A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure.

Complex III (CIII; ubiquinol cytochrome c reductase of the mitochondrial respiratory chain) catalyzes electron transfer from succinate and nicotinamide adenine dinucleotide-linked dehydrogenases to cytochrome c. CIII is made up of 11 subunits, of which all but one (cytochrome b) are encoded by nuclear DNA. CIII deficiencies are rare and manifest heterogeneous clinical presentations. Although pathogenic mutations in the gene encoding mitochondrial cytochrome b have been described, mutations in the nuclear-DNA-encoded subunits have not been reported. Involvement of various genes has been indicated in assembly of yeast CIII (refs. 8-11). So far only one such gene, BCS1L, has been identified in human. BCS1L represents, therefore, an obvious candidate gene in CIII deficiency. Here, we report BCS1L mutations in six patients, from four unrelated families and presenting neonatal proximal tubulopathy, hepatic involvement and encephalopathy. Complementation study in yeast confirmed the deleterious effect of these mutations. Mutation of BCS1L would seem to be a frequent cause of CIII deficiency, as one-third of our patients have BCS1L mutations.

Factor VIII procoagulant antigen in human tissues.

The localization of factor VIII procoagulant antigen (VIII:Ag) and factor VIII von Willebrand antigen (VWF:Ag) was investigated in human liver, lung, spleen, placenta and umbilical cord, by an immunoperoxidase technique using an avidin biotin complex (ABC). Positive staining for VIII:Ag was observed in the endothelial cells of liver sinusoids, veins and arteries, as well as in the endothelial cells of placenta, lung and spleen. VWF:Ag was detected in the vascular endothelial cells of all the organs explored. The staining intensity of both VIII:Ag and VWF:Ag varied in the different tissues and showed a distinctive pattern of distribution in the liver. VIII:Ag was also observed in the cytoplasm of dysplastic, foetal-like hepatocytes which infiltrated one liver specimen. Our results agree with the view that liver endothelial cells are a major site of Factor VIII (F VIII) storage and secondary release into the circulation. However, the bright staining intensity of VIII:Ag and VWF:Ag in the lung and placenta suggests that these two tissues might also be a substantial source of F VIII.

Human cultured skin fibroblasts survive profound inherited ubiquinone depletion.

Beside its role in electron transfer in the mitochondrial respiratory chain, ubiquinone is known to prevent lipid peroxidation and DNA damage by trapping cellular free radicals. Thanks to its antioxidant properties, ubiquinone may represent an important factor controlling both necrotic and apoptotic processes. We have investigated the consequences of a profound inherited ubiquinone depletion on cultured skin fibroblasts of a patient presenting with encephalomyopathy. Interestingly, cell respiration, mitochondrial oxidation of various substrates, and cell growth of ubiquinone-deficient fibroblasts were only partially decreased. Moreover, these cells did not apparently overproduce superoxide anions or lipoperoxides. Finally, apoptosis did not increase as compared to control, even after serum deprivation. These observations suggest that ubiquinone may not play a major role in the antioxidant defenses of cultured fibroblasts and that its role in controlling oxidative stress and apoptosis may greatly vary across cell types, especially as not all tissues were equally affected in the patient despite the widespread ubiquinone depletion in vivo.

Fructose-induced enhanced mitogenicity of diploid human cells: possible relationship with cell differentiation.

Fructose strongly stimulates the growth of normal diploid human skin fibroblasts (SFs) and induces marked changes in their morphology and lipid accumulation. This mitogenic effect occurs despite very low fructose consumption and depends on the presence of glutamine. The cell kinetics of cultured fructose-fed human skin fibroblasts were different from those fed on glucose: in the presence of fructose a high proliferative index persisted at Day 14 of culture and the duration of the total cell cycle and of the G1 + 1/2 M and S phases was slightly shorter. The mitogenic effect of fructose on SF was largest in the presence of human serum: it was small or undetectable when fibroblasts were cultured in media supplemented with dialyzed human serum, fetal bovine serum, or serum substitutes. This suggests that serum growth factor(s) mediate the mitogenic effect of fructose. Only normal diploid human cells seem to be sensitive to this mitogenic effect of fructose: the long-term growth of normal human liver cells on fructose was slightly better or similar to that on glucose. In contrast, fructose could only support limited growth of hamster fibroblastic Nil cells and of a transformed human fibroblastic line, which grew better with glucose.

Impaired hexose uptake by diploid skin fibroblasts from galactosaemic patients. Connection with cell growth and amino acid metabolism, and possible bearing on late-onset clinical symptoms.

