Mitochondrial DNA depletion and fatal infantile hepatic failure due to mutations in the mitochondrial polymerase γ (POLG) gene: a combined morphological/enzyme histochemical and immunocytochemical/biochemical and molecular genetic study.

Combined morphological, immunocytochemical, biochemical and molecular genetic studies were performed on skeletal muscle, heart muscle and liver tissue of a 16-months boy with fatal liver failure. The pathological characterization of the tissues revealed a severe depletion of mtDNA (mitochondrial DNA) that was most pronounced in liver, followed by a less severe, but still significant depletion in skeletal muscle and the heart. The primary cause of the disease was linked to compound heterozygous mutations in the polymerase γ (POLG) gene (DNA polymerase γ; A467T, K1191N). We present evidence, that compound heterozygous POLG mutations lead to tissue selective impairment of mtDNA replication and thus to a mosaic defect pattern even in the severely affected liver. A variable defect pattern was found in liver, muscle and heart tissue as revealed by biochemical, cytochemical, immunocytochemical and in situ hybridization analysis. Functionally, a severe deficiency of cytochrome-c-oxidase (cox) activity was seen in the liver. Although mtDNA depletion was detected in heart and skeletal muscle, there was no cox deficiency in these tissues. Depletion of mtDNA and microdissection of cox-positive or negative areas correlated with the histological pattern in the liver. Interestingly, the mosaic pattern detected for cox-activity and mtDNA copy number fully aligned with the immunohistologically revealed defect pattern using Pol γ, mtSSB- and mtTFA-antibodies, thus substantiating the hypothesis that nuclear encoded proteins located within mitochondria become unstable and are degraded when they are not actively bound to mtDNA. Their disappearance could also aggravate the mtDNA depletion and contribute to the non-homogenous defect pattern.

[Cholesterol ester storage disease: a rare disease or a rare diagnosis?]. / Cholesterinesterspeicherkrankheit: Eine seltene, weil zu selten diagnostizierte Krankheit?

We report the case of a 13-year-old boy with a longstanding history of unspecific hepatomegaly. The morphological investigations were diagnostic of a cholesterol ester storage disease (CESD), a rare autosomal recessive inherited disease with deficient activity of lysosomal acid lipase (LAL). The combination of hepatomegaly with accumulation of macrophages and ultrastructural evidence of lysosomal lipid storage are groundbreaking for the diagnosis. The probability of a underdiagnosis or false disease classification, for example as nonalcoholic steatohepatitis (NASH), is high, particularly with regard to genetic data which indicate a higher incidence of the disease.

Overexpression of cyclin D1, D3, and p21 in an infantile renal carcinoma with Xp11.2 TFE3-gene fusion.

Renal carcinomas harboring the TFE-3 translocation are rare and occur predominately in children and adolescents. Here, we report a case of infantile renal carcinoma with TFE3 translocation and show that the cell cycle is deregulated in this type of carcinoma. It is characterized by nuclear accumulation of cyclin D1 and D3 in combination with high levels of cyclin-dependent kinase inhibitor p21Cip1/Waf1 but without accumulation of p53, p16INK4a, or mdm2. The combined overexpression of p21, cyclin D1, and cyclin D3 was found exclusively in this type but not in other, more common types of renal carcinoma/oncocytoma (n=27). These results further underscore that renal carcinomas with Xp11. 2 translocations/TFE3-gene fusion represent a special type of renal neoplasm showing deregulation of specific cell cycle components. The analysis of further cases has to prove whether the derangement of the cell cycle is uniform and correlates with the specific type of molecular genetic derangement.

Aggressive scapular chondroblastoma with secondary metastasis--a case report and review of literature.

Chondroblastoma is a benign bone tumor, accounting for approximately one percent of all benign bone tumors. It mostly occurs in typical locations such as long bones. Malignant transformation including metastasis has been described in only a few cases. Therefore, we report a unique case of chondroblastoma with tumor manifestation in the 7th decade of life, location of the tumor in the scapula and occurrence of metastasis in the soft tissue of the mandible branch. Due to aggravation of the clinical course, a scapula en bloc resection was performed. The differential diagnosis is discussed and the current literature concerning malignant transformation of chondroblastoma is reviewed.

Benign symmetric lipomatosis with axonal neuropathy and abnormalities in specific mitochondrial tRNA regions.

Benign symmetric lipomatosis, also called Madelung's disease, is characterized by lipomata and fatty infiltrations. Involvement of the nervous system has occasionally been described, mitochondrial dysfunctions have been suggested. We report a 55 year old male suffering from benign symmetric lipomatosis with associated axonal neuropathy and hyperlipoproteinemia. He showed a remarkable phenotype of neuropathy i.e. no sensory disturbance, ubiquitous fasciculations and muscle cramps, furthermore reduced COX activity and abnormalities in specific mitochondrial tRNA regions.

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.

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.

Mitochondria and ageing.

This work reviews the role of mitochondria in the ageing process and summarizes pathomorphological biochemical and molecular genetic data. The pathophysiological mechanisms underlying the phenomenon of ageing are only partly understood. There is, however, increasing evidence that mitochondria are essentially involved. In various tissues of various species a decline in the respiratory chain capacity is seen with ageing. Enzyme histochemistry of cytochrome c oxidase (complex IV of the respiratory chain) has revealed an age-related increase of randomly distributed defective fibres/cells in the skeletal and heart muscle the random pattern probably indicating cellular heterogeneity of the ageing process. Observed deletions of mitochondrial DNA during ageing may represent one causative factor. Similar to primary mitochondrial myopathies point mutations and depletion of the mtDNA are probably also involved. There is some evidence that damage of the mitochondrial genome and of other mitochondrial structures might be due to increased oxygen radical production during ageing. The role of nuclear influences on the degeneration of mitochondrial function remains, however, also to be determined. Nevertheless, the decline of respiratory chain function with ageing represents an important factor for the decline of functional organ reserve capacity in senescence.

