Anna-Kaisa Niemi

Mitochondrial DNA and ACTN3 genotypes in Finnish elite endurance and sprint athletes

Differences in ACTN3 (alpha-actinin 3) genotypes have been reported among endurance and power athletes. Elite athletic performance in endurance sports should also depend on mitochondrial oxidative phosphorylation (OXPHOS) that produces ATP for muscle metabolism. We determined mitochondrial DNA (mtDNA) and ACTN3 genotypes in Finnish elite endurance (n=52) and sprint (n=89) athletes, and found that the frequencies of mtDNA haplogroups differed significantly between the two groups. Most notably, none of the endurance athletes belonged to haplogroup K or subhaplogroup J2, both of which have previously been associated with longevity. The frequency of ACTN3 XX genotype was higher and that of RR was lower among Finnish endurance athletes, and, in addition, none of the top Finnish sprinters had the XX genotype. Lack of mtDNA haplogroup K and subhaplogroup J2 among elite endurance athletes suggests that these haplogroups are ‘uncoupling genomes’. Such genomes should not be beneficial to endurance-type athletic performance but should be beneficial to longevity, since uncoupling of OXPHOS reduces the production of ATP, reduces the release of reactive oxygen species and generates heat.

A combination of three common inherited mitochondrial DNA polymorphisms promotes longevity in Finnish and Japanese subjects

Mitochondrial DNA (mtDNA) coding region polymorphisms, as well as the 150T polymorphism in the noncoding region, have been associated with longevity. We have studied here the association of 150T with longevity further and assessed differences in this association between various mtDNA haplogroups. We analysed a sample of 321 very old subjects and 489 middle-aged controls from Finland and Japan. 150T was more frequent among the very old than among the controls in both the Finnish and Japanese subjects. Interestingly, the association was not similar in all haplogroups, and a stratified analysis revealed that two additional common polymorphisms, 489C and 10398G, modified the association between 150T and longevity. These findings suggest that longevity is partly determined by epistatic interactions involving these three mtDNA loci.

Importance of culturing primary lymphocytes at physiological oxygen levels

Although studies with primary lymphocytes are almost always conducted in CO2 incubators maintained at atmospheric oxygen levels (atmosO2; 20%), the physiological oxygen levels (physO2; 5%) that cells encounter in vivo are 2–4 times lower. We show here that culturing primary T cells at atmosO2 significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO2 maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO2 than at physO2. This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO2 than at atmosO2; and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO2 that at atmosO2. Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO2 likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO2 because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.

A new LC-MS/MS method for the clinical determination of reduced and oxidized glutathione from whole blood.

Reduced levels of glutathione (γ-glutamylcysteinylglycine, GSH) and the ratio of GSH to glutathione disulfide (GSSG) can serve as important indicators of oxidative stress and disease risk. Measured concentrations of GSH and GSSG vary widely between laboratories, largely due to the instability of GSH during sample handling and variables arising from different analytical methods. We have developed a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring whole blood GSH and GSSG that minimizes preanalytic and analytic variability, reliably eliminates interference from ion suppression, and can easily be implemented in clinical laboratories. Samples were deproteinized with sulfosalicylic acid (SSA) and derivatized with N-ethylmaleimide (NEM) in a single preparative step, and the resulting supernatants combined with stable-isotope internal standards (GSH-(13)C, (15)N-NEM and GSSG-(13)C,(15)N), subjected to chromatographic separation using a Hypercarb column, and analyzed by MS/MS in the positive-ion mode. Results showed excellent linearity for both GSH and GSSG over the ranges of physiologic normal, with inter- and intra-assay CVs of 3.1-4.3% and accuracy between 95% and 101%. The lower limits of detection (LLOD) were 0.4μM for GSH and 0.1μM for GSSG and the lower limits of quantitation (LLOQ) were 1.5μM for GSH and 0.1μM for GSSG. Derivatized samples are stable for at least 3 years when stored at -80°C, and underivatized samples for at least 24h at either 4°C or room temperature. Reference intervals were determined for 59 control samples, and were (mean±SD): GSH 900±140μM; GSSG 1.17±0.43μM; GSH/GSSG 880±370.

Ectopia lentis as the presenting and primary feature in Marfan syndrome.

