Daniela Verrigni

A new simple and rapid LC–ESI-MS/MS method for quantification of plasma oxysterols as dimethylaminobutyrate esters. Its successful use for the diagnosis of Niemann–Pick type C disease

Two oxysterols, cholestan-3β,5α,6β-triol (C-triol) and 7-ketocholesterol (7-KC), have been recently proposed as diagnostic markers of Niemann-Pick type C (NP-C) disease, representing a potential alternative diagnostic tool to the more invasive and time consuming filipin test in cultured fibroblasts. Usually, the oxysterols are detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using atmospheric pressure chemical ionization (APCI) or electro-spray-ionization (ESI) sources, after a variety of derivatization procedures to enhance sensitivity. We developed a sensitive LC-MS/MS method to quantify the oxysterols in plasma as dimethylaminobutyrate ester, suitable for ESI analysis. This method, with an easy liquid-phase extraction and a short derivatization procedure, has been validated to demonstrate specificity, linearity, recovery, lowest limit of quantification, accuracy and precision. The assay was linear over a concentration range of 0.5-200ng/mL for C-triol and 1.0-200ng/mL for 7-KC. Intra-day and inter-day coefficients of variation (CV%) were <15% for both metabolites. Receiver operating characteristic analysis estimates that the area under curve was 0.998 for C-triol, and 0.972 for 7-KC, implying a significant discriminatory power for the method in this patient population of both oxysterols. In summary, our method provides a simple, rapid and non-invasive diagnostic tool for the biochemical diagnosis of NP-C disease.

COQ4 Mutations Cause a Broad Spectrum of Mitochondrial Disorders Associated with CoQ10 Deficiency

Primary coenzyme Q10 (CoQ10) deficiencies are rare, clinically heterogeneous disorders caused by mutations in several genes encoding proteins involved in CoQ10 biosynthesis. CoQ10 is an essential component of the electron transport chain (ETC), where it shuttles electrons from complex I or II to complex III. By whole-exome sequencing, we identified five individuals carrying biallelic mutations in COQ4. The precise function of human COQ4 is not known, but it seems to play a structural role in stabilizing a multiheteromeric complex that contains most of the CoQ10 biosynthetic enzymes. The clinical phenotypes of the five subjects varied widely, but four had a prenatal or perinatal onset with early fatal outcome. Two unrelated individuals presented with severe hypotonia, bradycardia, respiratory insufficiency, and heart failure; two sisters showed antenatal cerebellar hypoplasia, neonatal respiratory-distress syndrome, and epileptic encephalopathy. The fifth subject had an early-onset but slowly progressive clinical course dominated by neurological deterioration with hardly any involvement of other organs. All available specimens from affected subjects showed reduced amounts of CoQ10 and often displayed a decrease in CoQ10-dependent ETC complex activities. The pathogenic role of all identified mutations was experimentally validated in a recombinant yeast model; oxidative growth, strongly impaired in strains lacking COQ4, was corrected by expression of human wild-type COQ4 cDNA but failed to be corrected by expression of COQ4 cDNAs with any of the mutations identified in affected subjects. COQ4 mutations are responsible for early-onset mitochondrial diseases with heterogeneous clinical presentations and associated with CoQ10 deficiency.

Mitochondrial dysfunction in central nervous system white matter disorders

Defects of mitochondrial respiration and function had been proposed as a major culprit in the most common neurodegenerative diseases, including prototypic diseases of central nervous system (CNS) white matter such as multiple sclerosis. The importance of mitochondria for white matter is best exemplified in a group of defects of the mitochondria oxidative metabolism called mitochondria leukoencephalopathies or encephalomyopathies. These diseases are clinically and genetically heterogeneous, given the dual control of the respiratory chain by nuclear and mitochondrial DNA, which makes the precise diagnosis and classification challenging. Our understanding of disease pathogenesis is nowadays still limited. Here, we review current knowledge on pathogenesis and genetics, outlining diagnostic clues for the various forms of mitochondria disease. In particular, we underscore the value of magnetic resonance imaging (MRI) for the differential diagnosis of specific types of mitochondrial leukoencephalopathies, such as genetic defects on SDHFA1. The use of novel technologies for gene identification, such as whole‐exome sequencing studies, is expected to shed light on novel molecular etiologies, broadening prenatal diagnosis, disease understanding, and therapeutic options. Current treatments are mostly palliative, but very promising novel gene and pharmacologic therapies are emerging, which may also benefit a growing list of secondary mitochondriopathies, such as the peroxisomal disease adrenoleukodystrophy. GLIA 2014;62:1878–1894

