CoQ9 (1 μM, as internal standard, IS), Less invasive procedures such as obtaining lymphoblastoid and 800_μL of methanol were added to 100 μL of homog- cell lines, fibroblasts, or lymphocytes have been used for the enate. The results were expressed in nmol CoQ10/g protein diagnosis of CoQ10 deficiency (Rahman et al 2012;Montero or nmol CoQ10/Unit of citrate syntase (UCS). et al 2008; Arias et al 2012). For these reasons, our objective was to develop a meth- Viability test odology for the study of the endogenous biosynthesis of

CoQ10 in fibroblasts that may allow the identification of Viability tests were performed after 24, 48 and 72 h incu- 13 2 primary CoQ10 deficiencies. bation with C6-PHB or H3-MV at the concentrations above mentioned. Cells were washed with PBS and were incubated 3 h with 100 μL of MTT solution (0.5 mg/mL Materials and methods MTT in PBS). The purple MTT-formazan products were dissolved in dimethyl sulfoxide and optical densities of the Reagents solutions were measured by absorbance at 570 nm in an ELISA plate reader. Cells treated with 0.02 % SDS were 13 2 C6-PHB, H3-MV, non-labelled PHB, non-labelled MV, used as positive control. Untreated cells correspond to the cyclodextrine, 5,5′-ditio-bis[−2-nitrobenzoic acid] (DTNB), negative control. Cell viability was expressed as the optical oxaloacetate, tris(hydroxymethyl)aminomethane (Tris), density ratio of the treated cells respect to the negative saccharose, EDTA, coenzyme Q9 (CoQ9), CoQ10, thiazolyl control (% of control). Experiments were performed in blue tetrazolium bromide (MTT), sodium dodecyl sulphate triplicate. (SDS), dimethyl sulfoxide, ammonium bicarbonate and cy- cloheximide (CHX) were provided from Sigma-Aldrich HPLC-MS/MS analysis (Madrid, Spain). 13 2 Trypsin was from Thermo Scientific (Germany). Dulbecco’s CoQ10 and C6-CoQ10 or H3–CoQ10 (the two forms of modified Eagle’s medium (DMEM), fetal bovine serum (FBS), CoQ10 synthesized depending on whether the substrate is 13 2 penicillin (10,000 units/mL) and streptomycin (10,000 μg/mL) C6-PHB or H3-MV, respectively) were measured by were from PAA (Pasching, Austria). HPLC-MS/MS, as described in Arias et al (2012). The DNA and RNA extraction kits, QIAshredder and RNeasy HPLC (Alliance HT 2795, Waters) was equipped with a respectively, were from Qiagen (Germany). 2.1×50 mm Symmetry C18 HPLC column (3.5 μm particle All other solvents and chemicals were of analytical or size). The mobile phase consisted of 50 % methanol with liquid chromatography grade and were obtained from a 5 mM methylamine, 45 % 2-propanol and 5 % water acid- variety of sources. ified with formic acid (0.5 mL/L), at a flow rate of 0.2 mL/min and isocratic conditions. MS/MS analysis was performed in a

Biosynthesis of labelled CoQ10 in cultured fibroblasts Micromass Quattro micro™ (Waters/Micromass, Manchester, UK). The MS/MS was operated in the electrospray positive ion Our method was based on the previously reported method mode with CV and CE of 15 V and 20 eV respectively. The for radiolabelled substrates (Tekle et al 2008). Skin fibro- following multiple reaction monitoring (MRM) transitions 13 blasts were grown in DMEM containing 10 % FBS and 1 % were selected: m/z 900>203 and 897>197 for C6-CoQ10 or 2 penicillin-streptomycin. After culture, cells were rinsed with H3–CoQ10 respectively, 894>197 for the physiological CoQ10 phosphate buffered saline (PBS), trypsinized, centrifuged and 826>197 for CoQ9 (internal standard). Dwell time for each for 10 min at 252×g and cultured again in 6 well plates. transition was 200 ms and run-time was 16 min. Nitrogen (at At 60–70 % confluence the medium was changed for me- flow rate of 50 L/h) and argon (adjusted to obtain a vacuum of 13 2 −3 dium containing C6-PHB or H3-MV, and incubated for 3×10 bar) were used as nebulising and collision gas, 13 24, 48 or 72 h. C6-PHB was tested at 1.65 mmol/L and respectively. 2 3.3 mmol/L and H3-MV at 14 μmol/L, 28 μmol/L, The physiological content of CoQ10 for some fibroblast 42 μmol/L, 56 μmol/L, 112 μmol/L, 140 μmol/L and samples and for muscle tissue was determined by HPLC 280 μmol/L. After incubation, cells were trypsinized and with electrochemical detection as previously described washed twice with saline. Pelleted-cells were resuspended (Montero et al 2008). with 300μL of a buffer solution containing 0.25 mmol/L Intra-assay precision (CV) was evaluated in six parallel sucrose, 2 mmol/L EDTA, 10 mmol/L Tris and 100 UI/mL analyses of the same cell culture. To establish the inter-assay heparin, pH 7,4, and sonicated twice for 5 s. These homog- variability, one cell line was independently analysed on six enates were used to determine CoQ10 biosynthesis, total different days. 56 J Inherit Metab Dis (2014) 37:53–62

