Defective Mitochondrial Atpase Due to Rare Mtdna M.8969G>A Mutation—Causing Lactic Acidosis, Intellectual Disability, and Poor Growth

neurogenetics (2018) 19:49–53 https://doi.org/10.1007/s10048-018-0537-9

SHORT COMMUNICATION

Defective mitochondrial ATPase due to rare mtDNA m.8969G>A mutation—causing lactic acidosis, intellectual disability, and poor growth

Pirjo Isohanni1,2 & Christopher J. Carroll2 & Christopher B. Jackson2 & Max Pohjanpelto2 & Tuula Lönnqvist1 & Anu Suomalainen2,3

Received: 19 October 2017 /Accepted: 9 January 2018 /Published online: 19 January 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Mutations in mitochondrial ATP synthase 6 (MT-ATP6) are a frequent cause of NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) or Leigh syndromes, especially a point mutation at nucleotide position 8993. M.8969G>A is a rare MT- ATP6 mutation, previously reported only in three individuals, causing multisystem disorders with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia or IgA nephropathy. We present two siblings with the m.8969G>A mutation and a novel, substantially milder phenotype with lactic acidosis, poor growth, and intellectual disability. Our findings expand the phenotypic spectrum and show that mtDNA mutations should be taken account also with milder, stable phenotypes.

Keywords Mitochondrial diseases . Mitochondrial DNA . Intellectual disability . Lactic acidosis

Introduction lactic acidosis, and sideroblastic anemia) [8] and IgA nephropathy plus [9] phenotypes. Age-of-onset of these diseases MT-ATP6 in mitochondrial DNA (mtDNA) encodes subunit 6 varies from infant to late adulthood. of the F1Fo ATP synthase (complex V) of the oxidative phos- Point mutations at nucleotide position m.8696 are very rare phorylation system, which synthesizes ATP using the energy and been reported only with different multisystem phenotypes provided by the mitochondrial electrochemical gradient. [8, 9]. Here, we report a novel, substantially milder phenotype Mutations in MT-ATP6 gene are a frequent cause of NARP associated with the m.8969G>A mutation. (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) or Leigh syndrome phenotypes, with the most frequent cause being a point mutation at nucleotide position Materials and methods m.8993 T>C or T>G [1–3]. Other clinical presentations asso- MT-ATP6 ’ ciated with mutations include Leber s hereditary All samples were obtained for diagnostic purposes with in- optic neuropathy (LHON) [4], peripheral neuropathy [5], bi- formed consent from the parents or family members them- lateral striatal necrosis [6], hypertrophic cardiomyopathy [7], selves. The study was approved by the institutional ethics and the recently reported MLASA (mitochondrial myopathy, committee. Genomic DNA was extracted from peripheral blood or muscle tissue samples by standard procedures.

* Pirjo Isohanni Mitochondrial DNA sequencing and heteroplasmy [email protected] determination

1 Department of Child Neurology, Children’s Hospital, University of Mitochondrial DNA was sequenced as previously described Helsinki and Helsinki University Hospital, Helsinki, Finland [10]. We utilized next-generation sequencing (NGS) of 2 Research Programs Unit, Molecular Neurology, mtDNA to quantify mtDNA heteroplasmy (skeletal muscle Biomedicum-Helsinki, University of Helsinki, Helsinki, Finland of the index individual, blood from the other family mem- 3 Neuroscience Center, University of Helsinki, Helsinki, Finland bers). Amplification of mtDNA was performed using 50 Neurogenetics (2018) 19:49–53