Disease-Causing SDHAF1 Mutations Impair Transfer of Fe-S Clusters to SDHB

SDHAF1 mutations cause a rare mitochondrial complex II (CII) deficiency, which manifests as infantile leukoencephalopathy with elevated levels of serum and white matter succinate and lactate. Here, we demonstrate that SDHAF1 contributes to iron-sulfur (Fe-S) cluster incorporation into the Fe-S subunit of CII, SDHB. SDHAF1 transiently binds to aromatic peptides of SDHB through an arginine-rich region in its C terminus and specifically engages a Fe-S donor complex, consisting of the scaffold, holo-ISCU, and the co-chaperone-chaperone pair, HSC20-HSPA9, through an LYR motif near its N-terminal domain. Pathogenic mutations of SDHAF1 abrogate binding to SDHB, which impairs biogenesis of holo-SDHB and results in LONP1-mediated degradation of SDHB. Riboflavin treatment was found to ameliorate the neurologic condition of patients. We demonstrate that riboflavin enhances flavinylation of SDHA and reduces levels of succinate and Hypoxia-Inducible Factor (HIF)-1α and -2α, explaining the favorable response of patients to riboflavin.

Novel TTC19 mutation in a family with severe psychiatric manifestations and complex III deficiency

Complex III of the mitochondrial respiratory chain (CIII) catalyzes transfer of electrons from reduced coenzyme Q to cytochrome c. Low biochemical activity of CIII is not a frequent etiology in disorders of oxidative metabolism and is genetically heterogeneous. Recently, mutations in the human tetratricopeptide 19 gene (TTC19) have been involved in the etiology of CIII deficiency through impaired assembly of the holocomplex. We investigated a consanguineous Portuguese family where four siblings had reduced enzymatic activity of CIII in muscle and harbored a novel homozygous mutation in TTC19. The clinical phenotype in the four sibs was consistent with severe olivo–ponto–cerebellar atrophy, although their age at onset differed slightly. Interestingly, three patients also presented progressive psychosis. The mutation resulted in almost complete absence of TTC19 protein, defective assembly of CIII in muscle, and enhanced production of reactive oxygen species in cultured skin fibroblasts. Our findings add to the array of mutations in TTC19, corroborate the notion of genotype/phenotype variability in mitochondrial encephalomyopathies even within a single family, and indicate that psychiatric manifestations are a further presentation of low CIII.

A novel AIFM1 mutation expands the phenotype to an infantile motor neuron disease.

AIFM1 is a gene located on the X chromosome, coding for AIF (Apoptosis-Inducing Factor), a mitochondrial flavoprotein involved in caspase-independent cell death. AIFM1 mutations have been associated with different clinical phenotypes: a severe infantile encephalopathy with combined oxidative phosphorylation deficiency and the Cowchock syndrome, an X-linked Charcot-Marie-Tooth disease (CMTX4) with axonal sensorimotor neuropathy, deafness and cognitive impairment. In two male cousins with early-onset mitochondrial encephalopathy and cytochrome c oxidase (COX) deficiency, we identified a novel AIFM1 mutation. Muscle biopsies and electromyography in both patients showed signs of severe denervation. Our patients manifested a phenotype that included signs of both cortical and motor neuron involvement. These observations emphasize the role of AIF in the development and function of neurons.

Effects of levosimendan on mitochondrial function in patients with septic shock: A randomized trial

Mitochondrial dysfunction is key feature of septic shock and contributes to the development of sepsis related organ dysfunction. It is characterized by a variable reduction of the respiratory chain (RC) activities, altered mitochondrial morphology and reactive oxygen species production. Recent data have reported the efficacy of levosimendan, a calcium sensitizer, in improving heart performance and organ perfusion in critically ill patients. Moreover, it has been demonstrated that Levosimendan has antioxidant properties. Nevertheless, the effects of levosimendan on mitochondrial function are not fully elucidated. The objective of this study was therefore to evaluate the effect of levosimendan on mitochondria performance. Five mitochondrial parameters were screened: the redox status; the amount of scavenging enzymes; the activities of the RC complexes; the mitochondrial content; the steady state levels of the RC subunits; the mitochondrial biogenesis. Our results show that patients treated with levosimendan had a significant reduction of glutathionylated proteins and an increase in the amount of the antioxidant enzyme MnSOD, underlining its antioxidant properties. The activities of the RC complexes I, II and III were unchanged in the mitochondria of patients treated with levosimendan compared to controls whereas the mitochondrial content was significantly higher in levosimendan vs. control patients. Finally, evaluation of mitochondrial biogenesis did not show any significant difference in the two groups, although an overall increase in the amount of the RC subunits was observed in the levosimendan group. In conclusion, our study demonstrated that in septic shock patients, Levosimendan exerts antioxidant action by increasing antioxidant defense and lowering oxidative damage.

