Leigh syndrome caused by mutations in the flavoprotein (Fp) subunit of succinate dehydrogenase (SDHA).

Detailed clinical, neuroradiological, histological, biochemical, and genetic investigations were undertaken in a child suffering from Leigh syndrome. The clinical symptoms started at age five months and led to a severe progressive neurodegenerative disorder causing epilepsy, psychomotor retardation, and tetraspasticity. Biochemical measurement of skeletal muscle showed a severe decrease in mitochondrial complex II. Sequencing of SDHA revealed compound heterozygosity for a nonsense mutation in exon 4 (W119X) and a missense mutation in exon 3 (A83V), both absent in normal controls. In six additional patients--five with Leigh or Leigh-like syndrome and one with neuropathy and ataxia associated with isolated deficiency of complex II--mutations in SDHA were not detected, indicating genetic heterogeneity.

Mutations in mtDNA-encoded cytochrome c oxidase subunit genes causing isolated myopathy or severe encephalomyopathy.

We report on clinical, histological and genetic findings in two patients carrying novel heteroplasmic mutations in the mitochondrial cytochrome c oxidase subunit genes COII and COIII. The first patient, a 35 year-old man had a multisystemic disease, with clinical symptoms of bilateral cataract, sensori-neural hearing loss, myopathy, ataxia, cardiac arrhythmia, depression and short stature and carried a 7970 G>T (E129X) nonsense mutation in COII. A sudden episode of metabolic encephalopathy caused by extremely high blood lactate lead to coma. The second patient developed exercise intolerance and rhabdomyolysis at age 22 years. A heteroplasmic missense mutation 9789 T>C (S195P) was found in skeletal muscle, but not in blood and myoblasts pointing to a sporadic mutation. Our report of two patients with isolated COX deficiency and new mutations in COX subunit genes may help to draw more attention to this type of mtDNA defects and provide new aspects for counselling affected families.

Identification of expandable human hepatic progenitors which differentiate into mature hepatic cells in vivo.

BACKGROUND: Liver diseases include a wide spectrum of both acute and chronic conditions which are associated with significant morbidity and mortality worldwide. Hepatocyte transplantation has therapeutic potential in the treatment of liver diseases, but its clinical use is hampered by the lack of donor tissue. Generation of hepatocytes in vitro from adult or fetal liver cell progenitors or, alternatively, identification of a progenitor population which in vivo can generate mature liver cells could solve this problem. METHODS: CD117+/CD34+/Lin- human fetal liver cells were isolated by magnetic cell sorting and expanded in culture. Both freshly isolated and in vitro expanded cells in various passages were studied for their ability to be functional in hepatic parenchyma following d-galactosamine (GalN) induced injury in nude C57 black mice. RESULTS: Freshly isolated and in vitro expanded CD117+/CD34+/Lin- cells, when transplanted intrasplenically into GalN treated mice, morphologically and functionally differentiated into hepatocytes and cholangiocytes. Human specific albumin, alpha fetoprotein, cytokeratin 19, and antitrypsin mRNA were expressed in mouse liver. In addition, the human progenitor cells expressed glucose-6-phosphatase, glycogen, albumin, gamma glutamyl transpeptidase, and dipeptidyl peptidase IV after transplantation. Expanded cells in various passages maintained their capacity to differentiate into functional liver cells. CONCLUSIONS: Fetal liver CD117+/CD34+/Lin- progenitors and their progeny proliferated in vitro and also functionally differentiated into mature hepatic cells in an acute liver injury model. Successful in vitro expansion of liver progenitor cells provides a basis for developing cell therapy strategies, metabolic and toxicity testing systems, and may serve as a vehicle for gene therapy.

The pathophysiology of acute graft-versus-host disease.

Despite improvements in allogeneic stem cell transplantation, acute graft-versus-host disease (GVHD) remains a significant problem after transplantation, and it is still a major cause of post-transplant mortality. Disease progression is characterized by the differentiation of alloreactive T cells to effector cells leading to tissue damage, recruitment of additional inflammatory cell populations and further cytokine dysregulation. To make the complex process of acute GVHD more explicit, the pathophysiology of acute GVHD is often divided into three different phases. This review summarizes the mechanisms involved in the three phases of acute GVHD.

Congenital cataract, muscular hypotonia, developmental delay and sensorineural hearing loss associated with a defect in copper metabolism.

