J Neurol DOI 10.1007/s00415-016-8249-2
ORIGINAL COMMUNICATION
A novel mutation m.8561C>G in MT-ATP6/8 causing a mitochondrial syndrome with ataxia, peripheral neuropathy, diabetes mellitus, and hypergonadotropic hypogonadism Laura Kyto¨vuori1,2,3 • Joonas Lipponen1,2,3 • Harri Rusanen1,2,3 • Tuomas Komulainen4 Mika H. Martikainen5 • Kari Majamaa1,2,3
•
Received: 6 June 2016 / Revised: 27 July 2016 / Accepted: 28 July 2016 Ó Springer-Verlag Berlin Heidelberg 2016
Abstract Defects in the respiratory chain or mitochondrial ATP synthase (complex V) result in mitochondrial dysfunction that is an important cause of inherited neurological disease. Two of the subunits of complex V are encoded by MT-ATP6 and MT-ATP8 in the mitochondrial genome. Pathogenic mutations in MT-ATP6 are associated with the Leigh syndrome, the syndrome of neuropathy, ataxia, and retinitis pigmentosa (NARP), as well as with non-classical phenotypes, while MT-ATP8 is less frequently mutated in patients with mitochondrial disease. We investigated two adult siblings presenting with features of cerebellar ataxia, peripheral neuropathy, diabetes mellitus, sensorineural hearing impairment, and hypergonadotropic hypogonadism. As the phenotype was suggestive of mitochondrial disease, mitochondrial DNA was sequenced and a novel heteroplasmic mutation m.8561C[G in the overlapping region of the MT-ATP6 and MT-ATP8 was found. The mutation changed amino acids in both subunits. Mutation heteroplasmy correlated with the disease phenotype in five L. Kyto¨vuori and J. Lipponen contributed equally to the manuscript. & Kari Majamaa
[email protected] 1
Research Unit of Clinical Neuroscience, University of Oulu, P.O. Box 5000, 90014 Oulu, Finland
2
Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland
3
Department of Neurology, Oulu University Hospital, P.O. Box 20, 90029 Oulu, Finland
4
PEDEGO Research Unit, University of Oulu, P.O. Box 5000, 90014 Oulu, Finland
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Division of Clinical Neurosciences, University of Turku and Turku University Hospital, Kiinamyllynkatu 4-8, 20520 Turku, Finland
family members. An additional assembly intermediate of complex V and increased amount of subcomplex F1 were observed in myoblasts of the two patients, but the total amount of complex V was unaffected. Furthermore, intracellular ATP concentration was lower in patient myoblasts indicating defective energy production. We suggest that the m.8561C[G mutation in MT-ATP6/8 is pathogenic, leads biochemically to impaired assembly and decreased ATP production of complex V, and results clinically in a phenotype with the core features of cerebellar ataxia, peripheral neuropathy, diabetes mellitus, and hypergonadotropic hypogonadism. Keywords Mitochondrial disorders Gait disorders Neuromuscular disease Hypergonadotropic hypogonadism
Introduction Mitochondrial disorders arise from mutations in mitochondrial DNA (mtDNA) or from mutations in nuclear genes that encode factors involved in the oxidative phosphorylation (OXPHOS) and other functions of mitochondria. The mitochondrial genome itself is compact coding for 13 subunits of OXPHOS complexes, two rRNAs and 22 tRNAs. Overall, mutations in mtDNA are an important cause of neurological disease in adults, even though the prevalence of specific pathogenic mutations may remain low in the general population [1]. Ataxia is a frequent clinical manifestation in patients with mitochondrial disorder. A classic example of ataxia in a mitochondrial disorder is the neuropathy, ataxia and retinitis pigmentosa syndrome (NARP), commonly caused by m.8993T[G or m.8993T[C in the MT-ATP6 gene
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encoding subunit a of the mitochondrial ATP synthase (complex V). Since the discovery of the two mutations [2, 3], mutated MT-ATP6 has been increasingly reported to cause phenotypes ranging from the canonical NARP to adult-onset spinocerebellar ataxia or Charcot-Marie-Tooth type 2 (CMT2) [4–8]. In addition to the MT-ATP6, mtDNA contains MT-ATP8 coding for A6L, a second mitochondrial subunit of complex V. Indeed, the sequences of the two genes overlap by 46 base pairs making double mutations possible. In this study, we investigated the mitochondrial genome in two adult siblings, who presented with a phenotype suggesting a mitochondrial disease including cerebellar ataxia, peripheral neuropathy, diabetes mellitus, sensorineural hearing impairment, and hypergonadotropic hypogonadism. We describe a novel mutation m.8561C[G in the overlapping region of MT-ATP6 and MT-ATP8 and further demonstrate that the mutation results in impaired assembly and decreased cellular ATP suggesting dysfunction in energy production.
