A novel mutation in BCS1L associated with deafness, tubulopathy, growth retardation and microcephaly.

UNLABELLED: We report a novel homozygous missense mutation in the ubiquinol-cytochrome c reductase synthesis-like (BCS1L) gene in two consanguineous Turkish families associated with deafness, Fanconi syndrome (tubulopathy), microcephaly, mental and growth retardation. All three patients presented with transitory metabolic acidosis in the neonatal period and development of persistent renal de Toni-Debré-Fanconi-type tubulopathy, with subsequent rachitis, short stature, microcephaly, sensorineural hearing impairment, mild mental retardation and liver dysfunction. The novel missense mutation c.142A>G (p.M48V) in BCS1L is located at a highly conserved region associated with sorting to the mitochondria. Biochemical analysis revealed an isolated complex III deficiency in skeletal muscle not detected in fibroblasts. Native polyacrylamide gel electrophoresis (PAGE) revealed normal super complex formation, but a shift in mobility of complex III most likely caused by the absence of the BCS1L-mediated insertion of Rieske Fe/S protein into complex III. These findings expand the phenotypic spectrum of BCS1L mutations, highlight the importance of biochemical analysis of different primary affected tissue and underline that neonatal lactic acidosis with multi-organ involvement may resolve after the newborn period with a relatively spared neurological outcome and survival into adulthood. CONCLUSION: Mutation screening for BCS1L should be considered in the differential diagnosis of severe (proximal) tubulopathy in the newborn period. WHAT IS KNOWN: • Mutations in BCS1L cause mitochondrial complex III deficiencies. • Phenotypic presentations of defective BCS1L range from Bjornstad to neonatal GRACILE syndrome. What is New: • Description of a novel homozygous mutation in BCS1L with transient neonatal acidosis and persistent de Toni-Debré-Fanconi-type tubulopathy. • The long survival of patients with phenotypic presentation of severe complex III deficiency is uncommon.

False-positive reactions of some monoclonal anti-c reagents.

Cross-reactivities of monoclonal reagents for red blood cell (RBC) typing are seen very rarely, e.g. in some reagents for testing of ABO or Lewis antigens. We report on two patients in whom we observed weak reactions using a monoclonal anti-c reagent containing clone MS35, but no reactions with anti-c reagents containing other monoclonal or polyclonal antibodies. To resolve this discrepancy, we studied RHCE exon 1 (Ex1) and 2 from genomic DNA of one patient and performed quality controls of the false-positive reacting anti-c reagent. RHCE Ex1 showed no abnormalities as well as RH Ex2 examined with primers specific for Ex2 D/C. No amplification product of RH Ex2 was received with primers specific for c. A titration study with untreated, papain-treated and sialidase-treated adult and newborn RBCs showed anti-i reactivity of the false-positive reacting reagent. This reactivity could not be removed by absorption with c-negative newborn RBCs without reducing the anti-c titre in the same way, indicating a cross-reactivity. Some manufacturers give a remark on a possible cold agglutinin activity of their monoclonal anti-c reagents in the instruction leaflet, but in order to avoid such irritation, we recommend to remove this cross-reactivity by dilution during the manufacturing process.

Acute myopathy in a type 2 diabetic patient on combination therapy with metformin, fenofibrate and rosiglitazone.

AIMS/HYPOTHESIS: This report describes the case of a 75-year-old male type 2 diabetic Caucasian who was admitted to the clinical ward because of acute pain and cramps in both calf muscles. MATERIALS AND METHODS: Neuromuscular function was assessed by electromyography and electroneurography of the right leg. An open biopsy was taken from the left vastus lateralis muscle for histological and histochemical analyses. Southern blotting was performed to detect defects in mitochondrial DNA and tRNA. Cytochrome P450 (CYP-P450) polymorphisms were analysed in blood cells. RESULTS: Fifteen weeks before admission, the patient's lipid-lowering medication was switched from simvastatin to fenofibrate because of predominant hypertriglyceridaemia; this did not affect creatine kinase levels. Three weeks before admission, rosiglitazone was added to his existing metformin therapy because of worsening metabolic control. Upon admission, serum enzymes indicating myopathy were elevated (creatine kinase 6897 U/l, myoglobin 902 ng/ml) and kidney function was impaired (creatinine 0.116 mmol/l, blood urea nitrogen 2.3 mmol/l). Electrophysiology revealed myopathy and sensory polyneuropathy. Histology showed multiple damage of the myofibrillar architecture. There was no evidence of defects in mitochondrial DNA or tRNA. Furthermore, no functional limitations in CYP2C9, CYP2C19 and CYP2D6 were detected. Following withdrawal of the oral medication and intravenous hydration, clinical symptoms and laboratory parameters gradually decreased. CONCLUSIONS/INTERPRETATION: Until more data from controlled trials are available, we recommend that combination therapy with fibrates and thiazolidinediones should be monitored frequently by measurements of serum creatine kinase and creatinine, specifically in patients with pre-existing nephropathy and polyneuropathy.