REPLI-g mitochondrial DNA kit and sequenced with an Hearing was within normal range in audiological Illumina HiSeq2000 platform. Sequence reads were aligned examination. to the Human mtDNA Cambridge reference sequence Her older brother (individual III-1), the first child of the (NC_012920) and variants called as previously described family, was also born at 34 weeks of gestational age. He had [11]. Call depth was in all measurements above 3300×. mild intellectual disability and attention deficit hyperactivity disorder, was able to walk and run, was verbal with sentences, Blue-native polyacrylamide gel electrophoresis and attended special education at school. His blood lactate (BN-PAGE) and immunoblotting was normal whereas CSF lactate was elevated. His brain MRI and 1H-MRS, cardiologic, and audiological examina- BN-PAGE was performed as described [12]. Native protein tions were normal. He had tortuosity of the retinal vessels; extracts were separated on NativePAGE Novex 4–16% Bis- otherwise, ophthalmological findings were normal. At the Tris acrylamide gels (Invitrogen) and transferred to PVDF age of 9 years, his height was − 1.3 SD, weight − 24%, and membranes. Immunoblotting was performed with antibodies: head circumference − 1.5 SD. CI-NDUFA9 (Abcam, #14713), CII-SDHA (Abcam, Detailed clinical data are presented in Table 1 including #14715), CIV-COXI (Abcam, #ab14705), and CV-ATP5A comparison with the previously reported subjects. (Abcam, #ab14748). Mitochondrial ATP6 m.8969G>A mutation identified High-resolution respirometry as the genetic cause

High-resolution respirometry was performed as described 6 Sanger sequencing of the entire mtDNA revealed an apparent- [13]. Briefly, 2 × 10 digitonin-permeabilized fibroblast cells ly homoplasmic point mutation m.8969G>A [p.Ser148Asn] were used per sample in a substrate-uncoupler-inhibitor pro- in MT-ATP6 in the muscle of individual III-4. To determine tocol on an Oroboros oxygraph-2k. Oxygen consumption was the pathogenicity and accurate heteroplasmy of the mutation, determined in the presence of pyruvate-glutamate malate and we screened the muscle DNA of individual III-4, and the subsequent activities by addition of substrates +ADP (CI), blood DNA from the affected brother (III-1) and five healthy +succinate (CI + CII), maximal uncoupled respiration by family members by NGS. The affected individuals (III-4 and FCCP titration, and CIV by ascorbate +TMPD. III-1) had high mutant load (79–95%) whereas the healthy family members had low or near zero percentages of mutant Results mtDNA (Fig. 1a).

Clinical data Consequences of the m.8969G>A mutation

We studied a family with two affected children. The index case, BN-PAGE revealed normal levels of assembled ATP synthase individual III-4, was the fourth child of nonconsanguineous as well as complexes of the respiratory chain in fibroblasts and parents. She was born preterm at 34 weeks of gestational age muscle of individual III-4 (Fig. 1b). Long exposure revealed and was small for date. She received red blood cell transfusion only a small fraction of the total ATP synthase pool not to be once. Heart ultrasound revealed a patent foramen ovale. Her assembled, indicating that m. 8969G>A variant does not spe- growth had been poor and developmental delay was noticed cifically affect ATP synthase assembly as in other ATP6 mu- during the second year of life. No regression had been noticed, tations [14]. Functional analysis by oxygen consumption but her skills improved slowly. She learned to walk indepen- showed decreased ADP-stimulated respiration in patient fi- dently at 4.5 years of age and talked with only a few words. broblasts (Fig. 1c). As state 3 respiration was decreased in Psychological testing showed mild to moderate intellectual dis- patient cells (state 3/base ratio in patient 3.1 (control 5.0), ability. At the age of 5 years her height was − 3 SD, weight − residual oxygen consumption after uncoupling (FCCP) dem- 26%, and head circumference − 3.5 SD. onstrated that the oxidative phosphorylation capacity was lim- Lactate was elevated in the blood and CSF. Brain MRI and ited by complex V. proton magnetic resonance spectroscopy at 2 years of age were normal. Muscle biopsy sample showed normal histology, but respiratory chain enzyme analysis showed normal en- Discussion zyme activities with decreased ATP production. Steatosis was found in the liver biopsy sample. At the age of 4 years, oph- We describe here two siblings with the maternally inherited thalmological examination showed mild tortuosity of the ret- m. 8969G>A mtDNA variant and a stable phenotype with inal vessels, and cardiologic examination was normal. lactic acidosis, poor growth and intellectual disability. The Neurogenetics (2018) 19:49–53 51