Subjects and methods Case reports Patient II-5 The proband (II-5, Fig. 1a) is a 59-year-old woman, who has been diagnosed with migraine and ovarian dysgenesis with hypergonadotropic hypogonadism as well as secondary amenorrhea due to ovarian dysfunction at age 23 years. At age 24 years she began to experience dizziness and ataxia gradually developed over the following years. She underwent hysterectomy and oophorectomy at age 41 years because of metrorrhagia and dysmenorrhea. Diabetes mellitus was diagnosed at age 49 years. Clinical examination at age 58 years revealed ataxic gait, positive Romberg’s test, arm dysmetria and dysdiadochokinesia. Speech was dysarthric, but easy to understand. Tendon reflexes were absent in the legs. Babinski’s sign was positive. Muscle strength was slightly decreased, but muscle atrophy was observed only in distal upper limbs. No signs of retinitis pigmentosa were found in fundoscopy. Clinical evaluation using the Scale for the Assessment and Rating of Ataxia (SARA) [9] gave a score 15 out of 40 and the Inventory of Non-ataxia Symptoms (INAS) count [10] was 5 out of 16. Her height was 150 cm and weight was 83 kg. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) were elevated repeatedly during the followup since age 23 years. Blood lactate was normal. Brain MRI at age 36 years revealed cerebellar atrophy.
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Cerebrospinal fluid examination and electrophysiological examination of muscle and nerve were normal. Histological examination of muscle from vastus lateralis revealed type 2 muscle fiber atrophy. No cytochrome c oxidase negative or ragged-red fibers were detected. Ultrastructural analysis of mitochondria was normal. Patient II-4 The brother of the proband (II-4) is a 64-year-old man, who has had balance difficulties since childhood. His academic performance in primary school was poor. He experienced coordination difficulties of the lower limbs and walking problems at age 20 years and the difficulty to walk increased gradually thereafter. Moreover, he developed dysarthria and dysphagia. The patient was diagnosed with sensorineural hearing impairment and axonal sensorimotor polyneuropathy at age 39 years, and he was examined for erectile dysfunction at age 40 years. No organic cause was found, but endocrinological tests for hypogonadism at age 50 years showed increased FSH (21.9 U/l; laboratory reference \10 U/l) and low normal testosterone (9.1 nmol/l; laboratory reference 9–38 nmol/l). The patient was also diagnosed with ulcerative colitis at age 52 years, and type 2 diabetes at age 61 years. Clinical examination at age 62 years revealed that standing without support was not possible and he needed a walker and a wheelchair. Gait was severely ataxic and dysarthria, dysmetria and dysdiadochokinesia were noted. Fundoscopy did not reveal retinitis pigmentosa. Impaired sensation of vibration was observed in the legs and sensation to light touch was impaired distally in hands and feet. Tendon reflexes were absent in the lower extremities. Babinski’s sign was positive on both sides. Muscle strength was slightly decreased and mild muscle atrophy was observed in the legs distally. The SARA score was 19 and the INAS count was 5. His height was 168 cm and weight was 73 kg. Electrophysiological examination demonstrated distal sensorimotor axonal polyneuropathy. Brain CT showed cerebellar atrophy at age 40 years and brain MRI showed atrophy of putamina and cerebellum and mild frontoparietal cortical atrophy at age 59 years. Radiological studies and laboratory screening for other possible etiologies causing polyneuropathy and ataxia were normal. Cerebrospinal fluid examination was normal. Blood lactate at rest was slightly elevated (1.61 mmol/l; laboratory reference 0.331.33 mmol/l) on one occasion at age 53, but normal on other occasions. Histological and ultrastructural examination of muscle from vastus lateralis did not reveal any mitochondrial pathology. Transthoracic echocardiogram revealed that the left ventricle systolic function was within lower limit of normal with ejection fraction of 50 %.