Novel mutations in IBA57 are associated with leukodystrophy and variable clinical phenotypes

Defects of the Fe/S cluster biosynthesis represent a subgroup of diseases affecting the mitochondrial energy metabolism. In the last years, mutations in four genes (NFU1, BOLA3, ISCA2 and IBA57) have been related to a new group of multiple mitochondrial dysfunction syndromes characterized by lactic acidosis, hyperglycinemia, multiple defects of the respiratory chain complexes, and impairment of four lipoic acid-dependent enzymes: α-ketoglutarate dehydrogenase complex, pyruvic dehydrogenase, branched-chain α-keto acid dehydrogenase complex and the H protein of the glycine cleavage system. Few patients have been reported with mutations in IBA57 and with variable clinical phenotype. Herein, we describe four unrelated patients carrying novel mutations in IBA57. All patients presented with combined or isolated defect of complex I and II. Clinical features varied widely, ranging from fatal infantile onset of the disease to acute and severe psychomotor regression after the first year of life. Brain MRI was characterized by cavitating leukodystrophy. The identified mutations were never reported previously and all had a dramatic effect on IBA57 stability. Our study contributes to expand the array of the genotypic variation of IBA57 and delineates the leukodystrophic pattern of IBA57 deficient patients.

Pyruvate dehydrogenase deficiency presenting as isolated paroxysmal exercise induced dystonia successfully reversed with thiamine supplementation. Case report and mini-review

BACKGROUND Pyruvate dehydrogenase (PDH) deficiency is a disorder of energy metabolism with variable clinical presentations, ranging from severe infantile lactic acidosis to milder chronic neurological disorders. The spectrum of clinical manifestations is continuously expanding. METHODS AND RESULTS We report on a 19-year-old intelligent female with PDH deficiency caused by a Leu216Ser mutation in PDHA1. She presented with recurrent hemidystonic attacks, triggered by prolonged walking or running, as the unique clinical manifestation that manifested since childhood. Laboratory workup and neuroimages were initially normal but bilateral globus pallidum involvement appeared later on brain MRI. Dystonia completely remitted after high doses of thiamine, remaining free of symptoms after 3 years of follow up. We reviewed the literature for similar observations. CONCLUSIONS Dystonia precipitated by exercise may be the only symptom of a PDH deficiency, and the hallmark of the disease as high serum lactate or bilateral striatal necrosis at neuroimaging may be absent. A high index of suspicion and follow up is necessary for diagnosis. The clinical presentation of this patient meets the criteria for a Paroxysmal Exercise induced Dystonia, leading us to add this entity as another potential etiology for this type of paroxysmal dyskinesia, which is besides a treatable condition that responds to thiamine supplementation.

Persistent pulmonary arterial hypertension in the newborn (PPHN): a frequent manifestation of TMEM70 defective patients.

INTRODUCTION Mutations in the TMEM70 are the most common cause of nuclear ATP synthase deficiency resulting in a distinctive phenotype characterized by severe neonatal hypotonia, hypertrophic cardiomyopathy (HCMP), facial dysmorphism, severe lactic acidosis, hyperammonemia and 3-methylglutaconic aciduria (3-MGA). METHODS AND RESULTS We collected 9 patients with genetically confirmed TMEM70 defect from 8 different families. Six were homozygous for the c.317-2A>G mutation, 2 were compound heterozygous for mutations c.317-2A>G and c.628A>C and 1 was homozygous for the novel c.701A>C mutation. Generalized hypotonia, lactic acidosis, hyperammonemia and 3-MGA were present in all since birth. Five patients presented acute respiratory distress at birth requiring intubation and ventilatory support. HCMP was detected in 5 newborns and appeared a few months later in 3 additional children. Five patients showed a severe and persistent neonatal pulmonary hypertension (PPHN) requiring Nitric Oxide (NO) and/or sildenafil administration combined in 2 cases with high-frequency oscillatory (HFO) ventilation. In 3 of these patients, echocardiography detected signs of HCMP at birth. CONCLUSIONS PPHN is a life-threatening poorly understood condition with bad prognosis if untreated. Pulmonary hypertension has rarely been reported in mitochondrial disorders and, so far, it has been described in association with TMEM70 deficiency only in one patient. This report further expands the clinical and genetic spectrum of the syndrome indicating PPHN as a frequent and life-threatening complication regardless of the type of mutation. Moreover, in these children PPHN appears even in the absence of an overt cardiomyopathy, thus representing an early sign and a clue for diagnosis.