A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion.

Isolated mitochondrial myopathies (IMM) are either due to primary defects in mtDNA, in nuclear genes that control mtDNA abundance and structure such as thymidine kinase 2 (TK2), or due to CoQ deficiency. Defects in the TK2 gene have been found to be associated with mtDNA depletion attributed to a depleted mitochondrial dNTP pool in non-dividing cells. We report an unusual case of IMM, homozygous for the H90N mutation in the TK2 gene but unlike other cases with the same mutation, does not demonstrate mtDNA depletion. The patient's clinical course is relatively mild and a muscle biopsy showed ragged red muscle fibers with a mild decrease in complexes I and an increase in complexes IV and II activities. This report extends the phenotypic expression of TK2 defects and suggests that all patients who present with an IMM even with normal quantities of mtDNA should be screened for TK2 mutations.

Clinical presentations of mitochondrial cardiomyopathies.

UNLABELLED: To determine the clinical manifestations and interfamilial variability of patients diagnosed with a mitochondrial cardiomyopathy, we reviewed the charts of 14 patients with cardiomyopathy out of 59 patients with mitochondrial disorders who attended the mitochondrial disease clinic at Wolfson Medical Center from 1996 to 2001. All patients underwent a metabolic evaluation including blood lactate, pyruvate, carnitine, and amino acids and urine organic acids. Respiratory chain enzymes were assessed in 10 patients. The mitochondrial DNA (mtDNA) was assessed for mutations. The age at presentation ranged between 6 months and 24 years. Six of the patients died, 5 from heart failure. The cardiomyopathy was hypertrophic in 10 and dilated in 4. Conduction and rhythm abnormalities were present in 6. Eleven patients had family members with mitochondrial disorders. All the patients had additional involvement of one or more systems. Seven patients exhibited a deficiency of a respiratory chain enzyme in the muscle. The MELAS mtDNA point mutation (3243) was found in one patient. Blood lactic acid levels were increased in 5. Brain MRI abnormalities were observed in 4. CONCLUSIONS: Mitochondrial dysfunction frequently affects the heart and may cause both hypertrophic and dilated cardiomyopathy. The cardiomyopathy is usually a part of a multisystem involvement and may rarely be isolated. The course may be stable for many years, but rapid deterioration may occur. Understanding the biochemical and genetic features of these diseases will enable us to comprehend the clinical heterogeneity of these disorders.

Neonatal liver failure and Leigh syndrome possibly due to CoQ-responsive OXPHOS deficiency.

CoQ transfers electrons from complexes I and II of the mitochondrial respiratory chain to complex III. There are very few reports on human CoQ deficiency. The clinical presentation is usually characterized by: epilepsy, muscle weakness, ataxia, cerebellar atrophy, migraine, myogloblinuria and developmental delay. We describe a patient who presented with neonatal liver and pancreatic insufficiency, tyrosinemia and hyperammonemia and later developed sensorineural hearing loss and Leigh syndrome. Liver biopsy revealed markedly reduced complex I+III and II+III. Addition of CoQ to the liver homogenate restored the activities, suggesting CoQ depletion. Histological staining showed prominent bridging; septal fibrosis and widening of portal spaces with prominent mixed inflammatory infiltrate, associated with interface hepatitis, bile duct proliferation with numerous bile plugs. Electron microscopy revealed a large number of mitochondria, which were altered in shape and size, widened and disordered intercristal spaces. This may be the first case of Leigh syndrome with liver and pancreas insufficiency, possibly caused by CoQ responsive oxphos deficiency.

Identification of a common mutation (Gly194Cys) in both Arab Moslem and Ashkenazi Jewish patients with dihydrolipoamide dehydrogenase (E3) deficiency: possible beneficial effect of vitamin therapy.

Dihydrolipoamide dehydrogenase (E3) deficiency with a clinical phenotype and genotype (Gly194Cys homozygous) previously identified only in Ashkenazi Jewish patients, was diagnosed in two Palestinian Arab siblings and two unrelated Ashkenazi Jewish patients. While three of the four patients died in childhood without specific treatment, the surviving patient at age 18 years may have benefited from long-term daily supplementation with a cocktail of riboflavin, biotin, coenzyme Q and carnitine.