Screening for carnitine palmitoyltransferase II deficiency by tandem mass spectrometry.

Mitochondrial carnitine palmitoyltransferase II (CPT II) deficiency is the most common inherited disorder of lipid metabolism in adults. Currently the routine diagnosis is based on the determination of CPT enzyme activity in muscle tissue. We have analysed the tandem mass spectra of serum acylcarnitines of nine CPT II-deficient patients. These spectra were compared to those of a cohort of 99 patients with other neuromuscular disorders and metabolic conditions supposed to cause alterations of the long-chain acylcarnitines. The spectra in CPT II deficiency showed characteristic elevations of C16:0 and C18:1 acylcarnitines while acetylcarnitine C2 was not elevated. In the present study, the ratio (C16:0+C18:1)/C2 has detected all CPT II deficiencies and discriminated them from unspecific alterations of serum acylcarnitines. The ratios of CPT II-deficient patients showed virtually no overlap with those observed in patients with other neuromuscular disorders. We suggest mass spectrometry of serum acylcarnitines as a rapid screening test that should be included early in the diagnostic work-up of patients with recurrent myoglobinuria, recurrent muscular weakness and myalgia.

"Adult" form of muscular carnitine palmitoyltransferase II deficiency: manifestation in a 2-year-old child.

UNLABELLED: We describe a 6-year-old girl admitted with acute muscular weakness and pain which made her unable to walk. Her parents reported a 4-year history of similar episodes which occurred once or twice a year and always resolved spontaneously. Laboratory investigations showed elevated serum creatine kinase which peaked at day 2 of the attack with 18,600 U/l. Carnitine palmitoyltransferase-II deficiency was suspected based on the determination of serum acylcarnitines by tandem mass spectrometry which showed a characteristic elevation of long-chain C16 and C18:1 acylcarnitines. The diagnosis was confirmed by impaired in-vitro palmitate oxidation in blood and the detection of a homozygous substitution S113L in the carnitine palmitoyltransferase-II gene. CONCLUSION: Carnitine palmitoyltransferase-II deficiency should be included in the differential diagnosis of isolated muscular weakness even when manifesting in early childhood.

Role of the deafness dystonia peptide 1 (DDP1) in import of human Tim23 into the inner membrane of mitochondria.

Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1.hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1.hTim13 complex complements the function of the TIM8.13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction.

Detection of neonatal argininosuccinate lyase deficiency by serum tandem mass spectrometry.

Argininosuccinate lyase (ASL) deficiency (McKusick 207900) is an inborn error of the urea cycle. The leading symptom is progressive hyperammonaemia, which is a life-threatening condition, particularly in patients with a neonatal onset. Early diagnosis and treatment of the hyperammonaemia are necessary to improve survival and the long-term outcome of ASL-deficient patients. Currently, the diagnosis of ASL deficiency is based on the measurement of urea cycle intermediates and amino acids by automated quantitative ion exchange chromatography in plasma and urine. Here, we report a newborn presenting with coma and severe hyperammonaemia. ASL deficiency was suspected on the basis of an adapted tandem mass spectrometric (MS-MS) procedure which allows determination of argininosuccinate in addition to the amino acids in serum samples. MS-MS measurements revealed a characteristic increase of argininosuccinate, a moderate increase of citrulline, and lowered levels of arginine and ornithine in the serum of the patient. The diagnosis was confirmed by the detection of a novel homozygous frameshift mutation in exon 14 of the argininosuccinate lyase gene. We propose MS-MS as a diagnostic tool suitable for the rapid detection of specific alterations in the amino acid spectra caused by ASL deficiency.

Import of proteins into mitochondria: a novel pathomechanism for progressive neurodegeneration.

