Deregulation of Genes Related to Iron and Mitochondrial Metabolism in Refractory Anemia with Ring Sideroblasts

The presence of SF3B1 gene mutations is a hallmark of refractory anemia with ring sideroblasts (RARS). However, the mechanisms responsible for iron accumulation that characterize the Myelodysplastic Syndrome with ring sideroblasts (MDS-RS) are not completely understood. In order to gain insight in the molecular basis of MDS-RS, an integrative study of the expression and mutational status of genes related to iron and mitochondrial metabolism was carried out. A total of 231 low-risk MDS patients and 81 controls were studied. Gene expression analysis revealed that iron metabolism and mitochondrial function had the highest number of genes deregulated in RARS patients compared to controls and the refractory cytopenias with unilineage dysplasia (RCUD). Thus mitochondrial transporters SLC25 (SLC25A37 and SLC25A38) and ALAD genes were over-expressed in RARS. Moreover, significant differences were observed between patients with SF3B1 mutations and patients without the mutations. The deregulation of genes involved in iron and mitochondrial metabolism provides new insights in our knowledge of MDS-RS. New variants that could be involved in the pathogenesis of these diseases have been identified.     

Sequence analysis of the complete mitochondrial genome in patients with mitochondrial encephaloneuromyopathies lacking the common pathogenic DNA mutations

The purpose of this study was to identify novel mitochondrial deoxyribonucleic acid (mtDNA) mutations in a series of patients with clinical and/or morphological features of mitochondrial dysfunction, but still no genetic diagnosis. A heterogeneous group of clinical disorders is caused by mutations in mtDNA that damage respiratory chain function of cell energy production. We developed a method to systematically screen the entire mitochondrial genome. The sequence-data were obtained with a rapid automated system. In the six mitochondrial genomes analysed we found 20 variants of the revised Cambridge reference sequence [Nat. Genet. 23 (1999) 147]. In skeletal muscle nineteen novel mtDNA variants were homoplasmic, suggesting secondary pathogenicity or co-responsibility in determination of the disease. In one patient we identified a novel heteroplasmic mtDNA mutation which presumably has a pathogenic role. This screening is therefore useful to extend the mtDNA polymorphism database and should facilitate definition of disease-related mutations in human mtDNA.     

Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy

We have investigated nine children with infantile onset of mitochondrial myopathy and two adults with myoclonus epilepsy and ragged-red fibers (MERRF) and chronic progressive external ophthalmoplegia (CPEO), respectively. These patients lacked any of the previously known pathogenic tRNA mutations. Southern blot analysis of muscle mtDNA revealed no deletions. The tRNA genes of muscle mtDNA were sequenced. Restriction enxyme analysis of PCR fragments was performed to verify the presence of the mutations identified by automatic sequencing. Several tRNA mutations were found, but they were all homoplasmic. Furthermore, the mutations were either present in controls or did not change nucleotides conserved between species. This strongly suggests that none of the tRNA mutations identified in the 11 patients with mitochondrial encephalomyopathy was pathogenic. It can thus be concluded that mitochondrial tRNA mutations and mtDNA deletions probably are an infrequent cause of mitochondrial disorders in infants. Patients with MERRF and CPEO may lack both pathogenic point mutations of tRNA genes and deletions of mtDNA.     

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1–2 kb, digested in fragments of 90–600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA^Leu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA^Leu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.     

Role of mitochondrial DNA in diabetes Mellitus Type I and Type II

Morbidity and mortality from diabetes mellitus and associated illnesses is a major problem across the globe. Anti-diabetic medicines must be improved despite existing breakthroughs in treatment approaches. Diabetes has been linked to mitochondrial dysfunction. As a result, particular mitochondrial diabetes kinds like MIDD (maternally inherited diabetes & deafness) and DAD (diabetic autonomic dysfunction) have been identified and studied (diabetes and Deafness). Some mutations as in mitochondrial DNA (mtDNA), that encodes for a significant portion of mitochondrial proteins as well as mitochondrial tRNA essential for mitochondrial protein biosynthesis, are responsible for hereditary mitochondrial diseases. Tissue-specificity and heteroplasmy have a role in the harmful phenotype of mtDNA mutations, making it difficult to generalise findings from one study to another. There are a huge increase in the number for mtDNA mutations related with human illnesses that have been identified using current sequencing technologies. In this study, we make a list on mtDNA mutations linked with diseases and diabetic illnesses and explore the methods by which they contribute to the pathology's emergence.     