Table 1 Comparison of clinical characteristics of patients with m.8969G>A mutation

Subjects Burrage et al. Wen et al. Sallevelt This study This study et al. (individual III-1) (individual III-4)

Current age 6 y 14 y died at 9y 5y 7.5 mo Birth at 32+ NA NA 34+ 34+ gestational week Neonatal Respiratory, feeding NA NA Respiratory − problems Sensorineural + (neonatal) + (teen-age) NA –– hearing impairment Brain MRI Corpus callosum agenesia, gradus II IVH Brain atrophy NA Normal (7 y) Normal (2.5 y) (neonatal), metabolic stroke (4 y) Brain 1H–MRS NA Lactate peak (teen-age) NA Normal (7 y) Normal (2.5 y) Hematology Sideroblastic anemia, – NA – Neonatal anemia, one transfusion-dependent transfusion needed (2 mo →) Neurology Epilepsy (5 mo), severe developmental Epilepsy (teen-age), NA Mild intellectual Mild/moderate delay (non-ambulatory, non-verbal 6 y), muscle weakness disability (ambulant intellectual disability metabolic (teen-age) and verbal 9 y) (ambulant 5 y) strokes (4/6 y), muscle weakness and atrophy Renal problems NA IgA nephropathy NA –– Growth Failure to thrive, poor growth, gastrostomy NA NA Poor weight gain Poor weight gain for feeding Cardiology WPW WPW NA Normal Normal Lactate 6.1 (b) NA NA Normal (b) / 4.4 (csf) 6.5 (b) / 5.0 (csf) P-amino acids Alanine NA NA NE Alanine U- organic acids Fumarate, malate, B-hydroxybutyrate, NA NA NE Fumarate, lactate 3-hydroxybutyrate, lactate Muscle NA NA NA NE Normal histology Biochemical Cellular respiration decreased NA NA NE ATP production low analysis of muscle m.8969G>A 96% (b), 85% (f), 88% (m) 61% (b), 89% (k), 79% 95% (b, 79% (b) 96% (f), 95% (m) heteroplasmy (u) f, m) (tissue) Inheritance de novo maternal de novo maternal (heteroplasm- (49% /b, 57% /u) (9% /b) y /tissue) b blood, csf cerebro-spinal fluid, f fibroblasts, 1 H-MRS proton magnetic resonance spectroscopy, IVH intraventricular hemorrhage, k kidney, m muscle, mo months, NA not available NE not evaluated, p plasma, u urine, WPW Wolf-Parkinson-White syndrome, y years

phenotype in this family is much milder than in the previ- the m.8969G>A mutation, with a multi-organ disorder, man- ously reported cases. ifesting with IgA nephropathy at teen-age, progressing to ep- The patient reported by Burrage et al. had a severe prenatal- ilepsy, muscle weakness, hearing loss, brain atrophy, and elec- onset progressive disorder characterized by multi-organ in- trical abnormality of the heart (Wolff-Parkinson-White syn- volvement, including transfusion-dependent sideroblastic drome). Our subjects had stable developmental delay with anemia, failure to thrive, sensorineural hearing impairment, poor growth and elevated lactate, no exacerbations or other epilepsy, structural brain anomaly (agenesis of corpus organ involvements, and normal brain MRI. Interestingly, the callosum), severe developmental delay, and stroke-like epi- heteroplasmy of the variant in muscle was even higher in our sodes. Recently, Wen et al. reported a patient also carrying subject (individual III-4), 95%, compared to 88% of the 52 Neurogenetics (2018) 19:49–53

