HAVCR/KIM-1 activates the IL-6/STAT-3 pathway in clear cell renal cell carcinoma and determines tumor progression and patient outcome.

Renal cell carcinoma (RCC), the third most prevalent urological cancer, claims more than 100,000 lives/year worldwide. The clear cell variant (ccRCC) is the most common and aggressive subtype of this disease. While commonly asymptomatic, more than 30% of ccRCC are diagnosed when already metastatic, resulting in a 95% mortality rate. Notably, nearly one-third of organ-confined cancers treated by nephrectomy develop metastasis during follow-up care. At present, diagnostic and prognostic biomarkers to screen, diagnose, and monitor renal cancers are clearly needed. The gene encoding the cell surface molecule HAVCR1/KIM-1 is a suggested susceptibility gene for ccRCC and ectodomain shedding of this molecule may be a predictive biomarker of tumor progression. Microarray analysis of 769-P ccRCC-derived cells where HAVCR/KIM-1 levels have been upregulated or silenced revealed relevant HAVCR/KIM-1-related targets, some of which were further analyzed in a cohort of 98 ccRCC patients with 100 month follow-up. We found that HAVCR/KIM-1 activates the IL-6/STAT-3/HIF-1A axis in ccRCC-derived cell lines, which depends on HAVCR/KIM-1 shedding. Moreover, we found that pSTAT-3 S727 levels represented an independent prognostic factor for ccRCC patients. Our results suggest that HAVCR/KIM-1 upregulation in tumors might represent a novel mechanism to activate tumor growth and angiogenesis and that pSTAT-3 S727 is an independent prognostic factor for ccRCC.

An in vitro tool to assess cytochrome P450 drug biotransformation-dependent cytotoxicity in engineered HepG2 cells generated by using adenoviral vectors.

Many adverse drug reactions leading to hepatotoxicity are caused by the cytochrome P450-dependent activation of non-toxic drugs or chemicals into reactive metabolites. To this end, adenoviruses were used as a tool to efficiently deliver specific CYP genes into cultured cells (i.e., human hepatoma cell line HepG2). Recombinant-defective adenoviral vectors encoding for genes CYP3A4 (Adv-CYP3A4), CYP2E1 (Adv-CYP2E1), CYP2A6 (Adv-CYP2A6) and CYP1A2 (Adv-CYP1A2) were used to confer specific CYP drug metabolic capabilities to HepG2 cells. Upgraded cells transiently expressed single specific cytochrome P450 enzymatic activities in terms of the number of the infecting virus particles used in their transduction. HepG2 cells transduced with adenoviruses and wild HepG2 cells cultured in 96 well-plates were incubated in the presence of model compounds, some of which can be metabolized to reactive metabolites. After compound exposure, cell viability was assessed by the commonly used MTT assay. The results confirm that the cell-based assay is a valuable tool in toxicology assessments and high-throughput screenings to detect cytotoxicity mediated by cytochrome P450 biotransformation in preclinical drug development. The assay also has a potential applicability in other industrial sectors such as the chemical industry.

Defining new criteria for selection of cell-based intestinal models using publicly available databases.

BACKGROUND: The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. RESULTS: We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. CONCLUSIONS: This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.

Evaluation of uptake and transport of cationic and anionic ultrasmall iron oxide nanoparticles by human colon cells.

Nanoparticles (NPs) are in clinical use or under development for therapeutic imaging and drug delivery. However, relatively little information exists concerning the uptake and transport of NPs across human colon cell layers, or their potential to invade three-dimensional models of human colon cells that better mimic the tissue structures of normal and tumoral colon. In order to gain such information, the interactions of biocompatible ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) (iron oxide core 9-10 nm) coated with either cationic polyvinylamine (aminoPVA) or anionic oleic acid with human HT-29 and Caco-2 colon cells was determined. The uptake of the cationic USPIO NPs was much higher than the uptake of the anionic USPIO NPs. The intracellular localization of aminoPVA USPIO NPs was confirmed in HT-29 cells by transmission electron microscopy that detected the iron oxide core. AminoPVA USPIO NPs invaded three-dimensional spheroids of both HT-29 and Caco-2 cells, whereas oleic acid-coated USPIO NPs could only invade Caco-2 spheroids. Neither cationic aminoPVA USPIO NPs nor anionic oleic acid-coated USPIO NPs were transported at detectable levels across the tight CacoReady™ intestinal barrier model or the more permeable mucus-secreting CacoGoblet™ model.

Implementation challenges for designing integrated in vitro testing strategies (ITS) aiming at reducing and replacing animal experimentation.