Mitochondriale Erkrankungen im Erwachsenenalter

The most frequent manifestation of mitochondrial disease in adults is chronic progressive external ophthalmoplegia (CPEO) that can present with variable multisystemic involvement. Molecular genetically single mtDNA deletions are identified in more than half of the patients associated with mainly sporadic CPEO. There are also autosomal dominant and recessive forms of CPEO due to mutations in nuclear genes that are important for mtDNA replication resulting in multiple mtDNA deletions. Other common multisystemic disorders are MELAS syndrome and MERRF syndrome due to maternally inherited mtDNA point mutations. Leber??s hereditary optic neuropathy is a frequent mitochondrial disorder without multisystemic involvement, which is also due to mtDNA point mutations. In addition to classical mitochondrial disorders there are patients with mitochondrial disease showing non-characteristic sometimes monosymptomic phenotypes (e.g. myopathy or epilepsy) that are difficult to recognize.     

MtDNA mutations are a common cause of severe disease phenotypes in children with Leigh syndrome

Leigh syndrome is a common clinical manifestation in children with mitochondrial disease and other types of inborn errors of metabolism. We characterised clinical symptoms, prognosis, respiratory chain function and performed extensive genetic analysis of 25 Swedish children suffering from Leigh syndrome with the aim to obtain insights into the molecular pathophysiology and to provide a rationale for genetic counselling. We reviewed the clinical history of all patients and used muscle biopsies in order to perform molecular, biochemical and genetic investigations, including sequencing the entire mitochondrial DNA (mtDNA), the mitochondrial DNA polymerase (POLGA) gene and the surfeit locus protein 1 (SURF1) gene. Respiratory chain enzyme activity measurements identified five patients with isolated complex I deficiency and five with combined enzyme deficiencies. No patient presented with isolated complex IV deficiency. Seven patients had a decreased ATP production rate. Extensive sequence analysis identified eight patients with pathogenic mtDNA mutations and one patient with mutations in POLGA. Mutations of mtDNA are a common cause of LS and mtDNA analysis should always be included in the diagnosis of LS patients, whereas SURF1 mutations are not a common cause of LS in Sweden. Unexpectedly, age of onset, clinical symptoms and prognosis did not reveal any clear differences in LS patients with mtDNA or nuclear DNA mutations.     

The Role of the Iron Transporter ABCB7 in Refractory Anemia with Ring Sideroblasts

Refractory Anemia with Ring Sideroblasts (RARS) is an acquired myelodysplastic syndrome (MDS) characterized by an excess iron accumulation in the mitochondria of erythroblasts. The pathogenesis of RARS and the cause of this unusual pattern of iron deposition remain unknown. We considered that the inherited X-linked sideroblastic anemia with ataxia (XLSA/A) might be informative for the acquired disorder, RARS. XLSA/A is caused by partial inactivating mutations of the ABCB7 ATP-binding cassette transporter gene, which functions to enable transport of iron from the mitochondria to the cytoplasm. Furthermore, ABCB7 gene silencing in HeLa cells causes an accumulation of iron in the mitochondria. We have studied the role of ABCB7 in RARS by DNA sequencing, methylation studies, and gene expression studies in primary CD34⁺ cells and in cultured erythroblasts. The DNA sequence of the ABCB7 gene is normal in patients with RARS. We have investigated ABCB7 gene expression levels in the CD34⁺ cells of 122 MDS cases, comprising 35 patients with refractory anemia (RA), 33 patients with RARS and 54 patients with RA with excess blasts (RAEB), and in the CD34⁺ cells of 16 healthy controls. We found that the expression levels of ABCB7 are significantly lower in the RARS group. RARS is thus characterized by lower levels of ABCB7 gene expression in comparison to other MDS subtypes. Moreover, we find a strong relationship between increasing percentage of bone marrow ring sideroblasts and decreasing ABCB7 gene expression levels. Erythroblast cell cultures confirm the low levels of ABCB7 gene expression levels in RARS. These data provide an important link between inherited and acquired forms of sideroblastic anemia and indicate that ABCB7 is a strong candidate gene for RARS.     

Rapid identification of mitochondrial DNA (mtDNA) mutations in neuromuscular disorders by using surveyor strategy

Mutations of mitochondrial genome are responsible for respiratory chain defects in numerous patients. We have used a strategy, based on the use of a mismatch-specific DNA endonuclease named " Surveyor Nuclease", for screening the entire mtDNA in a group of 50 patients with neuromuscular features, suggesting a respiratory chain dysfunction. We identified mtDNA mutations in 20% of patients (10/50). Among the identified mutations, four are not found in any mitochondrial database and have not been reported previously. We also confirm that mtDNA polymorphisms are frequently found in a heteroplasmic state (15 different polymorphisms were identified among which five were novel).     

