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 nephrop- athy 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 histolo- gy, 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