M. Esther Gallardo

Clustering of Missense Mutations in the C-Terminal Region of Factor H in Atypical Hemolytic Uremic Syndrome

Hemolytic-uremic syndrome (HUS) is a microvasculature disorder leading to microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Most cases of HUS are associated with epidemics of diarrhea caused by verocytotoxin-producing bacteria, but atypical cases of HUS not associated with diarrhea (aHUS) also occur. Early studies describing the association of aHUS with deficiencies of factor H suggested a role for this complement regulator in aHUS. Molecular evidence of factor H involvement in aHUS was first provided by Warwicker et al., who demonstrated that aHUS segregated with the chromosome 1q region containing the factor H gene (HF1) and who identified a mutation in HF1 in a case of familial aHUS with normal levels of factor H. We have performed the mutational screening of the HF1 gene in a novel series of 13 Spanish patients with aHUS who present normal complement profiles and whose plasma levels of factor H are, with one exception, within the normal range. These studies have resulted in the identification of five novel HF1 mutations in four of the patients. Allele HF1 Delta exon2, a genomic deletion of exon 2, produces a null HF1 allele and results in plasma levels of factor H that are 50% of normal. T956M, W1183L, L1189R, and V1197A are missense mutations that alter amino acid residues in the C-terminal portion of factor H, within a region--SCR16-SCR20--that is involved in the binding to solid-phase C3b and to negatively charged cellular structures. This remarkable clustering of mutations in HF1 suggests that a specific dysfunction in the protection of cellular surfaces by factor H is a major pathogenic condition underlying aHUS.

Differences in reactive oxygen species production explain the phenotypes associated with common mouse mitochondrial DNA variants

Common mitochondrial DNA (mtDNA) haplotypes in humans and mice have been associated with various phenotypes, including learning performance and disease penetrance. Notably, no influence of mtDNA haplotype in cell respiration has been demonstrated. Here, using cell lines carrying four different common mouse mtDNA haplotypes in an identical nuclear background, we show that the similar level of respiration among the cell lines is only apparent and is a consequence of compensatory mechanisms triggered by different production of reactive oxygen species. We observe that the respiration capacity per molecule of mtDNA in cells with the NIH3T3 or NZB mtDNA is lower than in those with the C57BL/6J, CBA/J or BALB/cJ mtDNA. In addition, we have determined the genetic element underlying these differences. Our data provide insight into the molecular basis of the complex phenotypes associated with common mtDNA variants and anticipate a relevant contribution of mtDNA single nucleotide polymorphisms to phenotypic variability in humans.

Genetic basis of end‐stage hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by a heterogeneous presentation and clinical course. A minority of HCM patients develop end‐stage HCM and require cardiac transplantation. The genetic basis of end‐stage HCM is unknown but small series, isolated case reports and animal models have related the most aggressive heart failure course with the presence of multiple mutations.

Evolution Meets Disease: Penetrance and Functional Epistasis of Mitochondrial tRNA Mutations

About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m.4290T>C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNAIle that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both.

Mitochondrial haplogroups associated with end-stage heart failure and coronary allograft vasculopathy in heart transplant patients

AIMS Mitochondrial haplogroups are known to influence individual predisposition to a wide spectrum of metabolic and degenerative diseases, including ischaemic cardiovascular diseases. We have examined the influence of the mitochondrial DNA (mtDNA) background on the development of human end-stage heart failure (HF) in patients undergoing heart transplantation. The influence of mtDNA haplogroups on the incidence of transplant-related complications, mainly cardiac allograft vasculopathy (CAV), and on post-transplant survival was also studied. METHODS AND RESULTS The most common mitochondrial haplogroups in European populations were genotyped in 450 heart transplant recipients, 248 heart transplant donors, and 206 healthy controls. Mitochondrial haplogroups were determined by PCR amplification of short mtDNA fragments, followed by restriction fragment length polymorphism analysis. After adjustment for age and sex the frequency of haplogroup H was significantly higher in heart transplant recipients than in controls [OR: 1.86 (95% confidence intervals, CI: 1.27-2.74), P= 0.014], and in heart donors [OR: 1.47 (95% CI: 0.99-2.19), P= 0.032]. Likewise, haplogroup Uk was found significantly more frequently among CAV patients than in non-CAV heart allograft recipients [OR: 4.1 (95% CI: 1.51-11.42), P= 0.042]. Finally, heart donor haplogroups had no influence on the morbidity or mortality after heart transplantation. CONCLUSIONS Mitochondrial haplogroups behave like risk factors for the progress to end-stage HF in a Spanish cardiac transplant population. Mitochondrial DNA variants may have some influence on the appearance of cardiac transplant complications.

Mitochondrial tRNA valine as a recurrent target for mutations involved in mitochondrial cardiomyopathies.

The aim of this study was to identify the genetic defect in two patients having cardiac dysfunction accompanied by neurological symptoms, and in one case MRI evidence of cortical and cerebellar atrophy with hyperintensities in the basal ganglia. Muscle biopsies from each patient revealed single and combined mitochondrial respiratory chain deficiency. The complete mtDNA sequencing of both patients revealed two transitions in the mitochondrial tRNA(Val) gene (MT-TV) (m.1628C>T in Patient 1, and m.1644G>A in Patient 2). The functional and molecular analyses reported here suggest that the MT-TV gene should be routinely considered in the diagnosis of mitochondrial cardiomyopathies.

Generation of a human iPSC line from a patient with Leigh syndrome

Human iPSC line LND554SV.3 was generated from heteroplasmic fibroblasts of a patient with Leigh syndrome carrying a mutation in the MT-ND5 gene (m.13513GNA; p.D393N). Reprogramming factors Oct3/4, Sox2, Klf4,and cMyc were delivered using a non-integrative methodology that involves the use of Sendai virus.

Generation of a human iPSC line from a patient with a defect of intergenomic communication

Human iPSC line PG64SV.2 was generated from fibroblasts of a patient with a defect of intergenomic communication. This patient harbored a homozygous mutation (c.2243G>C; p.Trp748Ser) in the gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma gene (POLG). Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a non integrative methodology that involves the use of Sendai virus.

Generating Rho-0 cells using mesenchymal stem cell lines

Introduction The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. Methodology Two immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis. Results The results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic capacities were lower. Conclusion Among the drugs and conditions tested, the use of d4t was the best option for producing Rho-0 cells from hMSCs. Rho-0 cells are useful for studying the role of mitochondria in hMSC differentiation.

iPSCs, a Future Tool for Therapeutic Intervention in Mitochondrial Disorders: Pros and Cons

Mitochondrial disorders, although individually are rare, taken together constitute a big group of diseases that share a defect in the oxidative phosphorylation system. Up to now, the development of therapies for these diseases is very slow and ineffective due in part to the lack of appropriate disease models. Therefore, there is an urgent need for the discovery of new therapeutic interventions. Regarding this, the generation of induced pluripotent stem cells (iPSCs) has opened new expectations in the regenerative medicine field. However, special cares and considerations must be taken into account previous to a replacement therapy. J. Cell. Physiol. 231: 2317–2318, 2016.