Marfan syndrome (MFS) is a multisystem connective tissue disorder with primary involvement of the ocular, cardiovascular, and skeletal systems. We report on eight patients, all presenting initially with bilateral ectopia lentis (EL) during early childhood. These individuals did not have systemic manifestations of MFS, and did not fulfill the revised Ghent diagnostic criteria. However, all patients had demonstratable, disease‐causing missense mutations in the FBN1 gene. Based on molecular results, cardiovascular imaging was recommended and led to the identification of mild aortic root changes in seven of the eight patients. The remaining patient had mitral valve prolapse with a normal appearing thoracic aorta. The findings presented in this paper validate the necessity of FBN1 gene testing in all individuals presenting with isolated EL. As we observed, these individuals are at increased risk of cardiovascular complications. Furthermore, we also noted that the majority of our patient cohorts mutations occurred in the 5′ portion of the FBN1 gene, and were found to affect highly conserved cysteine residues, which may indicate a possible genotype–phenotype correlation. We conclude that in patients with isolated features of EL, FBN1 mutation analysis is necessary to aid in providing prompt diagnosis, and to identify patients at risk for potentially life‐threatening complications. Additionally, knowledge of the type and location of an FBN1 mutation may be useful in providing further clinical correlation regarding phenotypic progression and appropriate medical management.

Perinatal Features of the RASopathies: Noonan Syndrome, Cardiofaciocutaneous Syndrome and Costello Syndrome

The RASopathies are a family of developmental disorders caused by heritable defects of the RAS/MAPK signaling pathway. While the postnatal presentation of this group of disorders is well known, the prenatal and neonatal findings are less widely recognized. We report on the perinatal presentation of 10 patients with Noonan syndrome (NS), nine with Cardiofaciocutaneous syndrome (CFCS) and three with Costello syndrome (CS), in conjunction with the results of a comprehensive literature review. The majority of perinatal findings in NS, CS, and CFCS are shared: polyhydramnios; prematurity; lymphatic dysplasia; macrosomia; relative macrocephaly; respiratory distress; hypotonia, as well as cardiac and renal anomalies. In contrast, fetal arrhythmia and neonatal hypoglycemia are relatively specific to CS. NS, CS, and CFCS should all be considered as a possible diagnosis in pregnancies with a normal karyotype and ultrasound findings of a RASopathy. Recognition of the common perinatal findings of these disorders should facilitate both their prenatal and neonatal diagnosis.

Treatment of Methylmalonic Acidemia by Liver or Combined Liver-Kidney Transplantation

OBJECTIVE To assess biochemical, surgical, and long-term outcomes of liver (LT) or liver-kidney transplantation (LKT) for severe, early-onset methylmalonic acidemia/acid (MMA). STUDY DESIGN A retrospective chart review (December 1997 to May 2012) of patients with MMA who underwent LT or LKT at Lucile Packard Childrens Hospital at Stanford. RESULTS Fourteen patients underwent LT (n = 6) or LKT (n = 8) at mean age 8.2 years (range 0.8-20.7). Eleven (79%) were diagnosed during the neonatal period, including 6 by newborn screening. All underwent deceased donor transplantation; 12 (86%) received a whole liver graft. Postoperative survival was 100%. At a mean follow-up of 3.25 ± 4.2 years, patient survival was 100%, liver allograft survival 93%, and kidney allograft survival 100%. One patient underwent liver re-transplantation because of hepatic artery thrombosis. After transplantation, there were no episodes of hyperammonemia, acidosis, or metabolic decompensation. The mean serum MMA at the time of transplantation was 1648 ± 1492 μmol/L (normal <0.3, range 99-4420). By 3 days, post-transplantation levels fell on average by 87% (mean 210 ± 154 μmol/L), and at 4 months, they were 83% below pre-transplantation levels (mean 305 ± 108 μmol/L). Developmental delay was present in 12 patients (86%) before transplantation. All patients maintained neurodevelopmental abilities or exhibited improvements in motor skills, learning abilities, and social functioning. CONCLUSIONS LT or LKT for MMA eradicates episodes of hyperammonemia, results in excellent long-term survival, and suggests stabilization of neurocognitive development. Long-term follow-up is underway to evaluate whether patients who undergo early LT need kidney transplantation later in life.