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Fig. 1 Siblings with the m.8561C[G mutation in the overlapping region of the MT-ATP6 and MT-ATP8 genes. a Pedigree of the family. Open circle asymptomatic female; open square asymptomatic male; solid circle affected female; solid square affected male. b The m.8561C[G mutation in the sequence chromatograms of three family
members demonstrating variable heteroplasmy. c mtDNA spanning between the nucleotides 8527-8572 corresponding to amino acid residues 1–15 in subunit a and 55–68 in subunit A6L. The mutation leads to p.Pro12Arg replacement in subunit a and to p.Pro66Ala in subunit A6L of the complex V
The elder siblings (II-1, age 69 years; II-2, age 67 years; II-3, age 66 years) and their children were healthy with the exception of one sister (II-2) with coronary artery disease. The father (I-1) had had memory problems and had died at 88 years of age and the mother (I-2) had had diabetes and had died at 70 years of age. A written informed consent was requested from all study participants.
was purified with WizardÒ Genomic DNA purification kit (Promega Corporation, Madison, WI, USA). Mitochondrial DNA was amplified in twelve overlapping fragments using Phire Hot Start II polymerase (Thermo Fisher Scientific, Waltham, MA, USA) according to the provided protocol and sequenced using BigDyeTerminator v1.1 cycle sequencing kit (Applied BiosystemsTM, Thermo Fisher Scientific) and 3500xL Genetic Analyzer at Biocenter Oulu sequencing core facility. Primer sequences and detailed conditions of amplification reactions are available on request. The revised Cambridge mtDNA sequence (GenBank Accession number NC_012920) was used as the reference. One hundred control samples were screened for the m.8561C[G mutation using Cac8I restriction enzyme (New England Biolabs, Ipswich, MA, USA). Mitochondrial DNA haplogroup was determined using HaploGrep software [11, 12]. Pathogenicity evaluation was carried out using PredictSNP, which combines six common algorithms to produce the prediction [13]. To determine mutation heteroplasmy, different tissues and myoblasts were investigated by cloning using CloneJET PCR Cloning Kit with Subcloning EfficiencyÒ DH5a competent cells (Thermo Fisher Scientific) using the original protocols. A total of 100 bacterial colonies from each
Molecular genetic investigations Spinocerebellar ataxia type 1 (SCA1) and SCA8, Friedreich’s ataxia and inactivating point mutation (p.Ala189Val) of the human FSH receptor had previously been excluded in patient II-5 while SCA1, Friedreich’s ataxia, and mutations in the POLG gene had been excluded in patient II-4. As the phenotype of the patients was suggestive of a mitochondrial disorder, the entire mtDNA was sequenced. Sequencing of mtDNA, determination of mutation heteroplasmy and analyses in silico DNA was extracted from peripheral blood, buccal mucosa and myoblasts using QIAamp Blood Kit (QIAGEN, Hilden, Germany). In addition, DNA from muscle biopsies
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sample were collected and analyzed as previously using conformation sensitive gel electrophoresis and sequencing [14]. DNA from peripheral blood samples of the patients’ three healthy siblings were also analyzed by means of the aforementioned protocol. Myoblast cultures Primary myoblast cultures were established from muscle biopsies taken from the vastus lateralis of both patients [15]. The cells were then cultured in commercial myoblast medium (SMCGM with supplements, PromoCell, Heidelberg, Germany) with penicillin–streptomycin (100U/ 100 lg/ml, Corning, NY, USA) using pre-plating to remove fibroblasts. Cells were immunostained regularly with myoblast-specific desmin antibody (BioGenex, Fremont, CA, USA) to confirm the cell type (Fig. 2b). Control cells were GIBCOÒ human skeletal myoblasts (Life Technologies, Carlsbad, CA, USA) and fibroblasts from an adult control patient as the negative control. SDS-PAGE, blue native PAGE and 2D-SDS-PAGE Sample preparation for SDS-PAGE was done by collecting the myoblasts followed by solubilization using 1.5 % ndodecyl b-D-maltoside in 1 9 PBS w/o calcium and magnesium containing protease inhibitor. Commercial denaturing gels (NuPAGEÒ NovexÒ, Life Technologies) were electrophoresed and blotted according to the Fig. 2 Immunoblotting of subunits of the OXPHOS complexes in the patients with m.8561C[G. a Differences were not detected in subunits of complexes CI-CV between patient and control myoblasts. Tubulin was used as the loading control. b Fluorescence imaging of the cells. Arrow Hoechst 33342 staining of nucleus; double arrow desmin antibody. Upper panel patient myoblasts MB P; center panel myoblast control, MB C; lower panel fibroblast control, FB C
