The mitochondrial DNA variant m.9032T > C in MT-ATP6 encoding p.(Leu169Pro) causes a complex mitochondrial neurological syndrome
Introduction
Mitochondrial diseases are a heterogeneous group of diseases caused by pathogenic variants in either the nuclear or mitochondrial genome. Diagnosing mitochondrial disease due to changes in mitochondrial DNA (mtDNA) has been difficult due to the large diversity in clinical and biochemical phenotypes (Russell and Turnbull, 2014, Taylor and Turnbull, 2005). Diagnosing disease due to mitochondrial complex V variants has been particularly challenging given the limited availability of tools to confirm their functional significance. Complex V (ATP synthase EC 7.1.2.2) is composed of 15 structural and 2 assembly subunits, of which two, ATP6 and ATP8, are mtDNA-encoded (Jonckheere et al., 2012, Fernández-Vizarra et al., 2009). The mitochondrial DNA encoded MT-ATP6 gene product, ATPase subunit a, is composed of six transmembrane domains. It contributes to the proton pore of the complex and is essential to couple proton translocation to the rotation of the complex V enzymatic subunit involved in ATP synthesis (Schon et al., 2001). Recent reviews of all available cases of complex V deficiency due to pathogenic variants in MT-ATP6 (OMIM 500015) highlighted the great diversity of clinical and biochemical features that exists among genetic variants (Stendel et al., 2020, Ganetzky et al., 2019). Currently 33 MT-ATP6 variants are either known or suspected to cause disease, but 82% of all published cases are due to just 4 of these variants (Ganetzky et al., 2019). Defining the phenotype and the functional impact of those less well-described variants is of importance to better understand the disease pathophysiology, and to facilitate diagnosis in those with less specific phenotypes. Assays to evaluate the functional impact of a complex V variant have shown a decrease in complex V holoenzyme assembly or integrity, abnormal mitochondrial membrane potential (either increased or decreased), reduced ATP synthetic rate, ATP hydrolysis rate, ATP steady state levels, or an impact on oxygen consumption levels in the coupled versus uncoupled state (Ganetzky et al., 2019, Morava et al., 2006). The most reliable way today to prove the pathogenic nature of a variant is the identification in unrelated, yet similarly affected patients (Ganetzky et al., 2019).
In this report we describe a 7 year-old male with a complex phenotype due to m.9032T > C, p.(Leu169Pro) in MT-ATP6. This variant has been reported previously only in a single family of a proband and mother both with a neuropathy, ataxia, retinitis pigmentosa (NARP) phenotype (Schon et al., 2001). We document additional functional and biochemical findings providing functional confirmation of the pathogenic nature of this variant.
Section snippets
Clinical studies
All procedures were done in accordance with the standards of the ethics committee in accordance with the Helsinki Declaration of 1975 and as revised in 2000. The patient is enrolled in a Colorado Institutional Review Board approved study (COMIRB# 16-0146), and informed consent was obtained for this study. Fibroblasts were further studied in agreement with COMIRB# 18-0128. Clinical information was obtained via chart review of the patient's medical record, and neuroimaging was reviewed by a
Case report
The patient was born to non-consanguineous parents of mainly mixed European descent after a normal pregnancy by C-section for failure of progression of labor. After a normal neonatal period, he presented at age 2.5 months with myoclonic movements identified as right sided focal partial epilepsy and elevated lactic acid of up to 5.8 mM, but reportedly normal brain magnetic resonance imaging (MRI). He was initially treated with levetiracetam but developed choreatic movements that impaired fine
Mitochondrial genetic studies
Next generation sequencing of mtDNA revealed a variant m.9032T > C, p.(Leu169Pro) at a heteroplasmy level of 94.1% in blood and 96.6% in skeletal muscle. Studies of blood samples of the asymptomatic mother and sister did not detect this variant. The mtDNA content in liver was 54% of the mean value of age and tissue matched controls. The variant m.9032C is not present in 30,181 controls in mitowheel (www.mitowheel.org) and in 50,175 controls in mitomap (www.mitomap.org). It is predicted to be
Discussion
We report on a second independent patient with mitochondrial disease due to the m.9032T > C variant in MT-ATP6 encoding p.(Leu169Pro). The clinical characteristics of all reported patients are summarized in Table 2. In the previously reported family (López-Gallardo et al., 2014), the proband showed 70% heteroplasmy in blood. His history was notable for mild motor delays, mild ataxia and hypoactive deep tendon reflexes on exam and cerebellar vermian atrophy. He also had pigmentary retinopathy
Conclusions
We conclude that the pathogenic variant m.9032T > C causes a NARP phenotype at lower heteroplasmy level, and at high heteroplasmy level a more complex, slowly progressive ataxic phenotype with growth impairment and severe developmental delay. The varied phenotype and inability to directly assess the enzymatic function of complex V often makes MT-APT6-related complex V deficiency challenging to diagnose. We propose the use of blue native PAGE with in-gel activity staining be pursued in the
Contributions of authors
Kaz M. Knight: drafting first manuscript, functional laboratory studies, and critical rewriting of the manuscript, Emily Shelkowitz: drafting first manuscript, patient care data, and critical rewriting of the manuscript, Austin A. Larson: patient care data and critical rewriting of the manuscript, David M. Mirsky: patient care data and critical rewriting of the manuscript, Yue Wang: molecular laboratory studies and critical rewriting of the manuscript, Ting Chen: molecular laboratory studies
Ethics approval
Studies at the University of Colorado were done following IRB-approved protocols 16-0146 and 18-0128. Written informed consent was obtained prior to initiation of research as outlined in the IRB-approved study.
IACUC: No animals were used in this study.
Declaration of interest statement
JVH and AAL participate in clinical trials of mitochondrial disorders by Stealth Biotherapeutics, Inc. All other authors deny any real or apparent conflict of interest in the field of mitochondrial diseases.
Acknowledgments
Financial support for this study was received from Children’s Hospital Colorado Foundation, Summits for Samantha and Miracles for Mito (JVH, MWF, KMK). JVH, KMK, and MWF are supported by a grant from the National Institutes of Health, NIH U54NS078059 for the North American Mitochondrial Disease Consortium (NAMDC). NAMDC is part of Rare Diseases Clinical Research Network (RDCRN), an initiative of the Office of Rare Diseases Research (ORDR), NCATS. This consortium is funded through collaboration
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