In skin fibroblasts of patients presenting with galactosaemia, either from galactose 1-phosphate uridyltransferase or galactokinase deficiency, a deficit in extracellular glucose utilization was observed. This deficit was constant over 3 weeks of continuous cell growth in a medium containing 5.5 mmol/L glucose as the only hexose, and homologous serum. Levels of glucose utilization by deficient skin fibroblasts were stable at about 65-70% of the glucose utilization of control normal skin fibroblasts. Cell morphology was normal, and cell growth was subnormal during this period. However, the energy provision appeared sufficient for cellular needs since cell growth in this glucose medium was observed not to depend on the presence of extracellular glutamine. In contrast, glutamine was required for growth of galactosaemic fibroblasts cultured in medium containing 5.5 mmol/L galactose. If expressed in many cell types, this impaired glucose uptake would be expected seriously to damage highly glucose-dependent tissues such as the central nervous system. This might be of relevance to the persistent neurological damage observed in many galactosaemic patients in spite of their compliance with an early strict galactose-free diet.

Low efficiency of [14C]galactose incorporation by galactosemic skin fibroblasts: relationship with neurological sequelae.

The incorporation of radioactivity from [1-14C]-galactose into TCA-precipitable material was determined in skin fibroblasts derived from 11 galactosemic patients deficient in galactose 1-phosphate uridyl transferase (GALT-). "R" ratios (designated the R phenotype) were defined as the ratio between [14C]galactose incorporation and [3H]leucine incorporation. Results were expressed as a percentage of the controls. In the GALT-strains this ratio varied from strain to strain, presumably depending on the efficiency of the secondary route via the UDP-galactose pyrophosphorylase pathway. In 10 GALT-patients without late serious clinical manifestations, the R phenotype varied from 37 to 57% of the control value. In the 11th patient, the R phenotype was only 20% of the control. Thus, we obtained a significantly lower R phenotype in one patient who was distinguished from the others by having very severe delayed neurological complications, although compliance to galactose-free diet was good. We suggest that, in this patient, the development of the UDP-galactose pyrophosphorylase pathway was not sufficient to ensure the availability of enough galactose for the necessary synthesis of glycoproteins and glycolipids. Thus the R phenotype may be an indicator of the risk of late neurological complications. The determination of the R phenotype of GALT-patients may therefore be valuable. However, further investigations of galactosemic patients with neurological complications are required to confirm this relationship.

[Metabolic cooperation in cocultures of fibroblasts from patients with various abnormalities of galactose metabolism]. / Coopération métabolique dans des cocultures de fibroblastes issus de patients atteints d'anomalies différentes du métabolisme du galactose.

14C galactose incorporation into the TCA-precipitable material of cultures of fibroblasts deficient in galactokinase (GALK-) was nil. In cultures of fibroblasts deficient in uridyltransferase (GALT-), it was 30 to 75% of control incorporation. In cocultures of GALK and GALT-deficient fibroblasts, 14C incorporation was restored to near-normal levels. This restoration produced in the presence of close cellular contacts was not increased by polyethyleneglycol somatic hybridization. Our results indicate that metabolic cooperation occurred involving the transfer of galactose 1-phosphate from the GALT-deficient to the GALK-deficient cells via intercellular connections.

Cytosolic thymidine kinase activity in cultured human fibroblasts from individuals with galactokinase deficiency.

The structural genes for human galactokinase (GALK) and the human cytosolic form of thymidine kinase (TK1) are located on 17q21-q22. These two loci are tightly linked, and studies on Chinese hamster cell lines have shown that the expression of TK1 and GALK genes may alter simultaneously. We investigated the possibility of a dependent mutation of TK1 and GALK genes in cultured fibroblasts, obtained from two patients homozygous for the GALKG-deficient gene. Since we showed that the TK1 level varies as a function of the passage and the growth rate of a given strain, our experiments were performed on nonstored skin fibroblasts, between the third and the fifth passage for both controls and patients. We found that TK1 levels in GALK-deficient cells were almost 75% of those observed in control strains with a similar growth rate. Previous results in the literature have shown a pronounced decrease in TK1 activity in three GALK-deficient fibroblastic strains. We suggest that these disparities of TK1 levels in GALK-deficient fibroblasts may be related either to genetic heterogeneity of GALK deficiency or to differences in culture conditions.

Glutamine dependency of human skin fibroblasts: modulation by hexoses.