Citrate synthase and protein determinations muscle in some cases) but still with no definite genetic diagnosis: Group 1: three patients with a single mutation in one

CS activity was measured spectrophotometrically according COQ gene; Group 2: five patients responsive to CoQ10 sup- to the method described by Srere (1969), with 0.1 mM plementation, without mutations in the genes studied; Group

DTNB, 0.2 % Triton X100 and 30–50 μg protein in 3: eight patients with CoQ10 deficiency, without further ge- 500 μL total incubation volume. Proteins were quantified netic studies or documented response to treatment. All cell using Protein Assay kit (Bio-Rad Laboratories, EEUU) lines were analysed in two independent experiments. based on the Lowry method. Patients or parents provided informed consent. The study was approved by the Ethics Committee of the Hospital Clinic- Subjects Barcelona, Spain. All samples were obtained in accordance with the current revision of the Helsinki Declaration. Thirteen control fibroblast cell lines from the repository bank of our hospital were analysed to establish the reference values. Statistical analysis In order to validate the methodology, we studied seven patients with a definite diagnosis and CoQ10 deficiency in fibro- Statistical analysis was performed using the SPSS version blasts, including one patient homozygous for a COQ2 18.0.0 software. Kolmogorov-Smirnov test was used to (OMIM*609825) mutation (unpublished results) and six pa- check variables which were under a normal distribution. tients with other inborn errors of metabolism: multiple Acyl- The reference range was calculated as the mean±2 standard

CoA dehydrogenase deficiency (MADD; OMIM#231680), deviations. Pearson test was applied to correlate CoQ10 very long chain Acyl-CoA dehydrogenase deficiency biosynthesis between both substrates. (VLCADD; OMIM#201475), mitochondrial encephalopathy with complex III deficiency and a mtDNA mutation, Genetic studies and Niemann-Pick type C disease (NPC; OMIM#257220) (Table 1). Then, we investigated three further groups of pa- Prior to the introduction of the present methodology to tients (Table 2)withCoQ10 deficiency in fibroblasts (and in evaluate CoQ10 biosynthesis, incomplete studies of some

Table 1 CoQ10 biosynthesis in patients with definite diagnosis and CoQ10 deficiency in fibroblasts

a Patient Diagnosis Mutation Protein change CoQ10 concentration (nmol/UCS) Biosynthesis of CoQ10

2 13 Fibroblasts Muscle H3-MV C6-PHB

1 COQ2 c.437G>A and p.Ser146Asn and 0.4 ND 0.04 0.29 c.437G>A p.Ser146Asn b 2 MADD-ETFB c.124T>C and p.Cys42Arg and 1.2 ND 0.17 0.87 c.604_606delAAG p.Lys202del 3 MADD-ETFDH c.779T>C and p.Phe260Ser and 1.9 ND 0.13 0.96 c.41_42ins14pb p.Gln14Hisfs*11 4 VLCAD c.848T>C and p.Val283Ala and 1.9 ND 0.18 0.89 c.1748C>T p.Ser583Leu 5 Complex III m.3229A ins 1.7 1.8 0.17 0.91 deficiency 6 Niemann-Pick c.2932C>T and p.Arg978Cys and 1.3 ND 0.13 0.54 Type C c.983T>C p.Phe995Leuc 7 Niemann-Pick c.2746_2748delAAT p.Asn916del and 1.5 ND 0.10 0.60 Type C and c.3451G>A p.Ala1151Thr c Control mean 2.4 (2.0–2.8) 5.4 (2.7–8.5) 0.13 (0.09–0.17) 0.94 (0.84–1.0) (mean±2SD) n=number n=66 n=37 n=13 n=13 of controls