Deficiencies of different proteins involved in copper metabolism have been reported to cause human diseases. Well-known syndromes, for example, are Menkes and Wilson diseases. Here we report a patient presenting with congenital cataract, severe muscular hypotonia, developmental delay, sensorineural hearing loss and cytochrome-c oxidase deficiency with repeatedly low copper and ceruloplasmin levels. These findings were suggestive of a copper metabolism disorder. In support of this, the patient's fibroblasts showed an increased copper uptake with normal retention. Detailed follow-up examinations were performed. Immunoblotting for several proteins including ATP7A (MNK or Menkes protein), ATP7B (Wilson protein) and SOD1 showed normal results, implying a copper metabolism defect other than Wilson or Menkes disease. Sequence analysis of ATOX1 and genes coding for proteins that are known to play a role in the mitochondrial copper metabolism (COI-III, SCO1, SCO2, COX11, COX17, COX19) revealed no mutations. Additional disease genes that have been associated with cytochrome-c oxidase deficiency were negative for mutations as well. As beneficial effects of copper histidinate supplementation have been reported in selected disorders of copper metabolism presenting with low serum copper and ceruloplasmin levels, we initiated a copper histidinate supplementation. Remarkable improvement of clinical symptoms was observed, with complete restoration of cytochrome-c oxidase activity in skeletal muscle.

Two families with autosomal dominant progressive external ophthalmoplegia.

OBJECTIVES: We report here the clinical and genetic features of two new families with autosomal dominant progressive external ophthalmoplegia (adPEO). PATIENTS AND METHODS: The examination of index patients included a detailed clinical characterisation, histological analysis of muscle biopsy specimens, and genetic testing of mitochondrial and nuclear DNA extracted from muscle and leucocytes. RESULTS: Index patients in both families presented with PEO and developed other clinical disease manifestations, such as myopathy and cardiomyopathy (patient 1) and axonal neuropathy, diabetes mellitus, hearing loss, and myopathy (patient 2), later in the course of illness. Both patients had ragged red fibres on muscle histology. Southern blot of mtDNA from muscle of patient 2 showed multiple deletions. In this case, a novel heterozygous missense mutation F485L was identified in the nuclear encoded putative mitochondrial helicase Twinkle. The mutation co-segregated with the clinical phenotype in the family and was not detected in 150 control chromosomes. In the other index patient, sequencing of ANT1, C10orf2 (encoding for Twinkle), and POLG1 did not reveal pathogenic mutations. CONCLUSIONS: Our cases illustrate the clinical variability of adPEO, add a novel pathogenic mutation in Twinkle (F485L) to the growing list of genetic abnormalities in adPEO, and reinforce the relevance of other yet unidentified genes in mtDNA maintenance and pathogenesis of adPEO.

Reversion of hypertrophic cardiomyopathy in a patient with deficiency of the mitochondrial copper binding protein Sco2: is there a potential effect of copper?

Mutations in Sco2, a protein involved in copper trafficking to the terminal enzyme of the respiratory chain, cytochrome c oxidase, results in infantile hypertrophic cardioencephalomyopathy. We have recently shown that copper-histidine (Cu-his) supplementation of Sco2-deficient myoblasts rescues COX activity in vitro. Here, we report a patient with SCO 2 mutations and with resolution of severe hypertrophic cardiomyopathy. Weighing up the evidence, the most likely explanation for the improved cardiac function in this patient was the subcutaneous application of Cu-his.

Increased levels of immune transcript in patients with acute GVHD after allogeneic stem cell transplantation.

After allogeneic stem cell transplantation (SCT), donor T-cells are primarily responsible for the antihost activity, resulting in graft-versus-host disease (GVHD). Three effector pathways have been described for T-cell cytotoxicity: perforin/granzyme B; Fas/Fas ligand (FasL) and secreted molecules such as TNF-alpha. The goal of this pilot study was to utilize competitive reverse transcription (RT)-PCR to evaluate the pattern of granzyme B, perforin, FasL and TNF-alpha gene expression in peripheral blood in patients after SCT. Protein levels of granzyme B, soluble FasL (sFasL) and TNF-alpha in plasma were also analyzed. Eight patients who underwent allogeneic SCT were included; five were diagnosed with acute GVHD. In the patients diagnosed with acute GVHD, we found increased levels of granzyme B, perforin and FasL mRNA, although this did not correlate with the clinical severity. However, patients with increasing levels of gene expression during acute GVHD treatment may have an increased risk of developing severe acute GVHD, as two out of three patients with increasing immune transcript levels during GVHD therapy developed life-threatening acute GVHD. In conclusion, the quantitative RT-PCR of granzyme B, perforin and FasL may serve as a guide to the clinician in diagnosing acute GVHD and monitoring treatment.

Myofibrillar (desmin-related) myopathy: clinico-pathological spectrum in 3 cases and review of the literature.