The combined effects of carbohydrates and glutamine were investigated in diploid strains of normal human skin fibroblasts cultured for 21 days under eight different culture conditions: hexose-free medium or medium containing D-glucose, D-galactose, or D-fructose, with or without added glutamine. Cell growth, hexose consumption, lactate production, intracellular glycogen content and extracellular amino acid levels were measured every third to fourth day. In the presence of glutamine, cells reached a higher saturation density in fructose medium than in glucose or galactose medium but per cell consumption of fructose and galactose was much less than that of glucose. Consumption of all three carbohydrates per unit cell growth exhibited three distinct phases: Days 1-3, 3-10, and 10-20, respectively. In the absence of glutamine the rate of cell growth was not altered in glucose or galactose medium, but slowed down considerably in fructose medium. Glutamine deprivation also led to changes in hexose consumption. In hexose-free media the cell growth rate at first was very slow, but rose after 2 or 3 weeks of culture. The levels of extracellular nonessential amino acids varied according to medium and growth phase. One of the most exciting findings was that human fibroblasts are able to maintain a slight excess of glutamine in all media not supplemented with glutamine and, more surprisingly, to synthesize it in a medium containing galactose and glutamine.

Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads.

We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expanded on a plastic support. After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibrils. Electrophoretic analysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (pro alpha 1II, pc alpha 1II, and pn alpha 1II). After pepsin digestion a small amount of collagen type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the alpha 1(II) and alpha 1(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly alpha 1(II) mRNA, whereas alpha 1(I) mRNA transcripts were almost undetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to revert to a chondrocytic phenotype in this three-dimensional system. However, SV40-transformed chondrocytes were not able to redifferentiate in alginate as no alpha 1(II) mRNAs were detected. Total RNA was converted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of the complement regulatory protein (CRP)-like domain of aggrecan were originally identified in chondrocytes in monolayers. Like intact cartilage, chondrocytes in alginate expressed only the larger transcript with the CRP domain, whereas the two transcripts were equally expressed in SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondrocytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal chondrodysplasias. However it was unable to restore the chondrocytic phenotype in virally transformed cells.

Superoxide-induced massive apoptosis in cultured skin fibroblasts harboring the neurogenic ataxia retinitis pigmentosa (NARP) mutation in the ATPase-6 gene of the mitochondrial DNA.

The oxidative stress resulting from the neurogenic ataxia retinitis pigmentosa (NARP) mutation in the mitochondrial ATPase 6 gene was investigated in cultured skin fibroblasts from two patients presenting an isolated complex V deficiency. Taken as an index for superoxide overproduction, a huge induction of the superoxide dismutase (SOD) activity was observed in these fibroblasts harboring >90% of mutant mitochondrial DNA. The oxidative stress denoted by the high SOD activity was associated with increased cell death. In glucose-rich medium, apoptosis appeared as the main cell death process associated with complex V deficiency. Complex V-deficient fibroblasts, which showed a high SOD induction and stained positive for all studied apoptosis markers, were successfully rescued by perfluoro-tris-phenyl nitrone, an antioxidant spin-trap molecule. This established that the superoxide production associated with the ATPase deficiency triggered by the NARP mutation could be sufficient to override cell antioxidant defenses and to result in cell commitment to die. The potential participation of superoxides and/or their derivatives in the pathogenic mechanism of specific respiratory chain disorders makes them a promising target for therapy.

Quinone-responsive multiple respiratory-chain dysfunction due to widespread coenzyme Q10 deficiency.

BACKGROUND: The respiratory-chain deficiencies are a broad group of largely untreatable diseases. Among them, coenzyme Q10 (ubiquinone) deficiency constitutes a subclass that deserves early and accurate diagnosis. METHODS: We assessed respiratory-chain function in two siblings with severe encephalomyopathy and renal failure. We used high-performance liquid chromatography analyses, combined with radiolabelling experiments, to quantify cellular coenzyme Q10 content. Clinical follow-up and detailed biochemical investigations of respiratory chain activity were carried out over the 3 years of oral quinone administration. FINDINGS: Deficiency of coenzyme Q10-dependent respiratory-chain activities was identified in muscle biopsy, circulating lymphocytes, and cultured skin fibroblasts. Undetectable coenzyme Q10 and results of radiolabelling experiments in cultured fibroblasts supported the diagnosis of widespread coenzyme Q10 deficiency. Stimulation of respiration and fibroblast enzyme activities by exogenous quinones in vitro prompted us to treat the patients with oral ubidecarenone (5 mg/kg daily), which resulted in a substantial improvement of their condition over 3 years of therapy. INTERPRETATION: Particular attention should be paid to multiple quinone-responsive respiratory-chain enzyme deficiency because this rare disorder can be successfully treated by oral ubidecarenone.

Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan.

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.

Substitution of aspartic acid for glycine at position 310 in type II collagen produces achondrogenesis II, and substitution of serine at position 805 produces hypochondrogenesis: analysis of genotype-phenotype relationships.

Two different mutations were found in two unrelated probands with lethal chondrodysplasias, one with achondrogenesis type II and the other with the less severe phenotype of hypochondrogenesis. The mutations in the COL2A1 gene were identified by denaturing gradient gel electrophoresis analysis of genomic DNA followed by dideoxynucleotide sequencing and restriction site analysis. The proband with achondrogenesis type II had a heterozygous single-base mutation that substituted aspartate for glycine at position 310 of the alpha 1(II) chain of type II procollagen. The proband with hypochondrogenesis had a heterozygous single-base mutation that substituted serine for glycine at position 805. Type II collagen extracted from cartilage from the probands demonstrated the presence of type I collagen and a delayed electrophoretic mobility, indicating post-translational overmodifications. Analysis of CNBr peptides showed that, in proband 1, the entire peptides were overmodified. Examination of chondrocytes cultured in agarose or alginate indicated that there was a delayed secretion of type II procollagen. In addition, type II collagen synthesized by cartilage fragments from the probands demonstrated a decreased thermal stability. The melting temperature of the type II collagen containing the aspartate-for-glycine substitution was reduced by 4 degrees C, and that of the collagen containing the serine-for-glycine substitution was reduced by 2 degrees C. Electron microscopy of the extracellular matrix from the chondrocyte cultures showed a decreased density of matrix and the presence of unusually short and thin fibrils. Our results indicate that glycine substitutions in the N-terminal region of the type II collagen molecule can produce more severe phenotypes than mutations in the C-terminal region. The aspartate-for-glycine substitution at position 310, which was associated with defective secretion and a probable increased degradation of collagen, is the most destabilizing mutation yet reported in type II procollagen.

Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency.

Reduced nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest complex of the mitochondrial respiratory chain and complex I deficiency accounts for approximately 30% cases of respiratory-chain deficiency in humans. Only seven mitochondrial DNA genes, but >35 nuclear genes encode complex I subunits. In an attempt to elucidate the molecular bases of complex I deficiency, we studied the six most-conserved complex I nuclear genes (NDUFV1, NDUFS8, NDUFS7, NDUFS1, NDUFA8, and NDUFB6) in a series of 36 patients with isolated complex I deficiency by denaturing high-performance liquid chromatography and by direct sequencing of the corresponding cDNA from cultured skin fibroblasts. In 3/36 patients, we identified, for the first time, five point mutations (del222, D252G, M707V, R241W, and R557X) and one large-scale deletion in the NDUFS1 gene. In addition, we found six novel NDUFV1 mutations (Y204C, C206G, E214K, IVS 8+41, A432P, and del nt 989-990) in three other patients. The six unrelated patients presented with hypotonia, ataxia, psychomotor retardation, or Leigh syndrome. These results suggest that screening for complex I nuclear gene mutations is of particular interest in patients with complex I deficiency, even when normal respiratory-chain-enzyme activities in cultured fibroblasts are observed.

Molecular and enzymatic characterization of a unique carnitine palmitoyltransferase 1A mutation in the Hutterite community.

Hepatic carnitine palmitoyltransferase 1 (CPT1A) deficiency is a rare disorder of mitochondrial fatty acid oxidation inherited as an autosomal recessive trait. Symptomatology comprises attacks of hypoketotic hypoglycemia with risk of sudden death or neurological sequelae. Only one CPT1A mutation has been reported so far. Identification of the disease-causing mutations allows both insights into the structure-function relationships of CPT1A and management of the patients and their relatives. The molecular analysis of CPT1A deficiency in a large Hutterite kindred illustrates this point. Both cDNA and genomic DNA analysis demonstrate that the affected patients are homozygous for a 2129G>A mutation predicting a G710E substitution. Studies in fibroblasts from one patient as well as heterologous expression of the mutagenized CPT1A in yeast show that the G710E mutation alters neither mitochondrial targeting nor stability of the CPT1A protein. By contrast, kinetic studies conclusively establish that the mutant CPT1A is totally inactive, indicating that the G710E mutation dramatically impairs the catalytic function of CPT1A. Finally, due to a strongly suspected founder effect for the origin of CPT1A deficiency in this Hutterite kindred, identification of this disease-causing mutation allows the setup of a targeted DNA-based newborn screening in this at-risk population.