Altered results are outlined in bold. Each individual value is the mean of at least a duplicate determination ND not done a 2 13 Biosynthesis of CoQ10 was evaluated by measuring the corresponding labelled CoQ10 (nmol/UCS) generated both with H3-MV or C6-PHB as substrates b Mutation previously described by Diomedi-Camassei et al (2007) c Macías-Vidal et al (2011) J Inherit Metab Dis (2014) 37:53–62 57

13 COQ genes had been performed in some of the patients as 1.65 mmol/L C6-PHB when the incubation time was described beneath. The continuation of those studies was 24 h or 48 h. And, although the viability decreased slightly 13 conditioned to the demonstration of an altered biosynthesis (80 % residual) after 72 h incubation, the peak of C6- in the patients. CoQ10 was threefold the LLOQ (S/N>10) (data not shown). 13 Genomic DNA was extracted from blood, skin fibro- When incubating with C6-PHB, inter-assay variability 13 blasts or tissues using standard protocols, and mutational (CV) of the newly synthesized C6-CoQ10 was 19 % if screening of 13 COQ genes (PDSS1 (OMIM*607429), results were normalized to protein content and 10 % if 2 PDSS2 (OMIM*610564), COQ2 (OMIM*609825), COQ3 normalized to UCS. When incubating with H3-MV, inter- 2 (OMIM*605196), COQ4 (OMIM*612898), COQ5, COQ6 assay CV of the newly synthesized H3-CoQ10 was 16 % (OMIM*614647), COQ8 (OMIM*606980), COQ9 (OMIM: and 13 % related to protein and CS, respectively. 13 *612837), ADCK1, ADCK2, ADCK4 and ADCK5 was Concerning intra-assay CV, when incubating with C6- performed using self-designed oligonucleotides. PHB it was 10 % when expressed per g protein and 9 % if 2 Total RNA was isolated from cultured fibroblasts using expressed per UCS; and, when incubating with H3-MV, it QIAshredder and RNeasy kits and cDNA was synthesized was 12 % and 8 % per g protein and UCS, respectively. Due using standard protocols. We also isolated RNA from pa- to the lower imprecision when normalizing the results to tients’ fibroblasts that had been treated during 7 h with UCS we decided to use it instead of per protein content. 13 2 500 μg/mL CHX, in order to inhibit possible mRNA deg- The mean and reference range for C6-CoQ10 and H3- radation by nonsense-mediated decay (NMD). Overlapping CoQ10 biosynthesis were 0.94 (0.84–1.0) nmol/UCS and 0.13 segments of the COQ8, PDSS1, PDSS2 and COQ4 cDNAs (0.09–0.17) nmol/UCS, respectively (Table 1). The correla- 13 2 were PCR amplified and sequenced. tion between C6-CoQ10 and H3-CoQ10 biosynthesis in Patients from group 1 (Table 2)werestudiedforthefollow- fibroblasts, in the whole group of controls and patients, ing genes: PDSS1, PDSS2 and, COQ2-COQ9 in patient 8, and showed that the two variables tend to increase together and PDSS1, PDSS2, COQ2, COQ4, COQ5, COQ8 and COQ9 in 63 % of the variance was shared between them (Fig. 3c). patient 9. In addition, cDNA mutational screening was performed Patients studied to validate the methodology are summa- for the mentioned four genes in patients 9 and 10. rized in Table 1. Patient 1 shows significant CoQ10 deficien- Concerning patients of group 2, the 13 mentioned genes cy in fibroblasts is homozygous for a mutation in COQ2 and were screened in their genomic DNAs. presents deficient CoQ10 biosynthesis with both substrates 13 2 ( C6-PHB and H3-MV). In contrast, patients 2–5, with CoQ10 deficiency in fibroblasts and diagnosis of other in- Results born errors of metabolism, showed normal biosynthesis. Fibroblasts from patients 6 and 7, affected with NPC,