Reduced body fat and increased hepatic lipid synthesis in mice bearing interleukin-6-secreting tumor.

Chronic secretion of interleukin-6 (IL-6) in mice causes metabolic alteration in the liver, leading to increased synthesis of hepatic cholesterol and fatty acids (FA). Mice were injected with allogeneic tumor cells transduced with the murine IL-6 gene. During the 3 wk after tumor inoculation, elevated serum IL-6 levels were associated with increased spleen and liver weight. Histological examination of sections from the liver showed increased hepatocyte proliferation, resulting in liver enlargement. Body composition analysis revealed that IL-6 caused a significant loss in fat tissue without affecting lean body mass and water content. Hepatic de novo synthesis of FA and cholesterol, as measured by (3)H(2)O incorporation, was three to five times as high in mice secreting IL-6 (IL-6 mice) as in pair-fed mice bearing nonsecreting tumors. This increase in FA and cholesterol synthesis is sufficient to maintain hepatic triglyceride secretion at levels comparable with those of pair-fed mice bearing nonsecreting tumors and, presumably, is the main source of cholesterol and FA-phospholipids necessary for hepatocyte proliferation.

Familial mitochondrial intestinal pseudo-obstruction and neurogenic bladder.

Intestinal dysmotility and neurogenic bladder have been described as part of two autosomal-recessive mitochondrial disorders assumed to be due to a defect in communication between the nuclear and mitochondrial genomes: myoneurogastrointestinal encephalopathy (MNGIE) and diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (Wolfram syndrome). Partial cytochrome c oxidase deficiency has been described in both. We describe three Ashkenazi Jewish siblings with progressive intestinal dysmotility, neurogenic bladder, and autonomic manifestations but no central nervous system involvement. Cytochrome c oxidase deficiency was demonstrated in peripheral and multiple intestinal muscle biopsies. Mitochondrial DNA analysis of an intestinal biopsy of patient 1 showed heteroplasmy consisting of a normal 16.5-kb band and an approximately 28-kb band, suggestive of a duplication. Mitochondrial DNA analysis of a muscle biopsy of patient 2 showed multiple deletions, mainly 10- and 11-kb bands. We suggest that this unique combination of intestinal pseudo-obstruction and neurogenic bladder could comprise a new autosomal-recessive mitochondrial disorder.

Neurologic presentations of mitochondrial disorders.

This article describes the neurologic presentations of children with mitochondrial disorders. The charts of 42 children with highly suspect mitochondrial disorders were reviewed. Thirty-seven children were diagnosed as having definite mitochondrial disorders based on a suggestive clinical presentation and at least one accepted criteria, while in five patients the diagnosis remained probable. All patients had nervous system involvement, but it was the presenting symptom in 28 of 42. Eighteen children had normal intelligence and 24 had mental retardation or developmental delay at the onset of their disease. Twenty-five patients had either an acute regression or a progressive encephalopathy. The most frequent neurologic manifestations were abnormal tone, seizures, extrapyramidal movements, and autonomic dysfunction. The eyes were involved in 11 children. Nerve deafness was found in seven patients. Myopathy was found in only six patients. In conclusion, a complex neurologic picture, especially with other organ involvement, warrants a full mitochondrial evaluation.

Multiple presentation of mitochondrial disorders.

The aim of this study was to assess the heterogeneous clinical presentations of children with mitochondrial disorders evaluated at a metabolic neurogenetic clinic. The charts of 36 children with highly suspected mitochondrial disorders were reviewed. Thirty one children were diagnosed as having a mitochondrial disorder, based on a suggestive clinical presentation and at least one of the accepted laboratory criteria; however, in five children with no laboratory criteria the diagnosis remained probable. All of the patients had nervous system involvement. Twenty seven patients also had dysfunction of other systems: sensory organs in 15 patients, cardiovascular system in five, gastrointestinal system in 20, urinary system in four, haematopoietic system in four, and endocrine system in nine. The clinical presentation was compatible with an established syndrome in only 15 children. Severe lactic acidosis or ragged red muscle fibres were encountered in very few patients. These results suggest that mitochondrial disorders should be evaluated in children presenting with a complex neurological picture or multisystem involvement.

Adult polyglucosan body disease in Ashkenazi Jewish patients carrying the Tyr329Ser mutation in the glycogen-branching enzyme gene.

