Novel mutations in KARS cause hypertrophic cardiomyopathy and combined mitochondrial respiratory chain defect.

Mutations in KARS, which encodes for both mitochondrial and cytoplasmic lysyl-tRNA synthetase, have been so far associated with three different phenotypes: the recessive form of Charcot-Mary-Tooth polyneuropathy, the autosomal recessive nonsyndromic hearing loss and the last recently described condition related to congenital visual impairment and progressive microcephaly. Here we report the case of a 14-year-old girl with severe cardiomyopathy associated to mild psychomotor delay and mild myopathy; moreover, a diffuse reduction of cytochrome C oxidase (COX, complex IV) and a combined enzymatic defect of complex I (CI) and complex IV (CIV) was evident in muscle biopsy. Using the TruSight One sequencing panel we identified two novel mutations in KARS. Both mutations, never reported previously, occur in a highly conserved region of the catalytic domain and displayed a dramatic effect on KARS stability. Structural analysis confirmed the pathogenic role of the identified variants. Our findings confirm and emphasize that mt-aminoacyl-tRNA synthetases (mt-ARSs) enzymes are related to a broad clinical spectrum due to their multiple and still unknown functions.

A novel mutation in NDUFB11 unveils a new clinical phenotype associated with lactic acidosis and sideroblastic anemia.

NDUFB11, a component of mitochondrial complex I, is a relatively small integral membrane protein, belonging to the "supernumerary" group of subunits, but proved to be absolutely essential for the assembly of an active complex I. Mutations in the X-linked nuclear-encoded NDUFB11 gene have recently been discovered in association with two distinct phenotypes, i.e. microphthalmia with linear skin defects and histiocytoid cardiomyopathy. We report on a male with complex I deficiency, caused by a de novo mutation in NDUFB11 and displaying early-onset sideroblastic anemia as the unique feature. This is the third report that describes a mutation in NDUFB11, but all are associated with a different phenotype. Our results further expand the molecular spectrum and associated clinical phenotype of NDUFB11 defects.

DJ-1 modulates mitochondrial response to oxidative stress: clues from a novel diagnosis of PARK7.

DJ-1 mutations are associated to early-onset Parkinson's disease and accounts for about 1-2% of the genetic forms. The protein is involved in many biological processes and its role in mitochondrial regulation is gaining great interest, even if its function in mitochondria is still unclear. We describe a 47-year-old woman affected by a multisystem disorder characterized by progressive, early-onset parkinsonism plus distal spinal amyotrophy, cataracts and sensory-neural deafness associated with a novel homozygous c.461C>A [p.T154K] mutation in DJ-1. Patient's cultured fibroblasts showed low ATP synthesis, high ROS levels and reduced amount of some subunits of mitochondrial complex I; biomarkers of oxidative stress also resulted abnormal in patient's blood. The clinical pattern of multisystem involvement and the biochemical findings in our patient highlight the role for DJ-1 in modulating mitochondrial response against oxidative stress.

Effect of protein glutathionylation on neuronal cytoskeleton: a potential link to neurodegeneration.

Neurons are highly susceptible to oxidative stress and oxidation of cytoskeletal proteins is considered one of the first steps of neurodegeneration. Protein glutathionylation is a key event in the redox regulation of protein function and constitutes a sensor of tissue oxidative stress in patho-physiological conditions. In this study, we analyzed for the first time tubulin glutathionylation and its relation to neurites degeneration. For this purpose, we exposed motoneuronal cells to the physiological oxidant glutathione disulfide (GSSG) and we analyzed the extent and morphology of axonal changes caused by protein glutathionylation in these cells. Then we studied the effect of glutathionylation on the distribution of stable and dynamic microtubules in the same cells. Our results indicate that oxidative stress conditions determined by an increased intracellular level of oxidized glutathione may cause an alteration of the cytoskeleton organization and function leading to axon degeneration. These findings might contribute to understand the sequence of pathogenic events involved in the axonal degeneration that characterizes many diseases of the nervous system associated with oxidative stress.

Waist circumference correlates with liver fibrosis in children with non-alcoholic steatohepatitis.