Method setting up and validation showed deficient CoQ10 and normal biosynthesis with 2 H3-MV as precursor, while the rate was decreased using 13 CoQ10 biosynthesis in control fibroblasts (Fig. 2a) increased C6-PHB as substrate (Table 1). linearly with time (24–72 h) using either 1.65 mmol/L or 13 3.3 mmol/L C6-PHB as precursor, and the synthesized Patients’ results 13 C6-CoQ10 amounts were alike at both concentrations. 2 2 When using H3-MV as precursor, H3-CoQ10 biosynthesis The investigations in patients suspected of primary CoQ10 2 was also linear with time but increased with increasing H3- deficiency are summarized in Table 2, Fig. 3a and b.In MV from 14 to 56 μmol/L (Fig. 2b). For higher concentra- group 1 only patient 8 (with a mutation in COQ4) showed tions (112 μmol/L, 140 μmol/L and 280 μmol/L) during deficient CoQ10 biosynthesis. Table 2 also shows the 72 h incubation, CoQ10 biosynthesis decreased (Fig. 2c). results for patient 8’s parents; her father (number 25) gave After those results, the elected conditions were 1.65 mmol/L normal results for both substrates, while her mother’s 13 2 C6-PHB, 28 μmol/L H3-MV, and 72 h incubation. (number24) biosynthesis was at the lower control range 13 The quantity of cells grown in the wells and analysed for when the precursor was C6-PHB (0.84 nmol/UCS; con- each experiment was always similar, which is reflected by trols 0.84–1.0 nmol/UCS). the protein concentration measured in the preparations (0.63 Concerning group 2, all but patient 15 showed low

±0.07 mg/mL, n=102). Therefore, the assay conditions are CoQ10 biosynthesis, ranging from 0.04 to 0.09 nmol/UCS 2 comparable between cell lines. with H3-MV as substrate and from 0.55 to 0.78 nmol/UCS 2 13 Viability tests demonstrated that H3-MV at 28 μmol/L with C6-PHB (Table 2). does not affect fibroblasts stability at any incubation time Finally, CoQ10 biosynthesis was in the normal range in tested and neither was there effect on viability for four patients of group 3. In patients 16, 22 and 23, using 58

Table 2 CoQ10 biosynthesis in patients with CoQ10 deficiency in muscle or fibroblasts. Group 1: patients with a single detected mutation in a COQ gene. Group 2: patients with good clinical response to CoQ10. Group 3: patients with CoQ10 deficiency in fibroblasts, and no genetic studies or documented response to treatment

b Patient Clinical data [reference] Mitochondrial Respiratory CoQ10 concentration Biosynthesis of CoQ10 Altered COQ gene/ Chain activities (nmol/UCS) genotype

2 13 Fibroblasts Muscle Fibroblasts Muscle H3-Mevalonate C6-PHB

Group 1 8 Muscle hypotonia, weakness, psychomotor Normal Multiple 2.6a 1.2 0.06 0.54 COQ4 p.[Glu161Asp]+[=] retardation, rabdomyolysis, elevated CK, deficiencies fatty acids and ketones 9 Ataxia ND Normal 2.5a 1.3 0.23 0.91 COQ8 p.[Leu609Val]+[=] 10 Rabdomyolysis, myopathy; elevated CK Normal ND 1 ND 0.14 0.99 PDSS1 p.[Ala380Thr]+[=] Group 2 11 Ataxia, muscle weakness, cerebellar atrophy [32] Normal ↓CI+III, ↓CII+III 0.6a 2 0.04 0.63 NF 12 Ataxia, psychomotor retardation, myoclonias, Normal ND 1.8a ND 0.09 0.77 NF cerebellar atrophy [31] 13 Ataxia, psychomotor retardation, myoclonias, Normal ND 0.9a ND 0.07 0.55 NF cerebellar atrophy [31] 14 Ataxia, psychomotor retardation, epilepsy, Normal Normal 1.8a 1.6 0.08 0.78 NF convulsions [31] 15 Cerebellar atrophy, clumsiness, frequent falls, ND Normal 1.2a 7.9 0.14 1.05 NF nistagmus [32] Group 3 16 Hepatopathy, lactic and metabolic acidosis Normal ↓CII+III 1 1.6 0.15 0.7 NF 78 % mtDNA depletion [33] 17 Psychomotor retardation, ketosis and lactic acidosis ND ND 0.9 ND 0.12 0.82 NF 18 Ataxia, cerebellar syndrome ND ↓CI+III, ↓CII+III 1.1a 3.8 0.14 0.91 ND 19 Psycomotor retardation, myoclonias, elevated ND ↓CI+III, ↓CII+III, ↓CIV 2.9a 1.2 0.18 1.01 ND lactate and alanine, corpus callosum hypoplasia 20 Myopathy with exercise intolerance, high lactate Normal ↓CI+III, ↓CII+III 1.3a ND 0.12 0.96 ND and lactate/pyruvate ratio 21 Psychomotor retardation, cerebellar atrophy, ND Multiple 2.8a 1.3 0.11 0.86 ND hypotonia, ataxia, hypertrophic cardiomyopathy deficiencies; CS 22 Psychomotor retardation, ataxia, epilepsy, cerebellar ND ND 1.6 ND 0.12 0.65 ND atrophy. Retrospectively diagnosed of neuronal ceroid lipofuscinosis type 2 23 Psychomotor retardation, convulsions, ND ↓CI 1.1 ND 0.09 0.64 ND