The vast majority of mitochondrial proteins are encoded as precursors by the nuclear genome. A major aspect of mitochondrial biogenesis is therefore the transfer of nuclear-encoded, cytosplasmically synthesized precursor proteins across and into the mitochondrial membranes. During the past years the use of simple model organisms such as the yeasts S. cerevisiae and N. crassa has helped considerably to identify and unravel the structure and function of a substantial number of components involved in targeting of nuclear-encoded preproteins to mitochondria. Several pathways and a number of components were characterized that are involved in guiding mitochondrial preproteins to their specific sites of function. In particular, import of nuclear-encoded precursor proteins into and across the mitochondrial inner membrane is mediated by two distinct translocases, the TIM23 complex and the TIM22 complex. Both TIM complexes cooperate with the general preprotein translocase of the outer membrane, TOM complex. The TIM complexes differ in the their substrate specificity. While the TIM23 complex mediates import of preproteins with a positively charged matrix targeting signal, the TIM22 complex facilitates the insertion of a class of hydrophobic proteins with internal targeting signals into the inner membrane. Most recently the rapid progress of research has allowed elucidation of a new mitochondrial disease on the molecular level. This rare X-linked progressive neurodegenerative disorder, named Mohr-Tranebjaerg (MT syndrome), is caused by mutations in the DDP1 gene and includes sensorineural deafness, blindness, mental retardation and a complex movement disorder. The analysis of the novel pathomechanism is based on the homology of the affected DDP1 protein to a family of conserved yeast components acting along the TIM22 pathway. This contribution briefly summarizes the current knowledge of the pathways of protein import and proposes a mechanism to explain how defective import leads to neurodegeneration.

The role of the TIM8-13 complex in the import of Tim23 into mitochondria.

Tim8 and Tim13 are non-essential, conserved proteins of the mitochondrial intermembrane space, which are organized in a hetero-oligomeric complex. They are structurally related to Tim9 and Tim10, essential components of the import machinery for mitochondrial carrier proteins. Here we show that the TIM8-13 complex interacts with translocation intermediates of Tim23, which are partially translocated across the outer membrane but not with fully imported or assembled Tim23. The TIM8-13 complex binds to the N-terminal or intermediate domain of Tim23. It traps the incoming precursor in the intermembrane space thereby preventing retrograde translocation. The TIM8-13 complex is strictly required for import of Tim23 under conditions when a low membrane potential exists in the mitochondria. The human homologue of Tim8 is encoded by the DDP1 (deafness/dystonia peptide 1) gene, which is associated with the Mohr-Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder leading to deafness. It is demonstrated that import of human Tim23 is dependent on a high membrane potential. A mechanism to explain the pathology of MTS is discussed.

Tim23 links the inner and outer mitochondrial membranes.

Tim23, a key component of the mitochondrial preprotein translocase, is anchored in the inner membrane by its C-terminal domain and exposes an intermediate domain in the intermembrane space that functions as a presequence receptor. We show that the N-terminal domain of Tim23 is exposed on the surface of the outer membrane. The two-membrane-spanning topology of Tim23 is a novel characteristic in membrane biology. By the simultaneous integration into two membranes, Tim23 forms contacts between the outer and inner mitochondrial membranes. Tethering the inner membrane translocase to the outer membrane facilitates the transfer of precursor proteins from the TOM complex to the TIM23 complex and increases the efficiency of protein import.

Protein translocation into mitochondria: the role of TIM complexes.

Import of nuclear-encoded mitochondrial preproteins is mediated by a general translocase in the outer membrane, the TOM complex, and by two distinct translocases in the mitochondrial inner membrane, the TIM23 complex and the TIM22 complex. Both TIM complexes cooperate with the TOM complex but facilitate import of different classes of precursor proteins. Precursors with an N-terminal presequence are imported via the TIM23 complex, whereas mitochondrial carrier proteins require the TIM22 complex for insertion into the inner membrane. This review discusses recent advances in understanding the structure and function of the translocases of the inner membrane and the possible role of Tim proteins in the development of the Mohr-Tranebjaerg syndrome, a mitochondrial disorder leading to neurodegeneration.

Mitochondrial disorders. A diagnostic challenge in clinical chemistry.

Mitochondria play a pivotal role in cellular metabolism and in energy production in particular. Defects in structure or function of mitochondria, mainly involving the oxidative phosphorylation (OXPHOS), mitochondrial biogenesis and other metabolic pathways, have been shown to be associated with a wide spectrum of clinical phenotypes. The ubiquitous nature of mitochondria and their unique genetic features contribute to the clinical, biochemical and genetic heterogeneity of mitochondrial diseases. We will focus on the recent advances in the field of mitochondrial disorders and their consequences for an advanced clinical and genetic diagnostics. In addition, an overview on recently identified genetic defects and their pathogenic molecular mechanisms will be given.

The mitochondrial TIM22 preprotein translocase is highly conserved throughout the eukaryotic kingdom.