OBJECTIVE: Waist circumference is widely accepted as a risk factor for cardiovascular disease and metabolic syndrome. Non-alcoholic fatty liver disease (NAFLD) is a feature of the metabolic syndrome. A contribution of metabolic syndrome, and especially of waist circumference, to liver fibrosis in children with NAFLD is strongly suspected. DESIGN: Cross-sectional study. SETTING: Department of Hepatogastroenterology and Nutrition, Paediatric Hospital "Bambino Gesù", Rome, Italy. PATIENTS: 197 consecutive Caucasian children with NAFLD (136 males and 61 females) aged 3-19 years. MAIN OUTCOME MEASURES: Multivariable logistic regression models were used to examine the contribution of gender, age, body mass index (BMI) and metabolic syndrome components (waist circumference, high-density lipoprotein (HDL)-cholesterol, triglycerides, blood pressure and glucose) to the odds of liver fibrosis as detected by liver biopsy. RESULTS: 92% of the children had BMI > or = 85(th) percentile and 84% had a waist > or = 90(th) percentile for gender and age. Ten per cent of the children had metabolic syndrome and 67% had liver fibrosis, mostly of low degree. At multivariable analysis, waist was the only metabolic syndrome component to be associated with liver fibrosis. This was seen both when the components of the metabolic syndrome were coded as dichotomous (odds ratio (OR) = 2.40; 95% confidence interval (CI), 1.04 to 5.54) and continuous (OR = 2.07; 95% CI, 1.43 to 2.98 for a 5 cm increase). In the latter case, age was also associated with the outcome (OR = 0.70; 95% CI, 0.55 to 0.89 for a 1 year increase). CONCLUSIONS: Abdominal rather than generalised obesity contributes to liver fibrosis in children with NAFLD. Waist is also the only component of the metabolic syndrome to be associated with fibrosis in these children. Therefore, the presence of abdominal obesity is an additional criterion for the selection of children and adolescents who should undergo extensive investigation, including liver biopsy.

Effect of vitamin E on aminotransferase levels and insulin resistance in children with non-alcoholic fatty liver disease.

BACKGROUND: Few data are available on the effect of antioxidants in paediatric non-alcoholic fatty liver disease (NAFLD). AIM: To compare the effect of a nutritional programme alone or combined with alpha-tocopherol and ascorbic acid on alanine aminotransferase (ALT) levels, and insulin resistance (IR) in biopsy-proven NAFLD children. METHODS: IN a 12-month double-blind placebo study, 90 patients were prescribed a balanced calorie diet (25-30 cal/kg/d), physical exercise, and placebo (group A) or alpha-tocopherol 600 IU/day plus ascorbic acid 500 mg/day (group B). IR was estimated by the homeostasis model assessment (HOMA-IR). RESULTS: At month 12, ALT (32.67 +/- 8.09 vs. 32.18 +/- 11.39 IU/L; P = NS), HOMA-IR (1.52 +/- 0.66 vs. 1.84 +/- 0.95 IU/L; P = NS), and weight loss (32% vs. 35% of excessive body weight; P = NS) did not differ between the two arms. Among subjects who lost >or=20% of their excessive weight, ALT and body weight percentage changes were significantly related (r(o) = 0.260; P = 0.03). In subjects, who lost more than 1.0 kg, HOMA-IR significantly decreased (2.20 +/- 0.21 to 1.57 +/- 0.13 in group A (P

Antioxidant enzymes in blood of patients with Friedreich's ataxia.

BACKGROUND AND AIMS: Increased generation of reactive oxygen species and mitochondrial dysfunction may underlie the pathophysiology of Friedreich's ataxia, the most common inherited ataxia, due to GAA expansion in a gene coding for a mitochondrial protein (frataxin), implicated in the regulation of iron metabolism. Because iron overload would cause oxidative stress in Friedreich's ataxia, we investigated the enzyme antioxidant system in the blood of 14 patients by determining superoxide dismutase, glutathione peroxidase, and glutathione transferase catalytic activities. We also studied the glutathione S-transferase genotype polymorphism in order to evaluate its possible influence on enzyme activity. METHODS: Blood samples were obtained from 14 unrelated patients with Friedreich's ataxia and 21 age matched healthy subjects. Antioxidant enzyme determinations were spectrophotometrically assayed using specific substrates; the glutathione S-transferase genotype polymorphism was analysed by endonuclease restriction mapping of exon 5 and 6 amplification products. RESULTS: There was a significant elevation of the superoxide dismutase/glutathione peroxidase activity ratio (0.037 (0.01) v 0.025 (0.008) of controls) and an 83% rise of glutathione transferase specific activity (0.22 (0.1) v 0.12 (0.03) nmol/min/mg protein) in blood of patients with Friedreich's ataxia than in the controls. The genotype polymorphism of glutathione S-transferase enzyme did not show any relevant differences when compared to that of healthy subjects. CONCLUSIONS: Data show an impairment in vivo of antioxidant enzymes in patients with Friedreich's ataxia and provide evidence of an increased sensitivity to oxidative stress, supporting a consistent role of free radical cytotoxicity in the pathophysiology of the disease.