Adult polyglucosan body disease (APBD) is a late-onset, slowly progressive disorder of the nervous system caused by glycogen branching enzyme (GBE) deficiency in a subgroup of patients of Ashkenazi Jewish origin. Similar biochemical finding is shared by glycogen storage disease type IV (GSD IV) that, in contrast to APBD, is an early childhood disorder with primarily systemic manifestations. Recently, the GBE cDNA was cloned and several mutations were characterized in different clinical forms of GSD IV. To examine whether mutations in the GBE gene account for APBD, we studied 7 patients from five Jewish families of Ashkenazi ancestry. The diagnosis was based on the typical clinical and pathological findings, and supported by reduced GBE activity. We found that the clinical and biochemical APBD phenotype in all five families cosegregated with the Tyr329Ser mutation, not detected in 140 controls. As this mutation was previously identified in a nonprogressive form of GSD IV and was shown in expression studies to result in a significant residual GBE activity, present findings explain the late onset and slowly progressive course of APBD in our patients. We conclude that APBD represents an allelic variant of GSD IV, but the reason for the difference in primary tissue involvement must be established.

Mycoplasma fermentans glycolipid triggers inflammatory response in rat astrocytes.

Mycoplasma fermentans glycolipid (MfGL-II) is a major lipid in the membranes of this AIDS-associated mycoplasma and constituting up to 20% of the total phospholipids of this organism. It was recently shown that MfGL-II, mainly through its phosphocholine moiety, is responsible for the attachment of M. fermentans to host cells. We now show that MfGL-II is also associated with the secretion of inflammatory mediators by cells of the central nervous system. Stimulation of primary rat astrocytes by MfGL-II caused activation of protein kinase C, secretion of nitric oxide (NO) and prostaglandin E2, and augmented glucose utilization and lactate formation in a dose-dependent manner. In an attempt to define the minimal structural requirements for MfGL-II activity, the two O-acylated fatty acids in the molecule were removed. Deacylation pronouncedly reduced the stimulatory activity of the glycolipid, suggesting that the fatty acyl residues are essential. Incubation of MfGL-II with polyclonal anti-MfGL-II antiserum or with monoclonal anti-phosphocholine antibody diminished NO release, whereas incubation of MfGL-II with normal rabbit serum had no effect. It is, therefore, likely that the terminal phosphocholine moiety plays an important role in MfGL-IIs stimulation of glial cells.

Glycogen storage disease type 1a in Israel: biochemical, clinical, and mutational studies.

Glycogen storage disease type 1a (von Gierke disease, GSD 1a) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity which catalyzes the final common step of glycogenolysis and gluconeogenesis. The recent cloning of the G6Pase cDNA and characterization of the human G6Pase gene enabled the characterization of the mutations causing GSD 1a. This, in turn, allows the introduction of a noninvasive DNA-based diagnosis that provides reliable carrier testing and prenatal diagnosis. In this study, we report the biochemical and clinical characteristics as well as mutational analyses of 12 Israeli GSD 1a patients of different families, who represent most GSD 1a patients in Israel. The mutations, G6Pase activity, and glycogen content of 7 of these patients were reported previously. The biochemical data and clinical findings of all patients were similar and compatible with those described in other reports. All 9 Jewish patients, as well as one Muslim Arab patient, presented the R83C mutation. Two Muslim Arab patients had the V166G mutation which was not found in other patients' populations. The V166G mutation, which was introduced into the G6Pase cDNA by site-directed mutagenesis following transient expression in COS-1 cells, was shown to cause complete inactivation of the G6Pase. The characterization of all GSD 1a mutations in the Israeli population lends itself to carrier testing in these families as well as to prenatal diagnosis, which was carried out in 2 families. Since all Ashkenzai Jewish patients harbor the same mutation, our study suggests that DNA-based diagnosis may be used as an initial diagnostic step in Ashkenazi Jews suspected of having GSD 1a, thereby avoiding liver biopsy.

Interleukin-6 secretion in mice is associated with reduced glucose-6-phosphatase and liver glycogen levels.

