Localization of a New Type of X-Linked Liver Glycogenosis to the Chromosomal Region Xp22 Containing the Liver α-Subunit of Phosphorylase Kinase (PHKA2)

We describe here a new type of X-linked liver glycogen storage disease. The main symptoms include liver enlargement and growth retardation. The clinical and biochemical abnormalities of this glycogenosis are similar to those of classical X-linked liver glycogenosis due to phosphorylase kinase deficiency (XLG). However, in contrast to patients with XLG, the patients described here have no reduced phosphorylase kinase activity in erythrocytes and leukocytes, and no enzyme deficiency could be found. Linkage analysis of four families with this X-linked type of liver glycogenosis assigned the disease gene to Xp22. Lod scores obtained with the markers DXS987, DXS207, and DXS999 were 3.97, 2.71, and 2.40, respectively, all at 0% recombination. Multipoint linkage analysis localized the disease gene between DXS143 and DXS989 with a maximum lod score of 4.70 at θ = 0, relative to DXS987. As both the classical XLG gene and the liver α-subunit of PHK (PHKA2) are also located in Xp22, this variant type of XLG may be allelic to classical XLG, and both diseases may be caused by mutations in PHKA2. Therefore, we propose to classify XLG as XLG type I (the classical type of XLG) and XLG type II (the variant type of XLG).     

One mutation,three phenotypes: novel metabolic insights on MELAS,MIDD and myopathy caused by the m.3243A > G mutation

Metabolomics - Studies investigating crop resistance to abiotic and biotic stress have largely focused on plant responses to singular forms of stress and individual biochemical pathways that only...     

Biochemical examination of fibroblasts in the diagnosis and research of oxidative phosphorylation (OXPHOS) defects

The oxidative phosphorylation system (OXPHOS) is organized in five multi-protein complexes, comprising four complexes (I-IV) of the respiratory chain and ATP synthase (complex V). OXPHOS has a vital role in cellular energy metabolism and ATP production. Enzyme analysis of individual OXPHOS complexes in a skeletal muscle biopsy remains the mainstay of the diagnostic process for patients suspected of mitochondrial cytopathy. A fresh muscle biopsy is preferable to a frozen muscle biopsy because of the possibility to measure the overall capacity of the OXPHOS system. In about 25% of patients referred to our center for muscle biopsy, reduced substrate oxidation rates and ATP + creatine phosphate production rates were found without any defect in complex I-V and the pyruvate dehydrogenase complex. In a subset of patients it is necessary to investigate fibroblasts for diagnostic purposes. The indications for biochemical investigations in fibroblasts are: (a) If no muscle sample is available; (b) If prenatal diagnosis is required; (c) To clarify the results obtained in muscle tissue if no clear-cut diagnosis can be made; (d) If molecular-genetic investigations are required; (e) For research purposes. Fibroblasts are less suitable than fresh muscle for investigating respiratory chain disorders, for the following reasons: (i) A defect that is present in a muscle is not always expressed in fibroblasts. (ii) Exclusion of a defect in fibroblasts does not exclude the diagnosis with regard to muscle. (iii) A specific pattern of abnormalities demonstrated in fibroblasts may not be reflected in muscle tissue. (iv) Enzyme deficiencies found in muscle are generally more pronounced than in fibroblasts. An exact diagnosis of respiratory chain defects is a prerequisite for rational therapy and genetic counseling. Provided guidelines for specimen collection are followed, there are now reliable methods for identifying respiratory chain defects.     

Serial isoelectric focusing as an effective and economic way to obtain maximal resolution and high-throughput in 2D-based comparative proteomics of scarce samples: proof-of-principle

In 2D-based comparative proteomics of scarce samples, such as limited patient material, established methods for prefractionation and subsequent use of different narrow range IPG strips to increase overall resolution are difficult to apply. Also, a high number of samples, a prerequisite for drawing meaningful conclusions when pathological and control samples are considered, will increase the associated amount of work almost exponentially. Here, we introduce a novel, effective, and economic method designed to obtain maximum 2D resolution while maintaining the high throughput necessary to perform large-scale comparative proteomics studies. The method is based on connecting different IPG strips serially head-to-tail so that a complete line of different IPG strips with sequential pH regions can be focused in the same experiment. We show that when 3 IPG strips (covering together the pH range of 3-11) are connected head-to-tail an optimal resolution is achieved along the whole pH range. Sample consumption, time required, and associated costs are reduced by almost 70%, and the workload is reduced significantly.     

Decreased agonist-stimulated mitochondrial ATP production caused by a pathological reduction in endoplasmic reticulum calcium content in human complex I deficiency

Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.     

Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Lack of the mitochondrial protein acylglycerol kinase causes Sengers syndrome

Exome sequencing of an individual with congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis, all typical symptoms of Sengers syndrome, discovered two nonsense mutations in the gene encoding mitochondrial acylglycerol kinase (AGK). Mutation screening of AGK in further individuals with congenital cataracts and cardiomyopathy identified numerous loss-of-function mutations in an additional eight families, confirming the causal nature of AGK deficiency in Sengers syndrome. The loss of AGK led to a decrease of the adenine nucleotide translocator in the inner mitochondrial membrane in muscle, consistent with a role of AGK in driving the assembly of the translocator as a result of its effects on phospholipid metabolism in mitochondria.     

Pharmacological targeting of mitochondrial complex I deficiency: the cellular level and beyond

Complex I (CI) represents a major entry point of electrons in the mitochondrial electron transport chain (ETC). It consists of 45 different subunits, encoded by the mitochondrial (mtDNA) and nuclear DNA (nDNA). In humans, mutations in nDNA-encoded subunits cause severe neurodegenerative disorders like Leigh Syndrome with onset in early childhood. The pathophysiological mechanism of these disorders is still poorly understood. Here we summarize the current knowledge concerning the consequences of nDNA-encoded CI mutations in patient-derived cells, present mouse models for human CI deficiency, and discuss potential treatment strategies for CI deficiency.