Hypertrophic cardiomyopathy and mtDNA depletion. Successful treatment with heart transplantation.

Cardiomyopathy associated with a mitochondrial DNA depletion syndrome is a rare condition. We report on a child with a hypertrophic cardiomyopathy and a mitochondrial depletion syndrome who was successfully treated by heart transplantation, given the tissue-specific nature of her mitochondrial disorder.

Glutathione in blood of patients with Friedreich's ataxia.

BACKGROUND: Oxidative stress and mitochondrial dysfunction have long been considered to play a role in Friedreich's ataxia, a neurodegenerative disease due to a GAA expansion in a gene coding for a mitochondrial protein (frataxin), implicated in the regulation of iron metabolism. Since glutathione is an important antioxidant whose role has been recently proposed in the pathogenesis of some neurodegenerative diseases, we investigated glutathione metabolism in the blood of 14 patients with Friedreich's ataxia by measuring total, free and protein-bound glutathione concentrations. MATERIALS AND METHODS: Blood samples were obtained from 14 unrelated patients with Friedreich's ataxia (nine males, five females) and 20 age-matched healthy controls (10 males, 10 females). Total and free glutathione concentrations were determined by reverse-phase liquid chromatography with fluorescence detection; the glutathionyl-haemoglobin separation from healthy and pathological subjects was obtained by electrospray ionization-mass spectrometry. RESULTS: We consistently found a reduction of free glutathione levels (0.55 +/- 0.06 nmol mg(-1) haemoglobin, vs. 8.4 +/- 1.79 nmol mg(-1) haemoglobin, P < 0.001) in the blood of patients with Friedreich's ataxia, a total glutathione concentration comparable to the controls (15 +/- 2.6 nmol mg(-1) haemoglobin, vs. 15.4 +/- 1.4 nmol mg(-1) haemoglobin), and a significant increase of glutathione bound to haemoglobin (15 +/- 1.5 vs. 8 +/- 1.8%, P < 0.05) in erythrocytes. CONCLUSIONS: Our findings give evidence of an impairment in vivo of glutathione homeostasis in Friedreich's ataxia, suggesting a relevant role of free radical cytotoxicity in the pathophysiology of the disease; this study may also prove useful in the search for an oxidative stress marker in neurodegeneration.

Respiratory chain defects in hereditary spastic paraplegias.

Hereditary Spastic Paraplegias (HSPs) are heterogeneous neurodegenerative disorders whose etiopathogenesis is still unclear. The identification of pathogenic mutations in a gene (SPG7) encoding a mitochondrial metalloprotease suggested that oxidative phosphorylation (OXPHOS) alterations might underlie HSP in a subgroup of patients. We performed clinical, morphological, biochemical, and molecular genetic studies in six HSP patients and in six sporadic patients to investigate OXPHOS in muscle biopsies. Complicated and pure forms were included in our study. Morphological alterations of the type seen in OXPHOS-related disorders were found in three patients. Five patients showed an isolated defect of complex I activity. No mutations in the SPG7 gene were detected. Our results suggest that OXPHOS defects in HSP patients are more common than previously believed.

Fatal infantile leukodystrophy: a severe variant of CACH/VWM syndrome, allelic to chromosome 3q27.