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manufacturer’s protocol. Antibodies for SDS-PAGE blots were 30 kDa subunit (A21343, Molecular ProbesÒ, Life Technologies) of complex I, flavoprotein subunit of complex II (ab14715, Abcam, Cambridge, UK), core protein 2 of complex III (ab14745), subunit 4 of complex IV (ab14744), subunits alpha (ab14748) and beta (ab14730) of F1 of complex V, subunit d (ab110275), subunit a (EB12462, Everest Biotech, Oxfordshire, UK) and subunit A6L (sc-84231, Santa-Cruz Biotechnology, Dallas, TX, USA) of FO of complex V. The indication of oxidative stress was investigated using antibodies for superoxide dismutase (ab13533) and glutathione peroxidase 1 (ab22604). Alpha-tubulin (ab6160) was used as loading control. Samples for the BN-PAGE were prepared as described elsewhere [16]. For the first dimension BN-PAGE, the commercial gradient gels were used (NativePAGETM NovexÒ, Life Technologies) with running buffers prepared as previously [16]. The blue cathode buffer was replaced with colorless cathode buffer, when the blue line had migrated about two-thirds of the gel. After the blue line had migrated out from the gel, the electrophoresis was stopped and the lanes for the second dimension SDS-PAGE were separated. The second dimension was established in denaturing conditions and the gels were blotted as described in the manufacturer´s protocol (NovexÒ NuPAGE, Life Technologies). In addition, non-gradient BN-PAGE gel was carried out [17]. Western blotting was done at ?4 °C using transfer buffer containing 25 mM TRIS, 20 mM
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glycine, 0.2 % SDS and 20 % ethanol and nitrocellulose membrane with 90 V for 90 min. Antibodies for the blotted blue native gels were flavoprotein subunit of complex II (ab14715) used as the loading control, core protein 2 of complex III (ab14745), subunit 4 of complex IV (A21347) and alpha subunit of F1 (ab14748) of complex V. The intensity of signals was analyzed using Quantity one 1D software (Bio-Rad, Hercules, CA, USA).
accessed on 30 May 2016). PredictSNP, a consensus classifier combining six prediction algorithms, was used to provide predictions on pathogenic potential. The six prediction tools within PredictSNP consistently predicted the p.Pro12Arg change in subunit a to be pathogenic. The p.Pro66Ala mutation in subunit A6L was predicted to be neutral with the consensus algorithm, but three out of the six algorithms predicted against neutrality. The mtDNA of the patients belonged to haplogroup H4a1a1a.
ATP determination The baseline concentration of cellular ATP was measured in myoblasts cultured in growth medium. The effect of uncoupling the respiratory chain in cellular ATP was measured after treating the cells with 3 lM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, Cayman Chemical, Ann Arbor, MI, USA) for 10 min. ATP determination kit (Molecular ProbesÒ, Life Technologies) was used to measure cellular ATP content. ATP was first released by lysing the cells [18] followed by immediate freezing with liquid nitrogen. Reaction was started by adding 10 ll of cell lysate and 90 ll reaction buffer in measurement plate and incubating the mixture at 28 °C for 15 min. Luminescence was measured using FLUOStar Omega microplate reader (BMG Labtech, Ortenberg, Germany). Twenty-two samples from patients and control using four replicates of each sample were measured to determine the baseline ATP content. Nine samples from patient II-5 and control were used to investigate the uncoupling effect in cellular ATP. The amount of ATP after treatment was calculated in relation to the amount without FCCP treatment. The amount of ATP was normalized with the total amount of proteins determined by Bradford assay. The tests of normality, the paired-samples t-tests and related-samples Wilcoxon signed rank test were carried out using SPSS 20 (IBM, Chicago, IL, USA).