Mice bearing interleukin-6 (IL-6)-secreting tumor were used to study the chronic effect of IL-6 on carbohydrate metabolism. Mice were injected with allogeneic tumor cells transduced with the murine IL-6 gene. Serum IL-6 levels were correlated exponentially with tumor weight. Secretion of IL-6 from the developed tumors was associated with decreased food consumption, reduced body weight, and reduced blood glucose levels. Insulin levels did not change, and 2-deoxyglucose uptake was not affected in most tissues examined. A significant increase of 2-deoxyglucose uptake was measured in the liver. Glycogen content in the liver determined 0, 6, 12, and 18 days after tumor inoculation was 42, 23, 12, and 3 mg/g, respectively. The activity of phosphoenolpyruvate carboxykinase was not affected. The activity of glucose-6-phosphatase (G-6-Phase) determined 6, 12, and 18 days after tumor injection was 84, 70, and 50% of G-6-Pase activity in pair-fed mice bearing nonsecreting tumors, respectively. G-6-Pase mRNA levels were markedly reduced due to inhibition of G-6-Pase gene transcriptional rate.

Tumor necrosis factor inhibits the transcriptional rate of glucose-6-phosphatase in vivo and in vitro.

Recombinant human tumor necrosis factor-alpha (TNF) injection in mice was associated with a reduced blood glucose level, already manifest 6 hours following cytokine administration. Insulin levels were not affected. Glycogen content was decreased in a dose-dependent and time-response manner. The activity of glucose-6-phosphatase (G6Pase) was already reduced 6 hours after TNF injection and was sustained 12 hours afterward. Phosphoenolpyruvate carboxykinase (PEPCK) activity was not affected initially (6 hours after injection), but a 50% reduction was observed 12 hours following cytokine administration compared with levels in fasting controls. Both liver G6Pase and PEPCK mRNAs were markedly reduced due to an inhibition of the transcriptional rate. A direct inhibitory effect of TNF on G6Pase promoter activity was demonstrated using HuH-7 cells transiently transfected with G6Pase promoter, fused to a reporter gene.

Insulin resistance in the NIDDM model Psammomys obesus in the normoglycaemic, normoinsulinaemic state.

The desert gerbil Psammomys obesus ("sand rat"), a model of nutritionally induced insulin resistance and non-insulin-dependent diabetes mellitus, was treated after weaning with exogenous insulin implants in the normoglycaemic, normoinsulinaemic state. Albino rats matched for weight and age served as high energy diet adjusted reference animals. Insulin administration, elevating the serum insulin to 6000 pmol/l resulted in only a mild reduction in blood glucose levels in Psammomys, but caused a severe, often fatal hypoglycaemia in the albino rats. The hepatic response to insulin-induced hypoglycaemia in rats involved a significant loss in glycogen and suppression of phosphoenolpyruvate carboxykinase (PEPCK) activity. In Psammomys under similar hyperinsulinaemia no appreciable changes in liver glycogen and PEPCK activity were evident, indicating that blood glucose was replenished by continuing gluconeogenesis. Euglycaemic, hyperinsulinaemic clamp caused a complete shut-down of hepatic glucose production in albino rats. However, in both diabetes-prone and diabetes-resistant Psammomys lines, mean hepatic glucose production was reduced by only 62 to 53% respectively, despite longer lasting and higher levels of hyperinsulinaemia. These results indicate that Psammomys is characterized by muscle and liver insulin resistance prior to diet-induced hyperglycaemia and hyperinsulinaemia. This is assumed to be a species feature of Psammomys, exemplifying a metabolic adjustment to survival in conditions of food scarcity of both animal and human populations. It may reflect a propensity to insulin resistance and hyperglycaemia in population groups exposed to affluent nutrition.

Congenital lacticacidemia caused by lipoamide dehydrogenase deficiency with favorable outcome.

A 5-year-old boy had recurrent vomiting and lethargy with lacticacidemia and ketoacidemia since birth. Lipoamide dehydrogenase deficiency was found in muscle and fibroblasts. Therapy with sodium dichloroacetate, thiamine, and carnitine was associated with reduction of the severity and frequency of the decompensation episodes and near normal plasma lactate levels. At 5 years of age, the patient has normal cognitive function and moderate motor impairment.

The frequency of the C854 mutation in the aspartoacylase gene in Ashkenazi Jews in Israel.

