Mitochondrial complex V (CV) subunit gene mutations cause a variety of severe metabolic diseases that impair child health and development, with strokes, neuropathy, ataxia, vision loss, and cardiomyopathy. There is no clinically-approved assay of CV function, therefore, making the diagnosis is challenging. The discovery that metabolites downstream of Carbonic Anhydrase 5A (CA5A) are recurrently abnormal in patients with CV deficiency has profound implications on developing targeted diagnostic testing for this profound energy deficiency. We Hypothesize that (a) CV and Carbonic Anhydrase 5A (CA5A) physically associate, therefore, mutations in CV cause CA5A deficiency and (b) scrutiny of the metabolites downstream of CA5A will provide novel biomarkers for CV deficiency.
Specific Aims of this work are to [Aim 1] Evaluate incidence of CV deficiency among patients with a newborn screen consistent with CA5A deficiency; [Aim 2] Determine the presence and degree of CA5A impairment in human fibroblast, cybrid and liver cell line models of CV disease; and [Aim 3] Determine if there is a physical association between CA5A and CV. Methods will sequencing CV subunit genes in subjects with abnormal newborn screens consistent with CA5A dysfunction (prospective and retrospective cohort), direct measurement of CA5A function using stable isotope-labeled acetate and in vitro cellular assessment of targeted and untargeted metabolites (Orbitrap liquid chromatography/mass spectrometry) in fibroblasts and hepatic cell lines from healthy individuals, genetic-based CV diseases and pharmacologic inhibition (oligomycin). Targeted metabolomics will focus on metabolites downstream of CA5A: amino acids, particularly citrulline (ultra-high-performance liquid chromatrography) and the organic acids propionic acid and hydroxyisovaleric acid (gas chromatography/mass spectrometry). determination of CA5A function using stable isotope studies (13C-labeled acetate incubation, isotope-ratio mass spectrometry) in transmitochondrial cybrid, hepatic and fibroblast cell lines from healthy individuals, genetic-based CV diseases and pharmacologic CV inhibition (oligomycin). We will also investigate the in vitro physical interaction of CA5A and CV (co-immunoprecipitation, blue native gel) in normal and genetic-based CV disease hepatocyte cell- lines. These studies will rigorously investigate the interaction between CA5A function and CV deficiency and new potential biomarkers for diagnosing CV disease. Our long-term goal is to expand the diagnostic and treatment options available for human CV deficiency, for which no treatment currently exists. These studies will establish the foundation of which to develop future clinical diagnostic assays to rapidly and precisely diagnosis CV disease and will lead to a new understanding of the role of CV in coordinating mitochondrial biochemical pathways, providing a novel target for future therapies.
Genetic diseases that cause primary mitochondrial respiratory chain deficiency result in severe disease, affecting nearly every organ system, including brain, heart, liver and eyes. Making a diagnosis of primary respiratory chain deficiency is difficult, especially in the subset of diseases that affect Complex V (ATP Synthase; CV), the primary source of energy in the cell. This work seeks to understand how CV interacts with a critical mitochondrial enzyme, carbonic anhydrase 5A (CA5A), as a potential avenue for future diagnostic biomarkers and therapies in this disease.
