A Theoretical Model of Mitochondrial ATP Synthase Deficiencies. The Role of Mitochondrial Carriers

The m.8993T>G mutation of the mitochondrial MT-ATP6 gene is associated with NARP syndrome (neuropathy, ataxia and retinitis pigmentosa). The equivalent point mutation introduced in yeast Saccharomyces cerevisiae mitochondrial DNA considerably reduced the activity of ATP synthase and of cytochrome-c-oxidase, preventing yeast growth on oxidative substrates. The overexpression of the mitochondrial oxodicarboxylate carrier (Odc1p) was able to rescue the growth on the oxidative substrate by increasing the substrate-level phosphorylation of ADP coupled to the conversion of α-ketoglutarate (AKG) into succinate with an increase in Complex IV activity. Previous studies showed that equivalent point mutations in ATP synthase behave similarly and can be rescued by Odc1p overexpression and/or the uncoupling of OXPHOS from ATP synthesis. In order to better understand the mechanism of the ATP synthase mutation bypass, we developed a core model of mitochondrial metabolism based on AKG as a respiratory substrate. We describe the different possible metabolite outputs and the ATP/O ratio values as a function of ATP synthase inhibition.

A model of mitochondrial ATP synthase deficiencies. The role of mitochondrial carriers.

The m.8993T>G mutation of the mitochondrial MT-ATP6 gene is associated with NARP syndrome (Neuropathy, Ataxia and Retinitis Pigmentosa). The equivalent point mutation introduced in yeast Saccharomyces cerevisiae mitochondrial DNA considerably reduced the activity of ATP synthase and of cytochrome-C-oxidase preventing yeast growth on oxidative substrates. The overexpression of the mitochondrial oxodicarboxylate carrier (Odc1p) is able to rescue the growth on oxidative substrate in stimulating the substrate-level phosphorylation of ADP cou-pled to conversion of α-ketoglutarate (AKG) into succinate with an increase in Complex IV activity. In order to better understand the mechanism of ATP synthase mutation bypass, we developed a core model of mitochondrial metabolism based on AKG as respiratory substrate. We describe the different possible metabolite output and the ATP/O ratio values as a function of ATP synthase inhibition.

SEROLOGICAL AND MOLECULAR EVALUATION OF MIGRATORY TRICHINELLA SPIRALIS LARVAE IN BLOOD OF HUMANS IN KADUNA METROPOLIS, NIGERIA

Trichinellosis is an important food-borne zoonotic disease with public health implications and a worldwide distribution. In this study, Polymerase Chain Reaction (PCR) procedure using species specific ATP6 primers was used to detect the presence of migratory Trichinella spiralis larval mitochondrial ATP6 synthase F0 subunit (ATP6) gene, after detection of antibodies to Trichinella excretory-secretory (E/S) antigen using Enzyme-linked Immunosorbent Assay (ELISA), in blood of humans in Kaduna metropolis, Nigeria. The sera of 210 participants were tested for antibodies to Trichinella E/S antigen. Overall seroprevalence rate of 39% (82/210) was recorded using ELISA. Out of the 9 ELISA samples selected randomly, PCR detected migratory Trichinella spiralis larval ATP6 gene in 4 (44.4%) at the amplicon size of 250 base pairs using the whole blood of the participants. The 9 samples comprised 7 seropositive and 2 seronegative. The bands at lanes 1, 2, 3 and 4 were positive for ATP6 while lanes 5,6,7,8 and 9 were negative for ATP6. Lanes 4 and 5 were ELISA negative for anti-Trichinella antibodies. One in 5 of the 128 ELISA negative samples was positive for ATP6 representing a 25.6% prevalence rate by extrapolation. PCR using ATP6 gene as a genetic marker is valuable for the detection of T. spiralis migratory larvae in blood samples of humans and consequently the early diagnosis of trichinellosis in humans.

Case Report: Identification of a Novel Variant (m.8909T>C) of Human Mitochondrial ATP6 Gene and Its Functional Consequences on Yeast ATP Synthase

With the advent of next generation sequencing, the list of mitochondrial DNA (mtDNA) mutations identified in patients rapidly and continuously expands. They are frequently found in a limited number of cases, sometimes a single individual (as with the case herein reported) and in heterogeneous genetic backgrounds (heteroplasmy), which makes it difficult to conclude about their pathogenicity and functional consequences. As an organism amenable to mitochondrial DNA manipulation, able to survive by fermentation to loss-of-function mtDNA mutations, and where heteroplasmy is unstable, Saccharomyces cerevisiae is an excellent model for investigating novel human mtDNA variants, in isolation and in a controlled genetic context. We herein report the identification of a novel variant in mitochondrial ATP6 gene, m.8909T>C. It was found in combination with the well-known pathogenic m.3243A>G mutation in mt-tRNALeu. We show that an equivalent of the m.8909T>C mutation compromises yeast adenosine tri-phosphate (ATP) synthase assembly/stability and reduces the rate of mitochondrial ATP synthesis by 20–30% compared to wild type yeast. Other previously reported ATP6 mutations with a well-established pathogenicity (like m.8993T>C and m.9176T>C) were shown to have similar effects on yeast ATP synthase. It can be inferred that alone the m.8909T>C variant has the potential to compromise human health.

