**1. Introduction**

Mitochondria provide aerobic eukaryotes with cellular energy by generating adenosine triphosphate (ATP) through the process of oxidative phosphorylation (OXPHOS). As a first step, electrons

from nutrients (such as carbohydrates and fatty acids) are transferred by four complexes (CI-CIV) anchored to the mitochondrial inner membrane. This results in a transmembrane electrochemical proton gradient, which drives ATP synthesis from adenosine di-phosphate (ADP) and inorganic phosphate (Pi) by the ATP synthase (CV).

Defects of the OXPHOS system have been implicated in a broad spectrum of human diseases. Typical clinical traits include encephalopathies, cardiomyopathies, myopathies, visual/hearing, liver and renal dysfunctions [1–3]. Many of these diseases are caused by mitochondrial DNA (mtDNA) mutations. This DNA encodes 13 protein subunits of the OXPHOS system and a number of RNAs necessary for the synthesis of these proteins inside the organelle [4]. Pathogenic mtDNA mutations often co-exist with non-mutated mtDNA (heteroplasmy) and are highly recessive (only rare cases of dominancy were reported [5]), which makes it difficult to know how mitochondrial function is affected in patient's cells and tissues. Furthermore, because of the high mutability of the mitochondrial genome due to its exposure to damaging oxygen species (ROS) and the poor activity of mitochondrial DNA repair systems, it can be difficult to establish the pathogenicity of a mtDNA variant [4]. Additionally, the effects of deleterious mtDNA mutations may be aggravated by nucleotide changes in nuclear and mitochondrial DNA that are not pathogenic per se (the so-called modifier genes) [6,7].

Being amenable to mitochondrial genetic transformation [8], and owing to its good fermenting capability that enables survival after the loss of oxidative phosphorylation and inability to stably maintain heteroplasmy [9], *Saccharomyces cerevisiae* has been used as a model to investigate mtDNA mutations identified in patients. We have exploited these attributes to study *MT-ATP6* gene mutations found in patients. This gene encodes the subunit *a* of ATP synthase, which is involved in moving protons through the membrane domain (FO) of ATP synthase coupled to ATP synthesis [10–15]. These yeast-based studies helped to better define the functional consequences of subunit *a* mutations and provided support for the pathogenicity of rare alleles identified in only a limited number of cases like m.8851T>C [12] and m.8969G>A [16]. Importantly, the extent to which yeast ATP synthase was affected correlated with disease severity, which reflects the strong evolutionary conservation of the regions of subunit *a* where these mutations localize [17–26].

Herein we report the identification of a novel *MT-ATP6* variant, m.8909T>C. It converts into serine a highly conserved phenylalanine residue of subunit *a* (*aF128S* in humans; *aF145S* in yeast). We detected it by entirely sequencing the mtDNA of a patient with an extreme clinical presentation including brain, kidney and muscular dysfunctions leading to premature death at the age of 14. This patient also carried the well-known pathogenic m.3243A>G mutation in mt-tRNALeu(1) (*MT-TL1*). In a yeast model of the m.8909T>C variant, ATP synthase assembly/stability was significantly compromised to an extent comparable to that seen previously in yeast models of other *ATP6* mutations with a well-established pathogenicity. These findings indicate that alone the m.8909T>C variant has the potential to compromise human health.