Fig. 1 a Pedigree with percentages of the m.8969G>A variant in the but a subcomplex of CV was observed in individual III-4’sfibroblasts muscle of the index subject (individual III-4) and in blood of other only (asterisk). c Trace of oxygen consumption rates in fibroblasts from family members, as determined by next-generation sequencing. b Blue- individual III-4 (pmol/s/mio cells). ADP-stimulated respiration was native PAGE and immunoblotting of respiratory chain complexes in decreased in her fibroblasts compared to control fibroblasts, whereas fibroblasts and muscle from individual III-4. Total levels of ATP uncoupled (ATPase-independent) respiration was normal synthase (CV) and respiratory chain complexes were similar to control, individual of Burrage et al., although the phenotype of our of mutant mtDNA in the central nervous system, or protective subject was substantially milder. Another case with early modifier genes in this family. death at 7.5 months of age and similar high heteroplasmy of Our report expands the phenotypic spectrum caused by this this variant (95% in blood, fibroblasts, and muscle) was pub- rare MT-ATP6 variant. Childhood-onset mitochondrial disorders lished by Sallevelt et al., but unfortunately without any addi- are often progressive, devastating disorders, but they should be tional clinical data [15]. taken into account even with milder, stable phenotypes. Both histologic examination of the muscle biopsy sample and respiratory chain enzyme activities were normal in our Acknowledgements The authors would like to thank the family for par- subject, but ATP production was reduced as a sign of defect ticipation in the study. We acknowledge Brendan Battersby for discus- sions, and the Finnish Institute for Molecular Medicine (FIMM) for DNA in oxidative phosphorylation. It has been inferred that oxida- sequencing services. We wish to thank the following funding sources for tive phosphorylation capacity in this specific mutant is caused support: Foundation for Pediatric Research (PI), the special governmental by a slower ATP synthesis rate rather than less efficient cou- subsidy for health sciences research of the Helsinki University Hospital. pling [9],whichwereplicatedbydemonstratingthereduced (PI and TL), University of Helsinki (CJC and AS), Swiss National Foundation and Novartis Foundation (CJB), Sigrid Jusélius Foundation, ADP-stimulated oxygen consumption rate in the patient fibro- Academy of Finland, Jane and Aatos Erkko Foundation (AS). blasts. The milder neurological phenotype of our subjects compared to controls could be explained by lower amount Neurogenetics (2018) 19:49–53 53