cerebellar atrophy, epilepsy 37:53 (2014) Dis Metab Inherit J Parents 24 Asymptomatic ND ND 4.9a ND 0.16 0.84 NF 25 Asymptomatic ND ND 5.2a ND 0.16 1.02 COQ4 p.[Glu161Asp]+[=]

Control mean (mean±2SD) 2.4 (2.0–2.8) 5.4 (2.7–8.5) 0.13 (0.09–0.17) 0.94 (0.84–1.0) n=number of controls n=66 n=37 n=13 n=13

ND not done. NF not found a Determined by HPLC as previously described [18] b 2 13 Biosynthesis of CoQ10 was evaluated by measuring the corresponding labelled CoQ10 (nmol/UCS) generated, both with H3-Mevalonate or C6-PHB as substrates. Altered results are outlined in bold. Each individual value is the mean of at least a duplicate determination – 62 J Inherit Metab Dis (2014) 37:53–62 59 a 0.8

0.6

nmol/UCS 0.4 10 -CQ 6 0.2 C 13

0.0 1244872time (h)

13 13 C6-PHB 1,6 mmol/L C6-PHB 3,3 mmol/L b 0.25

0.20 /UCS l 0.15 nmo 0.10 10 -CQ

3 0.05 H 2 0.00 0244872time (h)

2 2 H3-MV 14 µmol/L H3-MV 42 µmol/L 2 2 H3-MV 28 µmol/L H3-MV 56 µmol/L c 72 h Incubation 0.5

0.4

0.3 nmol/UCS

10 0.2 -CQ 3 0.1 H 2

0.0 28 56 112 140 280 2 H3-MV concentration (µmol/L)

13 2 Fig. 2 CoQ10 biosynthesis using C6-PHB and H3-MV as substrates ’ 13 Fig. 3 Graphic representation of CoQ10 biosynthesis in controls at different concentrations and periods of incubation. a C6-CoQ10 ’ 13 2 13 and patients fibroblasts using C6-PHB and H3-MV as substrates. biosynthesis when using C6-PHB as the precursor, at 1.65 mmol/L or 13 2 2 a C6-CoQ10 biosynthesis. b H3-CoQ10 biosynthesis. c Correlation 3.3 mmol/L, it increases linearly with time. b H3− CoQ10 biosynthesis 13 2 2 between C6-CoQ10 and H3-CoQ10 biosynthesis in fibroblasts (Pearson when using H3-MV as the precursor at concentrations 14 to 56 μmol/L, 2 test, r=0.63;p<0.01). Numbers into the figure represent the corresponding it increases linearly with time. c H3− CoQ10 biosynthesis with higher 2 patient in the tables concentrations of H3-MV (28 μmol/L, to 280 μmol/L) and 72 h incubation; it decreases for concentrations greater than 56 μmol/L

2 2 H3-MV the amount of H3-CoQ10 synthesized was in the The same happened with patient 17 though his CoQ10 13 13 normal range, but with C6-PHB the biosynthesis was biosynthesis with C6-PHB was only slightly reduced deficient (0.70, 0.65 and 0.64 nmol/UCS, respectively). (0.82 nmol/UCS) (Fig. 3a). 60 J Inherit Metab Dis (2014) 37:53–62