OBJECTIVE: To describe clinical and neuropathologic studies and linkage analysis on two sisters with a severe form of leukodystrophy. METHODS: A detailed study was performed on the second sister. Genotyping markers for chromosome 3, including eight additional markers surrounding the vanishing white matter (VWM) locus, were used. RESULTS: During the first year of life, two sisters developed a severe neurologic condition after an intercurrent infection. It was accompanied by irritability and stupor with rapid loss of their motor abilities. Results of extensive metabolic studies were negative. Brain MRI showed severe and diffuse abnormalities of the encephalic white matter. Neuropathologic examination showed a severe lack of myelin with diffuse vacuolating white matter lesions in the brain, associated with an increased density of oligodendrocytes and a reduced number of astrocytes on morphometric analysis. In sharp contrast, the spinal cord white matter was preserved. The affected sibpairs shared a common haplotype for a broad region in chromosome 3. They were homozygous between markers D3S1565 and D3S3669, including the VWM locus. CONCLUSIONS: This condition is an unusual variant of childhood ataxia with diffuse central hypomyelination (CACH)/VWM, with characteristic shrinking and perivascular clustering of astrocytes. Haplotype analysis suggests that this variant is allelic to the VWM locus located on chromosome 3q27.

Cytochrome c oxidase-deficient patients have distinct subunit assembly profiles.

Cytochrome c oxidase (COX) deficiency is the most common respiratory chain defect in childhood and is clinically heterogeneous. We report a study of six patients with COX deficiencies. Two of the patients had as yet undefined defects, three patients had Surf-1 mutations, and one patient had a 15-base pair deletion in the COX III subunit. We show that quantitative measurements of steady-state levels of subunits by monoclonal antibody reactivity, when used in combination with a discontinuous sucrose gradient methods, provide an improved diagnosis of COX deficiencies by distinguishing between kinetic, stability, and assembly defects. The two mutants of undefined etiology had a full complement of subunits with one stable and the other partially unstable to detergent solubilization. Both are likely to carry mutations in nuclear-encoded subunits of the complex. The three Surf-1 mutants and the COX III mutant each had reduced steady-state levels of subunits but variable associations of the residual subunits. This information, as well as aiding in diagnosis, helps in understanding the genotype-phenotype relationships of COX deficiencies and provides insight into the mechanism of assembly of the enzyme complex.

The T9176G mtDNA mutation severely affects ATP production and results in Leigh syndrome.

The authors identified a novel mtDNA mutation (T9176G) in the ATPase 6 gene in a family in which a 10-year-old girl had a severe neurodegenerative disorder, her elder sister had died of Leigh syndrome (LS), and a maternal uncle had a spinocerebellar disorder. Biochemical studies disclosed a reduced rate of ATP synthesis in skin fibroblast cultures from the proposita as the likely explanation of her severe illness. The findings expand the genetic variants associated with LS.

A novel SURF1 mutation results in Leigh syndrome with peripheral neuropathy caused by cytochrome c oxidase deficiency.

We report on a 5-year-old boy with clinical and neuroradiological evidence of Leigh syndrome and peripheral neuropathy. Skeletal muscle biopsy showed decreased cytochrome c oxidase stain. Ultrastructurally, the nerve biopsy showed a defect of myelination. Biochemical analyses of muscle homogenate showed cytochrome c oxidase deficiency (15% residual activity). SURF1 gene analysis identified a novel homozygous nonsense mutation which predicts a truncated surf1 protein.

OXPHOS and mtDNA alterations in a family with spastic paraparesis.

OBJECTIVE: To study muscle biopsies in hereditary spastic paraparesis (HSP). METHODS: We analyzed oxidative phosphorylation activities and mtDNA in 3 individuals from an HSP family. RESULTS: We found histochemical evidence for mitochondrial proliferation and cytochrome c oxidase negative fibers. Biochemically, there was an important reduction of the activities of complexes I and IV in 3 patients. In addition, multiple mtDNA deletions (ranging 4.0-7.0 kb) were found in 2 cases by PCR but not by Southern blot. CONCLUSION: We suggest the use of a muscle biopsy when examining HSP patients. HSP can represent a disorder of nuclear-mitochondrial intercommunication.

Novel 7-DHCR mutation in a child with Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by minor facial anomalies, mental retardation, and multiple congenital abnormalities. Biochemically, the disorder is caused by deficient activity of 7-dehydrocholesterol reductase, which catalyzes the reduction of the Delta7 double bond of 7-dehydrocholesterol to produce cholesterol. Recently, mutations in the gene encoding 7-dehydrocholesterol reductase (7DHCR) were found to cause SLOS. We report the first molecular characterization of an Italian SLOS patient. Interestingly, his paternal 7DHCR allele, of Arab origin, harbored a novel P329L mutation which in combination with a maternal splice-site (IVS8-1 G>C) mutation resulted in a relatively milder phenotype.