Results Sequencing of the mitochondrial genome revealed a novel missense mutation m.8561C[G in the samples of the proband and her affected brother. This mutation was located in the MT-ATP6/8 overlapping region and leads to p.Pro12Arg substitution in the subunit a and to p.Pro66Ala in the subunit A6L (Fig. 1). Mutation heteroplasmy was C99 % in the blood, buccal mucosa and muscle as well as all the myoblast subcultures of the patients, but varied between 3 and 20 % in the blood of the three asymptomatic siblings. The m.8561C[G mutation was not found in healthy controls or in the 30,589 sequences of the MITOMAP database (http://www.mitomap.org/MITOMAP,
Quantification of complex V and analysis of assembly The quantity of complex V subunits was then examined in patient myoblasts and their assembly was evaluated. The amounts of the subunits of complex V or those of the other OXPHOS complexes were similar between the patient and control myoblasts in SDS-PAGE (Fig. 2a). Interestingly, the proportions of the assembly intermediates of complex V holoenzyme differed between the patient and control myoblasts in non-gradient BN-PAGE (Fig. 3a). An increased amount of subcomplex F1 was detected in patient myoblasts. After normalizing with complex II as the loading control, the mean relative amount of fully assembled complex V varied from 0.97 to 1.19 in the patients compared to the control, while the normalized mean intensities of subcomplex F1 were 1.7-fold in patient II-5 and 2.3-fold in patient II-4 than that in the controls. Furthermore, an additional assembly intermediate of complex V, referred as V*, was detected in patient myoblasts, but not in the control myoblasts. The additional assembly intermediate was recognized also in the second dimension analyses using antibodies for a and b of F1 (results not shown). Other OXPHOS complexes were similar between patients and control. Determination of ATP in myoblasts In comparison to the control myoblasts the baseline ATP concentration (pmol/lg protein) was lower in patient II-5 subcultures (p = 0.004; t test for paired samples) and in patient II-4 subcultures (p = 0.023) (Fig. 3b). The mean ATP concentration in patient II-5 myoblasts was 82.5 % of that in the control cells and 85.2 % in patient II-4. Uncoupling of the respiratory chain using FCCP led to significant decrease of ATP in patient II-5 myoblasts (p = 0.028; related-samples Wilcoxon signed rank test), while the concentration remained unaltered in control myoblasts after the treatment (Fig. 3c). Immunoblotting of superoxide dismutase (SOD2) or glutathione peroxidase 1 (GPX1) that were used as indicators of oxidative stress did not reveal differences between patients and controls (Fig. 3d).
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Fig. 3 Assembly of complex V and ATP determination in the myoblasts harboring m.8561C[G. a Immunoblotting of blue native PAGE. Additional assembly intermediate V* is present in patient myoblasts. The amount of subcomplex F1 is increased in the patients. Lane 1 control 25 lg of proteins; lane 2 patient II-5, sample 1 25 lg; lane 3 patient II-4, sample 1 25 lg; lane 4 patient II-5, sample 2 10 lg; lane 5 patient II-4, sample 2 10 lg. CV complex V, F1 subcomplex F1 of CV, V* additional assembly intermediate of CV,
CII complex II, loading control. b Boxplot of the baseline amount of cellular ATP (pmol/lg protein) in myoblasts. c The ratio of cellular ATP in myoblasts treated with 3 lM FCCP to that in untreated cells. Y axis the ratio; X axis nine separate experiments with patient and control myoblasts. d Immunoblotting with superoxide dismutase (SOD2) and glutathione peroxidase (GPX1). Tubulin was used as the loading control
Discussion
result from a joint effect. Mutation heteroplasmy correlated with the phenotype, as the mutation was close to homoplasmy in affected patients but present only at a low level in healthy siblings. The mutation was found in all the five siblings suggesting that it had arisen earlier in the maternal lineage. Unfortunately, no samples from the mother of the siblings were available. We found an abnormal assembly intermediate V* of complex V. Additional assembly intermediates of complex V have been reported in patients with mutations in the MTATP6 or MT-ATP8 and in cells lacking mtDNA [21, 24–26]. A decreased amount of complex V has previously been reported with mutations in either of the mitochondrial subunits [21, 24], but we did not find such a decrease of fully assembled complex V. In a family with the m.8993T[G mutation and atypical Leigh syndrome the decrease of fully assembled complex V was accompanied with increased subcomplex F1 and an additional assembly intermediate [24]. Similar findings have been detected in a patient with early-onset neuropathy and cardiomyopathy
We found a novel mutation m.8561C[G in the overlapping region of MT-ATP6 and MT-ATP8, which causes p.Pro12Arg substitution in subunit a and p.Pro66Ala substitution in subunit A6L of complex V. Complex V deficiency is rare among the cases with impaired oxidative phosphorylation, while defects in complexes I or IV are more common [19]. To date, mutations in MT-ATP6 have been associated with various phenotypes, most commonly Leigh syndrome and the NARP syndrome, but biochemical confirmation of the pathogenicity is lacking in some reported cases. Rare mutations in the MT-ATP8 have been reported to be associated with a disease [20, 21]. Mutations in the overlapping region of the two genes may lead to changes in both proteins, but disease-causing double mutations are extremely rare [22, 23]. In our study, the change in MT-ATP6 was predicted to be pathogenic and that in the MT-ATP8 was predicted to be neutral, but both changes were non-synonymous and the pathogenicity may