Myofibrillar or desmin-related myopathies encompass neuromuscular disorders with abnormal deposits of desmin and myofibrillar alterations. We report 3 unrelated patients presenting with proximal and distal myopathy, and, as a unique congenital syndrome, diffusely distributed myopathy, osteoporosis and myopia. Muscle biopsies shared cytoplasmic inclusions, rimmed vacuoles, and ragged-red-like fibers. Sarcoplasmic inclusions, either plaque-like or amorphous, strongly immunoreacted on dystrophin and variably for desmin, alphaB crystallin and ubiquitin. Cyclin-dependent kinases CDK1, CDK2 and CDK5 were overexpressed in affected fibers. Ultrastructurally, focal myofibrillar disruption was accompanied by tubulo-filamentous inclusions in one case and abundant glycogen and enlarged mitochondria displaying respiratory chain dysfunction at biochemistry in another case. Molecular analysis of the alphaB crystallin gene coding sequence and exons 4, 5 and 6 of the desmin gene did not reveal any mutation. The morphologic denominator of hyaline structures and areas of myofibrillar destruction occurs in heterogeneous conditions and may overlap with features of inclusion body myopathy and mitochondrial myopathy.

Cytochrome c oxidase deficiency due to mutations in SCO2, encoding a mitochondrial copper-binding protein, is rescued by copper in human myoblasts.

Mutations in SCO2, a cytochrome c oxidase (COX) assembly gene, have been reported in nine infants with early onset fatal cardioencephalomyopathy and a severe COX deficiency in striated muscle. Studies on a yeast homolog have suggested that human Sco2 acts as a copper chaperone, transporting copper to the Cu(A) site on the Cox II subunit, but the mechanism of action remains unclear. To investigate the molecular basis of pathogenesis of Sco2 defects in humans we performed genetic and biochemical studies on tissues, myoblasts and fibroblasts from affected patients, as well as on a recombinant human C-terminal Sco2 segment (22 kDa), bearing the putative CxxxC metal-binding motif. Recombinant Sco2 was shown to bind copper with a 1:1 stoichiometry and to form homomeric complexes in vitro, independent of the metal-binding motif. Immunohistochemistry using antibodies directed against different COX subunits showed a marked tissue-specific decrease in the Cox II/III subunits that form part of the catalytic core, consistent with the differential tissue involvement, but a more uniform distribution of Cox Vab, a nuclear-encoded subunit. Sco2 was severely reduced in patient fibroblasts and myoblasts by immunoblot analysis. Patient fibroblasts showed increased (64)Cu uptake but normal retention values and, consistent with this, the copper concentration was four times higher in Sco2-deficient myoblasts than in controls. COX activity in patient myoblasts was completely rescued by transduction with a retroviral vector expressing the human SCO2 coding sequence, and more interestingly by addition of copper-histidine (300 microM) to the culture medium. Whether the latter is accomplished by the very low residual levels of Sco2 in the patient cells, direct addition of copper to the Cu(A) site, or by another copper-binding protein remains unknown. Whatever the mechanism, this result suggests a possible therapy for the early treatment of this fatal infantile disease.

Homozygosity (E140K) in SCO2 causes delayed infantile onset of cardiomyopathy and neuropathy.

OBJECTIVE: To report three unrelated infants with a distinctive phenotype of Leigh-like syndrome, neurogenic muscular atrophy, and hypertrophic obstructive cardiomyopathy. The patients all had a homozygous missense mutation in SCO2. BACKGROUND: SCO2 encodes a mitochondrial inner membrane protein, thought to function as a copper transporter to cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. Mutations in SCO2 have been described in patients with severe COX deficiency and early onset fatal infantile hypertrophic cardioencephalomyopathy. All patients so far reported are compound heterozygotes for a missense mutation (E140K) near the predicted CxxxC metal binding motif; however, recent functional studies of the homologous mutation in yeast failed to demonstrate an effect on respiration. METHODS: Here we present clinical, biochemical, morphologic, functional, MRI, and MRS data in two infants, and a short report in an additional patient, all carrying a homozygous G1541A transition (E140K). RESULTS: The disease onset and symptoms differed significantly from those in compound heterozygotes. MRI and muscle morphology demonstrated an age-dependent progression of disease with predominant involvement of white matter, late appearance of basal ganglia lesions, and neurogenic muscular atrophy in addition to the relatively late onset of hypertrophic cardiomyopathy. The copper uptake of cultured fibroblasts was significantly increased. CONCLUSIONS: The clinical spectrum of SCO2 deficiency includes the delayed development of hypertrophic obstructive cardiomyopathy and severe neurogenic muscular atrophy. There is increased copper uptake in patients' fibroblasts indicating that the G1541A mutation effects cellular copper metabolism.

Valproic acid triggers acute rhabdomyolysis in a patient with carnitine palmitoyltransferase type II deficiency.