Compliance with ethical standards Wong L, Scaglia F (2014) Mitochondrial myopathy, lactic acidosis, and sideroblastic anemia (MLASA) plus associated with a novel de Conflict of interest The authors declare that they have no conflict of novo mutation (m.8969G>A) in the mitochondrial encoded ATP6 – interest. gene. Mol Genet Metab 113(3):207 212. https://doi.org/10.1016/j. ymgme.2014.06.004 9. Wen S, Niedzwiecka K, Zhao W, Xu S, Liang S, Zhu X, Xie H, Tribouillard-Tanvier D, Giraud M, Zeng C, Dautant A, Kucharczyk References R, Liu Z, di Rago J, Chen H (2016) Identification of G8969>A in mitochondrial ATP6 gene that severely compromises ATP synthase 1. Holt IJ, Harding AE, Petty RK, Morgan-Hughes JA (1990) A new function in a patient with IgA nephropathy. Sci Rep 6:36313. mitochondrial disease associated with mitochondrial DNA https://doi.org/10.1038/srep36313 heteroplasmy. Am J Hum Genet 46(3):428–433 10. Götz A, Isohanni P, Liljeström B, Rummukainen J, Nikolajev K, 2. Tatuch Y, Christodoulou J, Feigenbaum A, Clarke JT, Wherret J, Herrgård E, Marjavaara S, Suomalainen A (2012) Fatal neonatal Smith C, Rudd N, Petrova-Benedict R, Robinson BH (1992) lactic acidosis caused by a novel de novo mitochondrial G7453A – Heteroplasmic mtDNA mutation (T——G) at 8993 can cause tRNA-serine ((UCN)) mutation. Pediatr Res 72(1):90 94. https:// Leigh disease when the percentage of abnormal mtDNA is high. doi.org/10.1038/pr.2012.43 Am J Hum Genet 50(4):852–858 11. Sulonen A, Ellonen P, Almusa H, Lepistö M, Eldfors S, Hannula S, 3. de Vries DD, van Engelen BG, Gabreëls FJ, Ruitenbeek W, van Miettinen T, Tyynismaa H, Salo P, Heckman C, Joensuu H, Raivio Oost BA (1993) A second missense mutation in the mitochondrial T, Suomalainen A, Saarela J (2011) Comparison of solution-based ATPase 6 gene in Leigh’s syndrome. Ann Neurol 34(3):410–412. exome capture methods for next generation sequencing. Genome https://doi.org/10.1002/ana.410340319 Biol 12(9):R94. https://doi.org/10.1186/gb-2011-12-9-r94 4. Lamminen T, Majander A, Juvonen V, Wikström M, Aula P, 12. Leary SC, Sasarman F (2009) Oxidative phosphorylation: synthesis Nikoskelainen E, Savontous ML (1995) A mitochondrial mutation of mitochondrially encoded proteins and assembly of individual at nt 9101 in the ATP synthase 6 gene associated with deficient structural subunits into functional holoenzyme complexes. oxidative phosphorylation in a family with Leber hereditary optic Methods Mol Biol 554:143–162. https://doi.org/10.1007/978-1- neuroretinopathy. Am J Hum Genet 56(5):1238–1240 59745-521-3_10 5. Rantamäki MT, Soini HK, Finnilä SM, Majamaa K, Udd B (2005) 13. Courage C, Jackson CB, Hahn D, Euro L, Nuoffer J, Gallati S, Adult-onset ataxia and polyneuropathy caused by mitochondrial Schaller A (2017) SDHA mutation with dominant transmission 8993T–>C mutation. Ann Neurol 58(2):337–340. https://doi.org/ results in complex II deficiency with ocular, cardiac, and neurologic 10.1002/ana.20555 involvement. Am J Med Genet A 173(1):225–230. https://doi.org/ 6. Thyagarajan D, Shanske S, Vazquez-Memije M, De Vivo D, 10.1002/ajmg.a.37986 DiMauro S (1995) A novel mitochondrial ATPase 6 point mutation 14. Jackson CB, Hahn D, Schröter B, Richter U, Battersby BJ, Schmitt- in familial bilateral striatal necrosis. Ann Neurol 38(3):468–472. Mechelke T, Marttinen P, Nuoffer J, Schaller A (2017) A novel https://doi.org/10.1002/ana.410380321 mitochondrial ATP6 frameshift mutation causing isolated complex 7. Ware SM, El-Hassan N, Kahler SG, Zhang Q, Ma Y, Miller E, V deficiency, ataxia and encephalomyopathy. Eur J Med Genet Wong B, Spicer RL, Craigen WJ, Kozel BA, Grange DK, Wong 60(6):345–351. https://doi.org/10.1016/j.ejmg.2017.04.006 L (2009) Infantile cardiomyopathy caused by a mutation in the 15. Sallevelt SC, de Die-Smulders CE, Hendrickx AT, Hellebrekers overlapping region of mitochondrial ATPase 6 and 8 genes. J DM, de Coo IF, Alston CL, Knowles C, Taylor RW, McFarland Med Genet 46(5):308–314. https://doi.org/10.1136/jmg.2008. R, Smeets HJ (2017) De novo mtDNA point mutations are common 063149 and have a low recurrence risk. J Med Genet 54(2):73– 83. https:// 8. Burrage LC, Tang S, Wang J, Donti TR, Walkiewicz M, Luchak doi.org/10.1136/jmedgenet-2016-103876 JM, Chen L, Schmitt ES, Niu Z, Erana R, Hunter JV, Graham BH,