Ramon Martí

ALLOGENEIC STEM CELL TRANSPLANTATION CORRECTS BIOCHEMICAL DERANGEMENTS IN MNGIE

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a multisystemic autosomal recessive disease due to primary thymidine phosphorylase (TP) deficiency. To restore TP activity, we performed reduced intensity allogeneic stem cell transplantations (alloSCTs) in two patients. In the first, alloSCT failed to engraft, but the second achieved mixed donor chimerism, which partially restored buffy coat TP activity and lowered plasma nucleosides. Thus, alloSCT can correct biochemical abnormalities in the blood of patients with MNGIE, but clinical efficacy remains unproven.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): a disease of two genomes.

BackgroundMitochondrial encephalomyopathies are clinically and genetically heterogeneous because mitochondria are the products of 2 genomes: mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Among the mendelian-inherited mitochondrial diseases are defects of intergenomic communication, disorders due to nDNA mutations that cause depletion and multiple deletions of mtDNA. Review SummaryMitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder of intergenomic communication and is defined clinically by 1) severe gastrointestinal dysmotility; 2) cachexia; 3) ptosis, ophthalmoparesis, or both; 4) peripheral neuropathy; and 5) leukoencephalopathy. Skeletal muscle biopsies of patients have revealed abnormalities of mtDNA and mitochondrial respiratory chain enzymes. The disease is caused by mutations in the thymidine phosphorylase (TP) gene. TP protein catalyzes phosphorolysis of thymidine to thymine and deoxyribose 1-phosphate. In MNGIE patients, TP enzyme activity is reduced drastically, and plasma thymidine and deoxyuridine are elevated dramatically. We have hypothesized that alterations of nucleoside metabolism cause an imbalanced mitochondrial nucleotide pool that leads to depletion and deletions of mtDNA. ConclusionsMNGIE is a recognizable clinical syndrome caused by mutations in TP. The diagnosis can be confirmed by measuring TP activity in buffy coat or plasma levels of thymidine and deoxyuridine. Reduction of circulating thymidine and deoxyuridine in MNGIE patients may be therapeutic.

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

Unbalanced deoxynucleotide pools cause mitochondrial DNA instability in thymidine phosphorylase-deficient mice

Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP(-/-)UP(-/-)) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability.

Cell Cycle Control and HIV-1 Susceptibility Are Linked by CDK6-Dependent CDK2 Phosphorylation of SAMHD1 in Myeloid and Lymphoid Cells

Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain–containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67+) primary CD4+ T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability.

Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency

Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 microM (average 14.2) in MNGIE and were undetectable (<0.05 microM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE.

Infusion of platelets transiently reduces nucleoside overload in MNGIE

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by thymidine phosphorylase (TP) deficiency, which leads to toxic accumulations of thymidine (dThd) and deoxyuridine (dUrd). In this work, we report that infusion of platelets from healthy donors to patients with MNGIE restored transiently circulating TP and reduced plasma dThd and dUrd levels, suggesting that treatments to achieve permanent restoration of circulating TP such as allogeneic stem cell transplantation or gene transfer might be therapeutic.

Allogeneic hematopoietic SCT as treatment option for patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): a consensus conference proposal for a standardized approach.

Allogeneic hematopoietic SCT (HSCT) has been proposed as a treatment for patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). HSCT has been performed in nine patients using different protocols with varying success. Based on this preliminary experience, participants of the first consensus conference propose a common approach to allogeneic HSCT in MNGIE. Standardization of the transplant protocol and the clinical and biochemical assessments will allow evaluation of the safety and efficacy of HSCT as well as optimization of therapy for patients with MNGIE.

Quantification of mitochondrial DNA copy number: pre-analytical factors.

Mitochondrial DNA (mtDNA) content is important for understanding many cellular processes. Several pre-analytical factors, from sample collection to DNA extraction can affect measurement of mtDNA copy number. In the present study, whole blood samples yielded a higher mtDNA copy number than buffy coat samples. mtDNA content is affected by the cell separation method used and the time between blood withdrawal and cell separation. Thus, reference values must be established with the same type of sample. As to the DNA isolation and purification method, the manual phenol method can give randomly false high values. The QIAamp DNA Mini Kit provided the most highly reproducible mtDNA/nDNA yield.

Secure Integration of Distributed Medical Data Using Mobile Agents

Healthcare is information driven and knowledge driven. Good healthcare depends on making decisions at the right time and place, using the right patient data and applicable knowledge. Communication is of utmost relevance in todays healthcare settings, in that delivery of care, research, and management all depend on sharing information. The proposed system can securely gather, integrate, and display distributed medical information using mobile-agent technology and agent-driven security