A 47-year-old man suffering from a bipolar disorder and intermittent myoglobinuria presented with acute rhabdomyolysis with renal failure after starting therapy with valproic acid. On morphological examination, skeletal muscle revealed increased lipid storage. Biochemically, decreased enzyme activity of carnitine palmitoyltransferase (CPT) type II with carnitine levels in the lower limit was found. Genetic analysis detected the common Ser113Leu substitution on one allele of the CPT2 gene. We conclude that valproic acid should be avoided in patients with CPT type II deficiency.

Frequency of mitochondrial transfer RNA mutations and deletions in 225 patients presenting with respiratory chain deficiencies.

OBJECTIVE: To evaluate the frequency of pathogenic mtDNA transfer RNA mutations and deletions in biochemically demonstrable respiratory chain (RC) deficiencies in paediatric and adult patients. METHODS: We screened for deletions and sequenced mitochondrial transfer RNA genes in skeletal muscle DNA from 225 index patients with clinical symptoms suggestive of a mitochondrial disorder and with biochemically demonstrable RC deficiency in skeletal muscle. RESULTS: We found pathogenic mitochondrial DNA mutations in 29% of the patients. The detection rate was significantly higher in adults (48%) than in the paediatric group (18%). Only one pathogenic mutation was detected in the neonatal group. In addition, we describe seven novel transfer RNA sequence variations with unknown pathogenic relevance (six homoplasmic and one heteroplasmic) and 13 homoplasmic polymorphisms. One heteroplasmic transfer RNA(Leu(UUR)) A>G mutation at position 3274 is associated with a distinct neurological syndrome. CONCLUSIONS: We provide an estimation of the frequency of mitochondrial transfer RNA mutations and deletions in paediatric and adult patients with respiratory chain deficiencies.

The significance of graft-versus-host disease and pretransplantation minimal residual disease status to outcome after allogeneic stem cell transplantation in patients with acute lymphoblastic leukemia.

Relapse is the major cause of treatment failure after allogeneic stem cell transplantation (SCT) in patients with acute lymphoblastic leukemia. Minimal residual disease (MRD) was analyzed before SCT in 30 patients with acute lymphoblastic leukemia. The aim was to determine whether the level of MRD before transplantation was correlated with outcome. Fifteen patients were found to have high-level MRD (10(-2) to 10(-3)), 10 had low-level MRD (< 10(-3)), and 5 were MRD(-). Among MRD(-) patients the probability of relapse was 0 in 5, which was less than in MRD(+) patients (13 of 25) (P =.05). No major difference was found between the high- and low-level MRD(+) groups. Among the MRD(+) patients, only 2 of 11 with acute and chronic graft-versus-host disease had a relapse, versus 11 of 14 without (P =.005). In conclusion, for patients entering transplantation while they have residual disease, a combination of acute and chronic graft-versus-host disease may be needed to decrease the risk of relapse after SCT.

Nail-patella syndrome associated with respiratory chain disorder.

Nail-patella syndrome (NPS) has not been described to be associated with a respiratory chain disorder (RCD) before. In a 42-year-old man with the typical phenotype of an NPS, weakness and wasting of the shoulder girdle muscles, muscle cramps, fatigability, hyperhidrosis, chest pain and creatine kinase elevation were observed. Echocardiography revealed left ventricular hypertrabeculation. Needle electromyography was myopathic, lactate stress testing was abnormal, muscle biopsy showed typical features of an RCD and mtDNA analysis revealed the A3243G MELAS mutation. In conclusion, this case demonstrates that NPS may be randomly associated with RCD. NPS patients should undergo detailed cardiological and neurological investigations, in order not to overlook a double trouble partially mimicking NPS.

Characterization of human muscle type cofilin (CFL2) in normal and regenerating muscle.

Cofilins are actin binding proteins and regulate actin assembly in vivo. Numerous cofilin homologues have been characterized in various organisms including mammals. In mice, a ubiquitously expressed cofilin (CFL1) and a skeletal muscle specific cofilin (CFL2) have been described. In the present study, we identified and characterized a human CFL2 gene localized on chromosome 14, with high homology to murine CFL2. Furthermore, we provide evidence for differentially spliced CFL2 transcripts (CFL2a and CFL2b). CFL2b is expressed predominantly in human skeletal muscle and heart, while CFL2a is expressed in various tissues. Genetic defects of CFL2 were excluded for one human muscle disorder, the chromosome 14 linked distal myopathy MPD1, and shown to be only possible to be a rare cause of another, nemaline myopathy. In a mouse model of mechanically induced muscle damage the changes of cofilin expression were monitored during the first 10 days of regeneration, with dephosphorylated CFL2 being the major isoform at later stages of muscle regeneration. A similar predominance of dephosphorylated CFL2 was observed in chronically regenerating dystrophin-deficient muscles of Duchenne muscular dystrophy patients. Therefore, the CFL2 isoform may play an important role in normal muscle function and muscle regeneration.