Analysis of the mitochondrial DNA from patients with Wolfram (DIDMOAD) syndrome

Wolfram or DIDMOAD (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy and Deafness) syndrome, which has long been known as an autosomal-recessive disorder, has recently been proposed to be a mitochondrial-mediated disease with either a nuclear or a mitochondrial genetic background. The phenotypic characteristics of the syndrome resemble those found in other mitochondrial (mt)DNA mediated disorders such as Leber's hereditary optic neuropathy (LHON) or maternally inherited diabetes and deafness (MIDD). Therefore, we looked for respective mtDNA alterations in blood samples from 7 patients with DIDMOAD syndrome using SSCP-analysis of PCR-amplified fragments, encompassing all mitochondrial ND and tRNA genes, followed by direct sequencing. Subsequently, we compared mtDNA variants identified in this disease group with those detected in a group of LHON patients (n = 17) and in a group of 69 healthy controls. We found that 4/7 (57%) DIDMOAD patients harbored a specific set of point mutations in tRNA and ND genes including the so-called class II or secondary LHON mutations at nucleotide positions (nps) 4216 and 4917 (haplogroup B). In contrast, LHON-patients were frequently (10/17, 59%) found in association with another cluster of mtDNA variants including the secondary LHON mutations at nps 4216 and 13708 and further mtDNA polymorphisms in ND genes (haplogroup A), overlapping with haplogroup B only by variants at nps 4216 and 11251. The frequencies of both haplogroups were significantly lower in the control group versus the respective disease groups. We propose that haplogroup B represents a susceptibility factor for DIDMOAD which, by interaction with further exogeneous or genetic factors, might increase the risk for disease. (Mol Cell Biochem 174: 209–213, 1997)     

Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific Surveyor Nuclease

Mitochondrial DNA (mtDNA) mutations are responsible for mitochondrial diseases in numerous patients. But, until now, no rapid method was available for the identification of unknown deleterious point mutations. Here, we describe a new strategy for the rapid identification of heteroplasmic mtDNA mutations using mismatch-specific Surveyor Nuclease. This protocol involves the following three steps: (i) PCR amplification of the entire human mitochondrial genome in 17 overlapping fragments; (ii) localization of mtDNA mismatch(es) after digestion of the 17 amplicons by Surveyor Nuclease; and (iii) identification of the mutation by sequencing the region containing the mismatch. This Surveyor Nuclease-based strategy allows a systematic screening of the entire mtDNA to identify a mutation within 2 days. It represents an important diagnostic approach for mitochondrial diseases that can be routinely used in molecular diagnostic laboratories.     

Somatic mutations in the mitochondria of rheumatoid arthritis synoviocytes

Somatic mutations have a role in the pathogenesis of a number of diseases, particularly cancers. Here we present data supporting a role of mitochondrial somatic mutations in an autoimmune disease, rheumatoid arthritis (RA). RA is a complex, multifactorial disease with a number of predisposition traits, including major histocompatibility complex (MHC) type and early bacterial infection in the joint. Somatic mutations in mitochondrial peptides displayed by MHCs may be recognized as non-self, furthering the destructive immune infiltration of the RA joint. Because many bacterial proteins have mitochondrial homologues, the immune system may be primed against these altered peptides if they mimic bacterial homologues. In addition, somatic mutations may be influencing cellular function, aiding in the acquirement of transformed properties of RA synoviocytes. To test the hypothesis that mutations in mitochondrial DNA (mtDNA) are associated with RA, we focused on the MT-ND1 gene for mitochondrially encoded NADH dehydrogenase 1 (subunit one of complex I – NADH dehydrogenase) of synoviocyte mitochondria from RA patients, using tissue from osteoarthritis (OA) patients for controls. We identified the mutational burden and amino acid changes in potential epitope regions in the two patient groups. RA synoviocyte mtDNA had about twice the number of mutations as the OA group. Furthermore, some of these changes had resulted in potential non-self MHC peptide epitopes. These results provide evidence for a new role for somatic mutations in mtDNA in RA and predict a role in other diseases.     

Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases.

We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.     