Liver transplantation for urea cycle disorders in pediatric patients: A single-center experience

LT has emerged as a surgical treatment for UCDs. We hypothesize that LT can be safely and broadly utilized in the pediatric population to effectively prevent hyperammonemic crises and potentially improve neurocognitive outcomes. To determine the long‐term outcomes of LT for UCDs, charts of children with UCD who underwent LT were retrospectively reviewed at an academic institution between July 2001 and May 2012. A total of 23 patients with UCD underwent LT at a mean age of 3.4 yr. Fifteen (65%) patients received a whole‐liver graft, seven patients (30%) received a reduced‐size graft, and one patient received a living donor graft. Mean five‐yr patient survival was 100%, and allograft survival was 96%. Mean peak blood ammonia (NH3) at presentation was 772 μmol/L (median 500, range 178–2969, normal <30–50). After transplantation, there were no episodes of hyperammonemia. Eleven patients were diagnosed with some degree of developmental delay before transplantation, which remained stable or improved after transplantation. Patients without developmental delay before transplantation maintained their cognitive abilities at long‐term follow‐up. LT was associated with the eradication of hyperammonemia, removal of dietary restrictions, and potentially improved neurocognitive development. Long‐term follow‐up is underway to evaluate whether LT at an early age (<1 yr) will attain improved neurodevelopmental outcomes.

Degree of Glutathione Deficiency and Redox Imbalance Depend on Subtype of Mitochondrial Disease and Clinical Status

Mitochondrial disorders are associated with decreased energy production and redox imbalance. Glutathione plays a central role in redox signaling and protecting cells from oxidative damage. In order to understand the consequences of mitochondrial dysfunction on in vivo redox status, and to determine how this varies by mitochondrial disease subtype and clinical severity, we used a sensitive tandem mass spectrometry assay to precisely quantify whole blood reduced (GSH) and oxidized (GSSG) glutathione levels in a large cohort of mitochondrial disorder patients. Glutathione redox potential was calculated using the Nernst equation. Compared to healthy controls (n = 59), mitochondrial disease patients (n = 58) as a group showed significant redox imbalance (redox potential −251 mV±9.7, p<0.0001) with an increased level of oxidation by ∼9 mV compared to controls (−260 mV±6.4). Underlying this abnormality were significantly lower whole blood GSH levels (p = 0.0008) and GSH/GSSG ratio (p = 0.0002), and significantly higher GSSG levels (p<0.0001) in mitochondrial disease patients compared to controls. Redox potential was significantly more oxidized in all mitochondrial disease subgroups including Leigh syndrome (n = 15), electron transport chain abnormalities (n = 10), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (n = 8), mtDNA deletion syndrome (n = 7), mtDNA depletion syndrome (n = 7), and miscellaneous other mitochondrial disorders (n = 11). Patients hospitalized in metabolic crisis (n = 7) showed the greatest degree of redox imbalance at −242 mV±7. Peripheral whole blood GSH and GSSG levels are promising biomarkers of mitochondrial dysfunction, and may give insights into the contribution of oxidative stress to the pathophysiology of the various mitochondrial disorders. In particular, evaluation of redox potential may be useful in monitoring of clinical status or response to redox-modulating therapies in clinical trials.

Abnormal Hepatocellular Mitochondria in Methylmalonic Acidemia

Abstract Methylmalonic acidemia (MMA) is one of the most frequently encountered forms of branched-chain organic acidemias. Biochemical abnormalities seen in some MMA patients, such as lactic acidemia and increased tricarboxylic acid cycle intermediate excretion, suggest mitochondrial dysfunction. In order to investigate the possibility of mitochondrial involvement in MMA, we examined liver tissue for evidence of mitochondrial ultrastructural abnormalities. Five explanted livers obtained from MMA mut0 patients undergoing liver transplantation were biopsied. All patients had previous episodes of metabolic acidosis, lactic acidemia, ketonuria, and hyperammonemia. All biopsies revealed a striking mitochondriopathy by electron microscopy. Mitochondria were markedly variable in size, shape, and conformation of cristae. The inner matrix appeared to be greatly expanded and the cristae were diminutive and disconnected. No crystalloid inclusions were noted. This series clearly documents extensive mitochondrial ultrastructure abnormalities in liver samples from MMA patients undergoing transplantation, providing pathological evidence for mitochondrial dysfunction in the pathophysiology of MMA mut0. Considering the trend to abnormally large mitochondria, the metabolic effects of MMA may restrict mitochondrial fission or promote fusion. The correlation between mitochondrial dysfunction and morphological abnormalities in MMA may provide insights for better understanding and monitoring of optimized or novel therapeutic strategies.