Molecular Basis of the Pathogenic Mechanism Induced by the m.9191T>C Mutation in Mitochondrial ATP6 Gene

Probing the pathogenicity and functional consequences of mitochondrial DNA (mtDNA) mutations from patient’s cells and tissues is difficult due to genetic heteroplasmy (co-existence of wild type and mutated mtDNA in cells), occurrence of numerous mtDNA polymorphisms, and absence of methods for genetically transforming human mitochondria. Owing to its good fermenting capacity that enables survival to loss-of-function mtDNA mutations, its amenability to mitochondrial genome manipulation, and lack of heteroplasmy, Saccharomyces cerevisiae is an excellent model for studying and resolving the molecular bases of human diseases linked to mtDNA in a controlled genetic background. Using this model, we previously showed that a pathogenic mutation in mitochondrial ATP6 gene (m.9191T>C), that converts a highly conserved leucine residue into proline in human ATP synthase subunit a (aL222P), severely compromises the assembly of yeast ATP synthase and reduces by 90% the rate of mitochondrial ATP synthesis. Herein, we report the isolation of intragenic suppressors of this mutation. In light of recently described high resolution structures of ATP synthase, the results indicate that the m.9191T>C mutation disrupts a four α-helix bundle in subunit a and that the leucine residue it targets indirectly optimizes proton conduction through the membrane domain of ATP synthase.

The m.9143T>C Variant: Recurrent Infections and Immunodeficiency as an Extension of the Phenotypic Spectrum in MT-ATP6 Mutations?

Pathogenic variants in the MT-ATP6 are a well-known cause for maternally inherited mitochondrial disorders associated with a wide range of clinical phenotypes. Here, we present a 31- year old female with insulin-dependent diabetes mellitus, recurrent lactic acidosis and ketoacidosis recurrent infections with suspected immunodeficiency with T cell lymphopenia and hypogammaglobulinemia as well as proximal tetraparesis with severe muscle and limb pain and rapid physical exhaustion. Muscle biopsy and respiratory chain activities were normal. Single-exome sequencing revealed a variant in the MT-ATP6 gene: m.9143T>C. Analysis of further specimen of the index and mother (segregation studies) revealed the highest mutation load in muscle (99% level of mtDNA heteroplasmy) of the index patient. Interestingly, acute metabolic and physical decompensation during recurrent illness was documented to be a common clinical feature in patients with MT-ATP6 variants. However, it was not mentioned as a key symptom. Thus, we suggest that the clinical spectrum might be expanded in ATP6-associated diseases.

Mitochondrial genome variability in sudden cardiac death

Проведено исследование полиморфизма митохондриального генома и числа копий мтДНК в клетке при внезапной сердечной смерти (ВСС). Выявлено более низкое значение числа копий мтДНК в миокарде умерших ВСС. Анализ полиморфизма мтДНК показал более высокую частоту гаплогруппы Н1 в выборке ВСС, по сравнению с популяцией, а также более высокую частоту миссенс-варианта Ala177Thr в гене ATP6 у лиц, переживших остановку сердца. Результаты исследования указывают на связь полиморфизма мтДНК и числа копий мтДНК в клетке с риском внезапной сердечной смерти. We studied mitochondrial DNA polymorphism and mtDNA copy number in sudden cardiac death (SCD). In the SCD group, a lower mtDNA copy number was found in the myocardium. Analysis of mtDNA polymorphism revealed a higher frequency of H1 haplogroup in the SCD samples, compared with the population, as well as higher frequency of missense variant Ala177Thr in ATP6 gene in the group of cardiac arrest survivors. The results of the study suggest association of mtDNA polymorphism and mtDNA copy number with the risk of sudden cardiac death.

Association of a point mutation (m.9176T>G) of the MT-ATP6 gene with Leigh syndrome: A case report

Leigh Syndrome (LS) is an uncommon progressive neurodegenerative mitochondrial disorder. The condition is characterized by progressive mental and developmental disabilities (psychomotor regression) and commonly brings about death within a few years of diagnosis, more often due to respiratory failure. In a small number of patients the disorder does not manifest until adulthood. The principal indications of Leigh syndrome found in early stages typically are diarrhea, vomiting, and difficulty swallowing (dysphagia), which disturbs eating. These problems usually result in powerlessness to develop and put on weight under the normal rate (failure to thrive). Serious movement and muscle problems are basic in Leigh syndrome. In this case report, we introduce the molecular and clinical features of a 19-year-old female as proband, and also, we study other members of the family consequently. The m.9176T>G heteroplasmic mutation in the MT-ATP6 gene was detected by high-resolution melt (HRM) and DNA sequencing techniques. Similarly, the m.9176T>G was heteroplasmic in the mother. In conclusion, this report in compliance with previous studies underlines the necessity of further research on prenatal distinguishing proof of the responsible mutations and avoidance of the disease in families with known cases.