Discussion 2011). Previous authors (Schedin et al 1998) had observed that the accumulation of cholesterol in a NPC murine model

Method setting up and validation was paralleled by increased dolichol-P and decreased CoQ10 concentrations. The mevalonate pathway involves conden- We have developed a non-invasive, non-radioactive and sation of three molecules of acetyl-CoA to 3-hydroxy-3- sensitive method by HPLC-MS/MS to study the biosynthe- methylglutaryl-coenzyme A (HMG-CoA). HMG-CoA re- sis of CoQ10 in fibroblasts. Our results indicated that CoQ10 ductase then converts HMG-CoA to mevalonate (Fig. 1). biosynthesis increased linearly with time for both substrates, Cholesterol regulates HMG-CoA reductase by feedback 2 but H3-MV needed higher concentrations. We finally inhibition. In NPC, accumulation of cholesterol may 13 2 established 1.65 mmol/L C6-PHB, 28 μmol/L H3-MV down-regulate HMG-CoA reductase decreasing mevalonate 13 and 72 h of incubation. The concentration of C6-PHB and, consequently, CoQ10 synthesis (Turunen et al 2004). 2 used was as described by Tekle et al (2008) and for H3- Accordingly, patients 6 and 7 showed normal CoQ10 bio- 2 MV it was doubled. Although the conditions for MV were synthesis with H3-MV as precursor of the pathway, while it 13 not saturating they were judged adequate to obtain quanti- was decreased using C6-PHB, as expected if the MV fiable peaks of the labelled CoQ10 with limited substrate pathway was partially inhibited (Table 1, Fig. 3a, b). In costs and did not significantly affect cells’ viability. these cases we tried to improve CoQ10 biosynthesis by 13 2 13 Correlation studies between C6-CoQ10 and H3-CoQ10 adding non labelled mevalonate in the C6-PHB media showed that the biosynthesis of both products tend to in- but, surprisingly, no amelioration was obtained. Further crease together (Fig. 3c), for that reason and because they experiments should investigate this lack of response. measure different steps in the biosynthesis of CoQ10 we maintained the incubation with both substrates. Biosynthesis of CoQ10 in patients’ fibroblasts As expected for primary CoQ10 deficiency (Fig. 1), our results showed clearly decreased rates of CoQ biosynthesis As the previous results indicated that our method was suit- 13 2 in patient 1 either with C6-PHB or H3-MV as substrates able for recognizing alterations of CoQ10 biosynthesis, we (Table 1, Fig. 3a, b). Similar rates of CoQ10 biosynthesis applied it to different patients with decreased CoQ10 in were described for a patient harbouring a homozygous mu- fibroblasts (Table 2, Fig. 3a and b). tation in COQ2 with either 14C-PHB or 3H-decaprenyl- The three patients of group 1 carry a mutation in one diphosphate (Quinzii et al 2006). As expected, the rates of allele in one of the COQ genes; we failed to detect a second biosynthesis were normal for patients 2–5 with CoQ10 defi- mutation even after study of cDNA extracted from CHX ciency secondary to other inborn errors of metabolism treated fibroblasts. Only patient 8 (with a mutation in

(MADD, VLCAD and complex III deficiency). MADD is COQ4) showed deficient CoQ10 biosynthesis. Deficient caused by defects in components of flavin metabolism or CoQ10 content with diminished biosynthetic rate in cul- transport (Frerman and Goodman 2001) that, in mitochon- tured fibroblasts has been reported in a patient with dria, mediates the transfer of electrons from flavin to ubi- haploinsufficiency of COQ4 (Salviatietal2012). As the father quinone and the RC. Mutations in ETFDH, encoding for of patient 8 also carries the mutation, we studied the biosyn- electron transfer flavoprotein-ubiquinone oxidoreductase thesis in both parents. Results were normal for her father