At the IVTIP (in vitro testing industrial platform) meeting of November 26th 2009 entitled 'Toxicology in the 21st century ('21C')--working our way towards a visionary reality' all delegates endorsed the emerging concept of the '21C' vision as the way forward to enable a thorough, reliable and systematic approach to future toxicity testing without the use of animals. One of the emerging concepts focused on integrating a defined number of tests modelling in vivo-relevant and well-characterised toxicity pathways representing mechanistic endpoints. At this meeting the importance of Integrated Testing Strategies (ITS) as tools towards reduction and eventually replacement of the animals currently used for hazard identification and risk assessment was recognised. A follow-up IVTIP Spring 2010 meeting entitled 'Integrated In Vitro Testing Strategies (ITS)--Implementation Challenges' was organised to address pending questions about ITS. This report is not a review of the ITS literature, but a summary of the discussions triggered by presented examples on how to develop and implement ITS. Contrasts between pharmaceutical and chemical industry, as well as a list of general but practical aspects to be considered while developing an ITS emerged from the discussions. In addition, current recommendations on the validation of ITS were discussed. In conclusion, the outcome of this workshop improved the understanding of the participants of some important factors that may impact the design of an ITS in function of its purpose (e.g., screening, or early decision making versus regulatory), the context in which they need to be applied (e.g., ICH guidelines, REACH) and the status and quality of the available tools. A set of recommendations of best practices was established and the importance of the applicability of the individual tests as well as the testing strategy itself was highlighted.

Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity.

The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1(-) cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase γ, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.

Thymidine kinase 2 deficiency-induced mitochondrial DNA depletion causes abnormal development of adipose tissues and adipokine levels in mice.

Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS) are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA) is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT) and brown (BAT) adipose tissues in thymidine kinase 2 (Tk2) H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues.

Altered gene transcription profiles in fibroblasts harboring either TK2 or DGUOK mutations indicate compensatory mechanisms.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive disorder characterized by a reduced amount of mtDNA, which impairs synthesis of respiratory chain complexes. MDS has been classified into two main groups, the hepatocerebral form affecting liver and the central nervous system, and the myopathic form targeting the skeletal muscle. We have compared the molecular genetic characteristics of fibroblasts derived from two patients harboring TK2 mutations with two harboring mutations in DGUOK gene. Real-time PCR revealed mtDNA depletion in dGK-deficient fibroblasts (dGK-) but not in TK2-deficient cells (TK2-). Real-time RT-PCR and western blotting demonstrated significant differences in the expression of the human equilibrative nucleoside transporter 1 (hENT1) at the mRNA and protein levels. hENT1 transcript and protein were increased in quiescent control and TK2- fibroblasts relative to cycling cells. In contrast, hENT1 was stable in quiescent and cycling dGK- cells. Moreover, siRNA down-regulation of hENT1, but not of TK1, induced mtDNA depletion in TK2- fibroblasts indicating that hENT1 contributes to the maintenance of normal mtDNA levels in cells lacking TK2. Transcripts for thymidine phosphorylase, the mitochondrial transcription factor A (TFAM), and the polymerase gamma (Pol gamma), were reduced in dGK-, but not in TK2- cells while the mRNA expression of thymidylate synthase (TS) increased. Our results suggested differential gene expression in TK2 and dGK-deficient fibroblasts, and highlighted the importance of hENT1 as a compensatory factor in MDS disorder.

Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts.

Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.

Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2(-/-)) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2(-/-) animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

Selective muscle fiber loss and molecular compensation in mitochondrial myopathy due to TK2 deficiency.

A 12-year-old patient with mitochondrial DNA (mtDNA) depletion syndrome due to TK2 gene mutations has been evaluated serially over the last 10 years. We observed progressive muscle atrophy with selective loss of type 2 muscle fibers and, despite severe depletion of mtDNA, normal activities of respiratory chain (RC) complexes and levels of COX II mitochondrial protein in the remaining muscle fibers. These results indicate that compensatory mechanisms account for the slow progression of the disease. Identification of factors that ameliorate mtDNA depletion may reveal new therapeutic targets for these devastating disorders.

Altered expression of master regulatory genes of adipogenesis in lipomas from patients bearing tRNA(Lys) point mutations in mitochondrial DNA.

The mechanisms underlying the appearance of lipomas in patients bearing mutations in the tRNA(Lys) gene of mitochondrial DNA are unknown. We investigated changes in gene expression patterns in lipomas from three patients bearing A8344G or G8363A tRNA(Lys) gene mutations. Uncoupling protein-1 mRNA was detected in the lipomas, in contrast with undetectable expression in normal adipose tissue. However, expression of other markers of brown fat, such as PGC-1alpha, was unaltered. PPARgamma and retinoblastoma gene expression was down regulated in the lipomas, but C/EBPalpha mRNA was not affected. The expression of Pref-1 was dramatically down regulated. Thus, lipomatosis due to tRNA(Lys) mutations is associated with a pattern of altered expression of master regulators of adipogenesis consistent with enhanced proliferation but maintenance of adipocyte features, and with a distorted pattern of brown versus white adipocyte differentiation.

Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes.

Thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are the two key enzymes in mitochondrial DNA (mtDNA) precursor synthesis. Deficiencies in TK2 or dGK activity, due to genetic alteration, have been shown to cause tissue-specific depletion of mtDNA. In the case of TK2 deficiency, affected individuals suffer severe myopathy and, in the case of dGK deficiency, devastating liver or multi-systemic disease. Here, we report clinical and biochemical findings from two patients with mtDNA depletion syndrome. Patient A was a compound heterozygote carrying the previously reported T77M mutation and a novel mutation (R161K) in the TK2 gene. Patient B carried a novel mutation (L250S) in the dGK gene. The clinical symptoms of patient A included muscular weakness and exercise intolerance due to a severe mitochondrial myopathy associated with a 92% reduction in mtDNA. There was minimal involvement of other organs. Patient B suffered from rapidly progressive, early onset fatal liver failure associated with profoundly decreased mtDNA levels in liver and, to a lesser extent, in skeletal muscle. Site-directed mutagenesis was used to introduce the mutations detected in patients A and B into the TK2 and dGK cDNAs, respectively. We then characterized each of these recombinant enzymes. Catalytic activities of the three mutant enzymes were reduced to about 2-4% for TK2 and 0.5% for dGK as compared to the wild-type enzymes. Altered competition between dCyd and dThd was observed for the T77M mutant. The residual activities of the two mitochondrial enzymes correlated directly with disease development.

Transcriptomic and proteomic analysis of liver and muscle alterations caused by surgical stress in rats.

The metabolic response to injury includes major alterations in protein metabolism; however, little is known about alterations in the synthesis of individual proteins and their role in the stress response. Our aim was to study how individual proteins in liver and muscle are altered by abdominal surgery. Changes produced in mRNA and proteins by abdominal surgery were studied in rats using RAP (random arbitrary priming)-PCR, to investigate mRNA alterations, and standard or isotopic (with in vivo radioactive labelling of proteins) two-dimensional electrophoresis/MS proteomic analyses, to study differential expression of proteins. Many of the differentially expressed proteins identified in blood were specifically synthesized by the liver to participate in the stress response. The hepatic proteins (antioxidant proteins, serine protease inhibitors, acute-phase proteins and transport proteins) were secreted into the bloodstream to produce a systemic action, indicating the central role of the liver in the stress response. Overexpressed proteins identified in liver were associated with the glycolytic processes and the folding of nascent proteins, confirming the high metabolic activity of the liver after surgery. The role of skeletal muscle protein as an amino acid donor to fuel the processes involved in the stress response was shown by the decrease in high-molecular-mass myofibrillar proteins. Combined use of the three techniques studied, differential RAP-PCR and standard and isotopic proteome analysis, provided complementary information on the differentially expressed proteins in a rat model of surgical stress.

Hepatitis A virus receptor blocks cell differentiation and is overexpressed in clear cell renal cell carcinoma.

BACKGROUND: The molecular mechanisms underlying tumorigenesis and progression of clear cell renal cell carcinoma (ccRCC) are not well understood. We aimed to identify new molecular markers to provide insight into these processes. METHODS: This work reports on the identification of human hepatitis A virus cellular receptor 1 (hHAVcr-1) as a differentially expressed gene in ccRCC using RNA-based arbitrarily primed polymerase chain reaction (RAP-PCR). Results were further confirmed by Northern and Western blot assays. Carcinoma 769-P and normal HK-2 cells derived from proximal tubule epithelial cells, grown under different culture conditions, were used to understand the putative role of hHAVcr-1 in renal malignancy. hHAVcr-1 stable transfected clones and dipeptidyl peptidase IV (DPPIV) assays allowed assessing its involvement in cell differentiation. RESULTS: The hHAVcr-1 is overexpressed in eight out of 13 ccRCC and its expression neglected in benign oncocytomas. In culture, hhavcr-1 is dramatically overexpressed in normal and tumor cell lines that, having acquired the fully differentiated phenotype, are induced to de-differentiate by means of phorbol ester phorbol 12-myristate-13-acetate (PMA) treatment. Similarly, differentiation prevention by addition of PMA to confluent cells also increases hhavcr-1 expression. hHAVcr-1 stable transfected 769-P cells proved that hhavcr-1 itself blocks differentiation. Since hhavcr-1 is expressed at higher levels in tumor cells, we used an African green monkey cell model to show that immunotoxins directed against the monkey homologue of hhavcr-1 could kill kidney cells. CONCLUSION: Our results showed that hHAVcr-1 blocks differentiation of proximal tubule epithelial cells and that it could be used as a target for therapy of kidney carcinomas.