Mitochondrial genome mutations and neuronal dysfunction of induced pluripotent stem cells derived from patients with Alzheimer's disease

ObjectivesPatient‐derived induced pluripotent stem cells (iPSCs) are materials that can be used for autologous stem cell therapy. We screened mtDNA mutations in iPSCs and iPSC‐derived neuronal cells from patients with Alzheimer''s disease (AD). Also, we investigated whether the mutations could affect mitochondrial function and deposition of β‐amyloid (Aβ) in differentiated neuronal cells.Materials and MethodsmtDNA mutations were measured and compared among iPSCs and iPSC‐derived neuronal cells. The selected iPSCs carrying mtDNA mutations were subcloned, and then their growth rate and neuronal differentiation pattern were analyzed. The differentiated cells were measured for mitochondrial respiration and membrane potential, as well as deposition of Aβ.ResultsMost iPSCs from subjects with AD harbored ≥1 mtDNA mutations, and the number of mutations was significantly higher than that from umbilical cord blood. About 35% and 40% of mutations in iPSCs were shared with isogenic iPSCs and their differentiated neuronal precursor cells, respectively, with similar or different heteroplasmy. Furthermore, the mutations in clonal iPSCs were stable during extended culture and neuronal differentiation. Finally, mtDNA mutations could induce a growth advantage with higher viability and proliferation, lower mitochondrial respiration and membrane potential, as well as increased Aβ deposition.ConclusionThis study demonstrates that mtDNA mutations in patients with AD could lead to mitochondrial dysfunction and accelerated Aβ deposition. Therefore, early screening for mtDNA mutations in iPSC lines would be essential for developing autologous cell therapy or drug screening for patients with AD.

mtDNA mutations were found in induced pluripotent stem cells derived from patients with Alzheimer''s disease. The mutations could induce growth advantage due to their high viability and proliferation. Differentiated neuronal cells with mtDNA mutations exhibited mitochondrial and neuronal dysfunction, as well as increased Aβ deposition.     

Relationship between mitochondrial DNA mutations and clinical characteristics in human lung cancer

Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations, some of which are related to various diseases, including cancers. However, roles of mutations and polymorphisms in some diseases are among heated debate, especially for cancer. To investigate the possible role of mtDNA mutations in lung cancer, we sequenced complete mtDNA of lung cancer tissues, corresponding normal (i.e., non-cancerous) lung tissues, and peripheral blood samples from 55 lung cancer patients and examined the relationship between mtDNA mutations or polymorphisms and clinical parameters. We identified 56 mutations in 33 (60%) of the 55 patients, including 48 point mutations, four single-nucleotide insertions, and four single-nucleotide deletions. Nineteen of these mutations resulted in amino acid substitution. These missense mtDNA mutations were distributed in 9 of 13 mitochondrial DNA coding genes. Three hundred eighty eight polymorphisms were identified among the 55 patients. Seventy-three polymorphisms resulted in amino acid substitution. There was no association of incidence of specific mtDNA mutation or polymorphism with patients' gender, age at diagnosis, smoking history, tumor type or tumor stage (P>0.05). This study revealed a variety of mtDNA mutations and mtDNA polymorphisms in human lung cancer, some of which might be involved in human lung carcinogenesis.     

Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu

Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)-encoded RNAs and nuclear DNA-encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis.     

Production of transmitochondrial cybrids containing naturally occurring pathogenic mtDNA variants

The human mitochondrial genome (mtDNA) encodes polypeptides that are critical for coupling oxidative phosphorylation. Our detailed understanding of the molecular processes that mediate mitochondrial gene expression and the structure–function relationships of the OXPHOS components could be greatly improved if we were able to transfect mitochondria and manipulate mtDNA in vivo. Increasing our knowledge of this process is not merely of fundamental importance, as mutations of the mitochondrial genome are known to cause a spectrum of clinical disorders and have been implicated in more common neurodegenerative disease and the ageing process. In organellar or in vitro reconstitution studies have identified many factors central to the mechanisms of mitochondrial gene expression, but being able to investigate the molecular aetiology of a limited number of cell lines from patients harbouring mutated mtDNA has been enormously beneficial. In the absence of a mechanism for manipulating mtDNA, a much larger pool of pathogenic mtDNA mutations would increase our knowledge of mitochondrial gene expression. Colonic crypts from ageing individuals harbour mutated mtDNA. Here we show that by generating cytoplasts from colonocytes, standard fusion techniques can be used to transfer mtDNA into rapidly dividing immortalized cells and, thereby, respiratory-deficient transmitochondrial cybrids can be isolated. A simple screen identified clones that carried putative pathogenic mutations in MTRNR1, MTRNR2, MTCOI and MTND2, MTND4 and MTND6. This method can therefore be exploited to produce a library of cell lines carrying pathogenic human mtDNA for further study.