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and a nonsense mutation in MT-ATP8 [21]. Even though the clinical phenotype is less severe in our patients, the biochemical consequences resemble those in patients with confirmed pathogenic mutations. The mutation was homoplasmic in our patient myoblasts, but the amount of subunit a or A6L did not differ between the patients and control suggesting that the mutant proteins were not degraded. Indeed, the amount of complex V was normal in both patients. Thus, mutated subunits were incorporated in the enzyme complex leading to an altered function of complex V that was supported by the measurements of cellular ATP. Decreased activity of complex V containing mutant subunits has been reported in cybrids carrying the m.8993T[G mutation in MT-ATP6 [25]. The synthesis of ATP was decreased in these cybrid cell lines, even though some of them almost lacked the abnormal subassemblies and the amount of complex V was similar to that in the control cells. In addition to the decrease of baseline cellular ATP in our study, the patient myoblasts were more sensitive to the uncoupling by FCCP. This finding may suggest that patient myoblasts are overdriving the respiratory chain as a consequence of complex V dysfunction, and hence, are more vulnerable for uncoupling. However, there was no change in the amounts of SOD2 or GPX1 suggesting unaltered level of ROS. The measurement of ATP using the luciferase-based chemiluminescence assay requires highly standardized assay conditions. Therefore, all the mutant-containing subcultures were tested with a substantial number of samples and replicates that enabled a confident demonstration of the overall ATP decrease in the myoblasts. Similar to the ATP measurements, heterogeneity was detected in the ratio of the assembly intermediates of complex V within the subcultures. Nevertheless, the normalized amount of subcomplex F1 was clearly increased and the intermediate V* was observed in all samples prepared from patient subcultures. The mutation was homoplasmic in subcultures excluding the possibility that heteroplasmy would explain the differences. The patients reported here presented with cerebellar ataxia and peripheral neuropathy, both of which are common features in mitochondrial disease associated with mutations in MT-ATP6. Furthermore, both of them presented with diabetes mellitus and patient II-4 with sensorineural hearing impairment, which are frequently encountered in mitochondrial disease. It is notable that patients with MT-ATP6 mutations often have normal findings in muscle biopsy and that the mutation heteroplasmy required for clinical manifestations is relatively high. An interesting feature in the phenotype of the patients harboring the m.8561C[G mutation was hypergonadotropic hypogonadism. Hypergonadotropic hypogonadism had been diagnosed previously in patient II-5,
whereas patient II-4 had no diagnosis of primary hypogonadism, but a history of elevated FSH, low normal testosterone and erectile dysfunction. Both hypogonadotropic and, less commonly, hypergonadotropic hypogonadism have occasionally been reported in association with various forms of mitochondrial disease [14, 27–31]. However, it is possible that the prevalence of hypogonadism in mitochondrial disease is under-appreciated, as other features commonly dominate the clinical picture [31]. In conclusion, we described a novel mutation m.8561C[G in the MT-ATP6/8 gene in two siblings with a phenotype consisting of cerebellar ataxia, peripheral neuropathy with upper motor signs, diabetes mellitus, and hypergonadotropic hypogonadism. In addition, one patient presented also with sensorineural hearing impairment. The mutation was heteroplasmic and mutation heteroplasmy correlated with the clinical phenotype. Impaired assembly of complex V and altered ATP production were demonstrated. The biochemical defect is similar to that in patients with other confirmed pathogenic mutations in mitochondrial genes encoding subunits of complex V. We suggest that m.8561C[G is pathogenic and wish to highlight hypogonadism as a feature of mitochondrial disease that has probably been under-appreciated in clinical practice. Acknowledgments The authors gratefully acknowledge the expert technical assistance of Ms. Anja Heikkinen and Ms. Pirjo Kera¨nen. The study was supported by grants from the Sigrid Juselius Foundation, Medical Research Center, University of Oulu and Oulu University Hospital, and State research funding from Oulu University Hospital. Compliance with ethical standards Ethical standards A written informed consent was requested from all study participants. The study protocol was approved by the Oulu University Hospital ethics committee. High standard of ethics according to the WMA Declaration of Helsinki was applied in all investigations and clinical work described in this manuscript. Conflict of interest The authors declare that they have no conflict of interest.
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