Mitochondrial DNA abnormalities and autistic spectrum disorders.

OBJECTIVES: To further characterize mtDNA defects associated with autistic features, especially the A3243G mtDNA mutation and mtDNA depletion.Study design Five patients with autistic spectrum disorders and family histories of mitochondrial DNA diseases were studied. We performed mtDNA analysis in all patients and magnetic resonance spectroscopy in three. RESULTS: Three patients manifested isolated autistic spectrum features and two had additional neurologic symptoms. Two patients harbored the A3243G mutation. In two others, the A3243G mutation was not found in accessible tissues but was present in tissues from their mothers. The fifth patient had 72% mtDNA depletion in skeletal muscle. CONCLUSIONS: Autistic spectrum disorders with or without additional neurologic features can be early presentations of the A3243G mtDNA mutation and can be a prominent clinical manifestation of mtDNA depletion. Mitochondrial dysfunction should be considered in patients who have autistic features and associated neurologic findings or who have evidence of maternal inheritance.

Lack of paternal inheritance of muscle mitochondrial DNA in sporadic mitochondrial myopathies.

In 2002, paternal inheritance of muscle mitochondrial DNA (mtDNA) was reported in a patient with exercise intolerance and a mitochondrial DNA (mtDNA) mutation restricted to skeletal muscle. To evaluate whether paternal inheritance is a common phenomenon, we studied 10 sporadic patients with skeletal muscle-restricted mtDNA mutations: five harbored mtDNA point mutations in protein-coding genes and five had single mtDNA deletions. We performed haplotype analysis and direct sequencing of the hypervariable regions 1 and 2 of the D-loop in muscle and blood from the patients and, when available, in blood from their parents. We did not observe paternal inheritance in any of our patients.

Higher processing rates of Alu-containing sequences in kidney tumors and cell lines with overexpressed Alu-mRNAs.

Tumor cell growth and differentiation involve several molecular mechanisms that control gene expression and define specific genomic molecular profiles in cancer cells. Among these mechanisms, it has been shown that Alu-repetitive sequences are capable of regulating gene expression at transcriptional and posttranscriptional levels, and also of modulating cellular growth, differentiation and tumor suppression. Furthermore, repetitive sequences have also been implicated in alternative RNA splicing, although the specific mechanisms involved remain unknown. Nonetheless, exactly what the involvement of Alu-containing sequences in tumor cell growth and differentiation is or to what extent they might be related to tumorigenesis or to alternative splicing is not yet clear. In order to address some of these issues, we analyzed the level of expression of Alu-containing sequences in renal tumors and cell lines and their association with immunoprecipitated ribonucleoprotein splicing complexes in nuclear RNA fractions. Over-expression of Alu-containing sequences was detected in the poly(A)-RNA fractions of all analyzed tumors and cell lines. Furthermore, Alu-sequences were associated with tumor cell growth and differentiation and found overexpressed in purified small nuclear ribonucleoprotein fractions. Overall, our results suggest the involvement of Alu-sequences in the overexpression of Alu-containing-mRNAs in human tumors, and also higher processing rates of Alu-containing sequences at the spliceosome associated with tumor cell growth and differentiation.

Alteration of nucleotide metabolism: a new mechanism for mitochondrial disorders.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). TP deficiency alters the metabolism of the nucleosides thymidine and deoxyuridine, which, in turn, produces abnormalities of mitochondrial DNA (mtDNA) including depletion, deletions, and point mutations. MNGIE is the best characterized of the expanding number of mitochondrial disorders caused by alterations in the metabolism of nucleosides/nucleotides. Because mitochondria contain their own machinery for nucleoside and nucleotide metabolism and have physically separate nucleotide pools, it is not surprising that disorders of these pathways cause human diseases. Other diseases in this group include mtDNA depletion syndromes caused by mutations on the nuclear genes encoding the mitochondrial thymidine kinase and deoxyguanosine kinase; autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA due to mutations in the genes encoding the muscle-isoform of mitochondrial ADP/ATP translocator; and mitochondrial DNA depletion due to toxicities of nucleoside analogues. Mutations in the deoxynucleotide carrier, a transporter of deoxynucleoside diphosphates, have been identified as a cause of congenital microcephaly. However, alterations of mtDNA have not yet been established in this disorder. Future studies are likely to reveal additional diseases and provide further insight into this new subject.