Canavan disease (CD) is an infantile neurodegenerative disease that is transmitted in an autosomal recessive manner and has mainly been reported in Ashkenazi Jewish families. The primary enzymatic defect is aspartoacylase deficiency, and an A-to-C transition at nucleotide 854 of the cDNA has recently been reported. We screened 18 patients with CD and 879 healthy individuals, all Israeli Ashkenazi Jews, for the mutation. All 18 patients were homozygotes for the mutation, and 15 heterozygotes were found among the healthy individuals. The results disclose a carrier rate of 1:59 and suggest that a screening for the mutation is warranted among Ashkenazi Jewish couples.

Late-onset muscular weakness in phosphofructokinase deficiency due to exon 5/intron 5 junction point mutation: a unique disorder or the natural course of this glycolytic disorder?

Late-onset muscle weakness is rare in glycolytic disorders. There are two reports in the literature of phosphofructokinase (PFK)-deficient Ashkenazi Jews with severe vacuolar myopathy manifesting in late adulthood. The genetic abnormality in these patients is unknown. We report a third patient with a similar syndrome: early-onset exercise intolerance in young childhood and progressive weakness in a limb-girdle distribution appearing at 57 years of age, leading to severe incapacity. Muscle histology showed diffuse vacuolar changes, and muscle fibers contained excess glycogen-like material. Muscle biochemistry was diagnostic for PFK deficiency. DNA analysis from the patient and his family showed that he was homozygous for a recently identified point mutation at the exon 5/intron 5 junction (a G-to-A change); two other family members were heterozygous for this mutation. It is not clear whether late-onset weakness is the natural course for all PFK-deficient patients or whether the exon 5 mutation carries increased risk for this severe myopathy.

Modulation of fetal and placental metabolic pathways in response to maternal thyroid and glucocorticoid hormone excess.

Triiodothyronine (T3) treatment of pregnant rats for 6 days, 10 micrograms/100 g, resulted in a pronounced induction of enzymes related to gluconeogenesis and lipogenesis and of mitochondrial FAD-glycerophosphate dehydrogenase in the maternal liver, as previously observed in male rats. There was virtually no change in the activity of these enzymes in the placenta. However, there was a distinct induction of these enzymes in the fetal liver, even if increments in fetal serum and liver T3 were much smaller than on the maternal side. This indicates that changes in hepatic enzyme activities are a more sensitive index of fetal hyperthyroidism than T3 levels. The increased lipogenic capacity was expressed by greater incorporation of a tritium tracer into fatty acids. Administration of triamcinolone, 2 mg/100 g, for the last 5 days of gestation resulted in marked induction of maternal hepatic enzymes of lipogenesis, gluconeogenesis and of aspartate aminotransferase (ASAT), known to occur in male rats, as well as in a metabolic pattern of insulin resistance. The response of placental enzymes was limited to a small elevation in ASAT and phosphoenolpyruvate carboxykinase (PEPCK) activity. In the fetal liver there was no stimulation of lipogenic enzymes, but a marked induction of PEPCK and ASAT. The changes in the lipogenic capacity were confirmed by tritium incorporation into serum and liver fatty acids. These results demonstrate the marked sensitivity of specific fetal enzyme systems to the maternal iatrogenic hyperthyroidism or hypercorticism. The limited alterations in placental enzyme activities are in accord with the concept that placental metabolic stability fulfils a protective function toward the fetus.

Metabolic effects of nicotine on human adipose tissue in organ culture.

Fragments of human adipose tissue were maintained in culture for 1 week in a medium containing 1 mU/ml insulin and 100 ng/ml dexamethasone. Under these conditions lipoprotein lipase activity was present in human adipose tissue fragments which converted [14C]glucose to 14CO2 and [14C]triglyceride. Both metabolic parameters studied were affected by human tumor necrosis factor and brefeldin A. When fragments of human adipose tissue after 1 week in culture were incubated with nicotine tartrate for 20 h, a slight but significant increase in lipoprotein lipase activity was observed, and an increased conversion of [14C]glucose to 14CO2 and [14C]triglyceride occurred. Nicotine was taken up by human adipose tissue, but no conversion to cotinine was observed. Our data demonstrate a direct effect of nicotine on human adipose tissue metabolism. Furthermore, it is suggested that weight loss in smokers is a multifactorial phenomenon, and one of the important factors to be considered is the direct effect of nicotine within the tissue.