(ETF-QO), have been reported to cause secondary CoQ10 (number 25), while the mother’s rate (number 24) was at the 13 deficiency (Gempel et al 2007; Liang et al 2009). We also lower control range with C6-PHB as substrate. Therefore, the studied fibroblasts from a patient with VLCADD. The as- COQ4 mutation in our patient may not be enough to reduce sociation between VLCADD and CoQ10 deficiency had CoQ10 production. Our observations might be hypothetically previously been noted in one patient (Laforêt et al 2009). explained by an additional maternal mutation in an unknown Finally, some patients with mtDNA deletions or mt-tRNA point of the pathway that lowers the rate of synthesis without point mutations may show secondary CoQ10 deficiencies clinical effect in heterozygotes. Consequently, patient 8 would (Rahman et al 2012; Sznajer et al 2007; Matsuoka et al carry two genetic alterations (the mutation in COQ4 and that 1991), as happens with patient 5, with deficient complex hypothetical mutation) that together cause the deficiency. III and a homoplasmic mutation in mtDNA. All the men- Whole exome sequencing is on course in this family. The other tioned diseases are mitochondrial dysfunctions that may twopatientsofgroup1(patients9and10)showednormalRC hypothetically increase degradation of CoQ10 or decrease activities and CoQ biosynthesis. We may conclude that these its ATP-dependent transport in some patients, but the actual patients’ CoQ10 deficiency is secondary and that their hetero- mechanism of the secondary deficiency in all these condi- zygous mutations are not the cause of the deficiency. tions remains unknown (Rahman et al 2012). Therefore, a normal CoQ10 biosynthesis rules out a primary Additionally, to validate our method, we have studied defect but, as exemplified with patients of group 2, the oppo- patients 6 and 7 affected with NPC (Macías-Vidal et al site is not always true. In fact, all patients of this group but J Inherit Metab Dis (2014) 37:53–62 61