The Mohr-Tranebjaerg syndrome (MTS), a neurodegenerative syndrome characterized by progressive sensorineural hearing loss, dystonia, mental retardation and blindness, is a mitochondrial disease caused by mutations in the deafness/dystonia peptide 1 (DDP1) gene. DDP1 shows similarity to the yeast proteins Tim9, Tim10 and Tim12, components of the mitochondrial import machinery for carrier proteins. Here, we show that DDP1 belongs to a large family of evolutionarily conserved proteins. We report the identification, chromosomal localization and expressional analysis of six human family members which represent further candidate genes for neurodegenerative diseases.

Genetic and structural characterization of the human mitochondrial inner membrane translocase.

Translocation of nuclear-encoded mitochondrial preproteins is mediated by translocases in the outer and inner membranes. In the yeast Saccharomyces cerevisiae, translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p. We have cloned and functionally analyzed the human homologues of Tim17, Tim23 and Tim44. In contrast to yeast, two TIM17 genes were found to be expressed in humans. TIM44, TIM23 and TIM17a genes were mapped to chromosomes 19p13.2-p13.3, 10q11. 21-q11.23 and 1q32. The TIM17b gene mapped to Xp11.23, near the fusion point where an autosomal region was proposed to have been added to the "ancient" part of the X chromosome about 80-130 MY ago. The primary sequences of the two proteins, hTim17a and hTim17b, are essentially identical, significant differences being restricted to their C termini. They are ubiquitously expressed in fetal and adult tissues, and both show expression levels comparable to that of hTim23. Biochemical characterization of the human Tim components revealed that hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane. hTim23 is organized into two distinct complexes in the inner membrane, one containing hTim17a and one containing hTim17b. Both TIM complexes display a native molecular mass of 110 kDa. We suggest that the structural organization of TIM23.17 preprotein translocases is conserved from low to high eukaryotes.

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins.

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.

Functional cooperation and stoichiometry of protein translocases of the outer and inner membranes of mitochondria.

The qualitative relationship between preprotein translocases in the mitochondrial outer and inner membranes was determined by both a functional analysis and a determination of characteristic components of the translocases. Translocation contact sites of isolated mitochondria were saturated with intermediates of a matrix-targeted precursor of the beta-subunit of the F1-ATPase (pF1beta), and import of preproteins into the different mitochondrial subcompartments was monitored. A strong inhibition (75-95%) was observed for preproteins with an N-terminal matrix targeting signal, indicating that a significant portion of the contact sites was blocked by accumulated F1beta. Insertion of preproteins into the outer membrane and import into the intermembrane space of preproteins without matrix targeting signals was inhibited by about 45%, indicating that functional outer membrane translocases were available despite saturation of contact sites. Similarly, import of members of the mitochondrial carrier family into the inner membrane was only partly inhibited (40-50%), demonstrating that functional Tim22 translocases were available to cooperate with the Tom machinery in the import of carrier proteins. The stoichiometry of Tom40, Tim23, and Tim22 in mitochondria was determined to be 5:1:0.22. We conclude that translocases of the outer membrane are present in excess over translocases of the inner membrane.

Yeast mitochondrial F1F0-ATPase: the novel subunit e is identical to Tim11.

We report here the identification of the novel subunit of the mitochondrial F1F0-ATPase from Saccharomyces cerevisiae, ATPase subunit e. Yeast ATPase subunit e displays significant similarities in both amino acid sequence, properties (hydropathy and predicted coiled-coil structure) and orientation in the inner membrane, with previously identified mammalian ATPase subunit e proteins. Estimation of its native molecular mass and ability to be co-immunoprecipitated with a subunit of the F1-ATPase, demonstrate that subunit e is a subunit of the F1F0-ATPase. Stable expression of subunit e requires the presence of the mitochondrially encoded subunits of the F0-ATPase. Subunit e had been previously identified as Tim11 and was proposed to be involved in the process of sorting of proteins to the mitochondrial inner membrane.

Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22.

Translocation of mitochondrial preproteins across the inner membrane is facilitated by the TIM machinery. Tim23 binds to matrix targeting signals and initiates membrane potential-dependent import. Tim23 and Tim17 are constituents of a translocation channel across the inner membrane. Tim44 is associated with this channel at the matrix side, and Tim44 recruits mitochondrial Hsp70 and its co-chaperone Mgel, which drive protein translocation into the matrix using ATP as an energy source. Tim22 is a new component of the import machinery of mitochondria, which shares sequence similarity with both Tim23 and Tim17. Here we report that Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane. Members of the yeast AAC family are synthesized without matrix targeting signals. Tim22 is in an assembly of high relative molecular mass that is distinct from the Tim23-Tim17 complex. Import of proteins of the AAC family is independent of Tim23, and import of matrix targeting signals containing preproteins is independent of Tim22.