patient 15 showed deficient CoQ10 biosynthesis. However, References screening for COQ genes failed to detect pathological changes. Patient 15 is the father of patient 11; they have Aeby A, Sznajer Y, Cavé H, Rebuffat E, Van Coster R, Rigal O, Van previously been described (Pineda et al 2010;Artuchetal Bogaert P (2007) Cardiofaciocutaneous (CFC) syndrome associated 2006). He presented slight clinical alterations including action with muscular coenzyme Q10 deficiency. J Inherit Metab Dis 30:827 tremor, mild modification of fluency, transient nystagmus and Arias A, García-Villoria J, Rojo A, Buján N, Briones P, Ribes A (2012) Analysis of coenzyme Q10 in lymphocytes by HPLC–MS/MS. J slight saccadic pursuit that were corrected with treatment. Chromatogr B Anal Technol Biomed Life Sci 908:23–26 CoQ10 levels were low in his fibroblasts, but muscle CoQ10 Artuch R, Brea-Calvo G, Briones P et al (2006) Cerebellar ataxia with and RC activities, as well as fibroblast CoQ biosynthesis, coenzyme Q10 deficiency: diagnosis and follow-up after coen- 10 – were normal. In contrast, his daughter (patient 11), with a zyme Q10 supplementation. J Neurol Sci 246:153 158 Crane FL, Hatefli Y, Lester RL, Widmer C (1957) Isolation of a more severe ataxia and biochemical alterations, showed quinone from beef heart mitochondria. Biochim Biophys Acta clearly deficient CoQ10 biosynthesis (Artuch et al 2006). 25:220–221 The disease in this family may be caused by a heterozy- Dallner G, Sindelar PJ (2000) Regulation of ubiquinone metabolism. – gous mutation in an unidentified gene that causes mild Free Radic Biol Med 29:285 294 Diomedi-Camassei F, Di Giandomenico S, Santorelli FM et al (2007) alterations as seen in the father while the more severe COQ2 nephropathy: a newly described inherited mitochondriopathy disease in the girl is due to homozygosity (or compound with primary renal involvement. J Am Soc Nephrol 18:2773–2780 heterozygosity). We cannot exclude that their CoQ deficien- Festenstein GN, Heaton FW, Loewe JS, Morton RA (1955) A constit- 10 λ cy is secondary and, in this case, it might only be a modulator uent of the unsaponifiable portion of animal tissue lipids ( max 272mμ). Biochem J 59:558–566 of the phenotype, exacerbating the clinical picture in the Frerman FE (1987) Reaction of electron transfer flavoprotein ubiqui- daughter. none oxidoreductase with the respiration chain. Biochim Biophys – Four patients of group 3 presented CoQ10 biosynthesis in Acta 893:161 169 the normal range. Therefore, they are most probably sec- Frerman FE, Goodman SI (2001) Defects of electron transfer flavo- ’ protein and electron transfer flavoprotein-ubiquinone oxidoreduc- ondary CoQ10 deficiencies. Conversely, patient 23 s biosyn- tase: glutaric academia type II. In: Scriver CR, Beaudet AL, Sly 2 thesis was in the lower control range with H3-MV and WS, Valle D (eds) The metabolic and molecular bases of inherited 13 – decreased with C6-PHB. We could infer that this patient’s disease. McGraw-Hill, New York, pp 2357 2365 deficiency is primary, and mutations in COQ genes should Gempel K, Topaloglu H, Talim B et al (2007) The myopathic form of coenzyme Q10 deficiency is caused by mutations in the electron- be investigated. Results in patients 16 (Montero et al 2009), transferring-flavoprotein dehydrogenase (ETFDH) gene. Brain 17 and 22 are difficult to conclude because the amount of 130:2037–2044 2 CoQ10 synthesized using H3-MV is in the normal range, Horvath R, Schneiderat P, Schoser BG et al (2006) Coenzyme Q10 – but with 13C -PHB, the biosynthesis is slightly low or deficiency and isolated myopathy. Neurology 66:253 255 6 Laforêt P, Acquaviva-Bourdain C, Rigal O et al (2009) Diagnostic deficient. This points to some altered or inhibited step in assessment and long-term follow-up of 13 patients with Very the biosynthesis of mevalonate, as observed in NPC disease. Long-Chain Acyl-Coenzyme A dehydrogenase (VLCAD) defi- In fact, patient 22 has recently been diagnosed with neuronal ciency. Neuromuscul Disord 19:324–329 ceroid lipofuscinosis type 2 (OMIM#204500). To our Liang WC, Ohkuma A, Hayashi YK et al (2009) ETFDH mutations, CoQ10 levels, and respiratory chain activities in patients with knowledge, the relationship between this disease and riboflavin-responsive multiple acyl-CoA dehydrogenase deficien- CoQ10 deficiency had not been reported previously and cy. Neuromuscul Disord 19:212–216 should be further investigated. López LC, Quinzii CM, Area E, Naini A, Rahman S, Schuelke M, In conclusion, we have developed a non-invasive non- Salviati L, DiMauro S, Hirano M (2010) Treatment of CoQ10 deficient fibroblasts with ubiquinone, CoQ analogs, and vitamin radioactive method suitable for the detection of defects in C: time- and compound-dependent effects. PLoS One 5:e11897 CoQ10 biosynthesis, which offers a good tool for the Macías-Vidal J, Rodríguez-Pascau L, Sánchez-Ollé G et al (2011) stratification of these treatable mitochondrial diseases. Molecular analysis of 30 Niemann-Pick type C patients from Additionally, our method might be of interest to study Spain. Clin Genet 80:39–49 Matsuoka T, Maeda H, Goto Y, Nonaka I (1991) Muscle coenzyme Q10 unknown aspects about the subcellular turnover of newly in mitochondrial encephalomyopathies. Neuromuscul Disord synthesized CoQ. 1:443–447 Montero R, Sánchez-Alcázar JA, Briones P et al (2008) Analysis of Acknowledgments We acknowledge the technical support of Carlota coenzyme Q10 in muscle and fibroblasts for the diagnosis of – Ogg, Sonia Moliner, Patricia Alcala and Cristina Fernandez. Nuria CoQ10 deficiency syndromes. Clin Biochem 41:697 700 Buján is a PhD student of the University of Girona. The Centro de Montero R, Sánchez-Alcázar JA, Briones P et al (2009) Coenzyme Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER) Q10 deficiency associated with a mitochondrial DNA depletion is an initiative of the Instituto de Salud Carlos III (Ministerio de Ciencia e syndrome: a case report. Clin Biochem 42:742–745 Innovación, Spain). This research was supported in part by grants Ogasahara S, Engel AG, Frens D, Mack D (1989) Muscle coenzyme Q PI08/0307, PI08/0663, and PI11/02350, PI12/01138 from Fondo de deficiency in familial mitochondrial encephalomyopathy. Proc Investigación Sanitaria. Natl Acad Sci U S A 86:2379–2382 Pineda M, Montero R, Aracil A et al (2010) Coenzyme Q(10)-responsive Conflict of interest None. ataxia: 2-year-treatment follow-up. Mov Disord 25:1262–1268 62 J Inherit Metab Dis (2014) 37:53–62

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