Small in-frame deletions and missense mutations in CADASIL: 3D models predict misfolding of Notch3 EGF-like repeat domains.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is a hereditary microangiopathic condition causing stroke in young adults. The responsible gene has recently been identified as the Notch3 gene. Notch3 encodes a large transmembrane receptor with 34 extracellularly localised epidermal growth factor-like (EGF) repeat domains. We screened 71 unrelated CADASIL families for mutations in two exons coding for the first five EGF-like repeats and found mutations in 70% of the families (n = 50). Two types of mutations were identified: 48 families (96%) had missense mutations and two families (4%) had small in-frame deletions. Seven mutations occurred multiple times. All of them are C to T transitions that affect CpG dinucleotides, suggesting that their multiple occurrence is due to the hypermutability of this sequence. All mutations, including the two deletions, result in the gain or loss of a cysteine residue, thus substantiating the pivotal role of an uneven number of cysteine residues within EGF-like repeat domains of Notch3 in the pathogenesis of CADASIL. To study the potential effects of these mutations 3D homology models of the first six EGF domains were generated on the basis of NMR data from human fibrillin-1. These models predict domain misfolding for a subset of mutations.

Cytochrome c oxidase defects of the human substantia nigra in normal aging.

Based on morphological, biochemical, and molecular biologic analyses, degeneration of the dopaminergic nigrostriatal system has been reported to occur with normal aging. In the present study, the substantia nigra of 36 human brains with normal aging was investigated by means of morphometry and immunohistochemistry. The anteromedial (Am), anterointermediolateral (Ail), posteromedial (Pm), and posterolateral (Pl) nuclei of the substantia nigra were analyzed using antibodies directed against the subunits II/III of cytochrome c oxidase (COX), the complex IV of the respiratory chain. The numerical density of melanin-positive neurons with COX defects was significantly increased in the four investigated nuclei, namely Am, Ail, Pm, and Pl. These cells did not show any histologic signs of degeneration. The numerical density of melanin-positive neurons without COX defects was decreased with aging. The data of the present study indicate that complex IV defects of neurons in the substantia nigra might be one cause of neuronal dysfunction occurring during aging.

The point mutation of mitochondrial DNA characteristic for MERRF disease is found also in healthy people of different ages.

The A-to-G transition mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA), characteristic for the maternally inherited MERRF syndrome (myoclonic epilepsy with ragged red fibers), has been identified by point mutation-specific polymerase chain reaction in extraocular muscle from 11 of 16 healthy people of different ages. No mutation was found in navel-string samples from 5 newborns, in HeLa cells, and in 2 individuals younger than 20 years. On the other hand, the mutation is present in all 5 tested 74-89-year-old individuals and in 6 of 9 20-70-year-old individuals. The amount of mutated from total mtDNA was estimated by 'mispairing PCR' in extraocular muscle of 2 individuals of 74 and 89 years to 2.0 and 2.4%, respectively. In most tissue samples the MERRF mutation occurs together with the 'common deletion' of mtDNA, which was previously shown to accumulate in healthy individuals with increasing age. It is proposed that during aging, deletions and point mutations of mtDNA accumulate, which could impair mitochondrial energetics.

Respiratory chain activity in tissues from patients (MELAS) with a point mutation of the mitochondrial genome [tRNA(Leu(UUR))].

A heteroplasmic point mutation (transition A to G at position 3243 in the mitochondrial tRNA(Leu(UUR)) gene is indicative for myo-encephalopathy with lactic acidosis and stroke-like episodes (MELAS). Decreased respiratory chain complex activities measured in different tissues from four patients with MELAS syndrome do not correlate with the proportion of mutated mitochondrial genome.

Defects of the respiratory chain in various tissues of old monkeys: a cytochemical-immunocytochemical study.

The aim of the present study was to evaluate if defects of the respiratory chain known to occur in humans, also exist in lower primates. Cytochemical-immunocytochemical studies of the respiratory chain enzymes in five monkeys (10-25 years of age) showed defects of ubiquinone cytochrome-c-oxidoreductase (complex III), of cytochrome-c-oxidase (complex IV) and of ATP-synthase (complex V) in the limb muscles, diaphragm, heart muscle and extraocular muscles of three old animals (about 25 years) and also in the heart muscle of two younger animals (10 and 15 years). Characteristically, the defects were randomly distributed and there was no loss of succinate-dehydrogenase (complex II) in the fibres. Ultracytochemistry-immunocytochemistry of complex IV disclosed that in an involved fibre segment all the mitochondria exhibited the defect. The highest number of defects was observed in the extraocular muscle (up to 340/cm2) while the lowest defect density was present in the limb muscles (2-5/cm2). Defects of complex IV occurred two to three times more often than defects of complex III and besides isolated defects of complex III and IV, combined defects of both complexes were also observed. Defects of complex V occurred exclusively in combination and were rarely seen. Using subunit specific antisera against complex IV, it could be demonstrated at light and electron microscopic level that loss of activity of cytochrome-c-oxidase was associated with a loss both of mitochondrially and nuclearly coded subunits of the enzyme. In summary, aging in lower primates and humans is characterised by a highly similar defect expression of the respiratory chain enzymes, with intercellular and interorgan differences of the aging process, underlining the universal nature of the involved pathogenetic mechanisms.

De novo missense mutation in a constitutively expressed exon of the slow alpha-tropomyosin gene TPM3 associated with an atypical, sporadic case of nemaline myopathy.

We describe an atypical case of nemaline myopathy with an unusual distribution of muscle weakness who presented at 14 years of age with kyphoscoliosis. In this patient, we demonstrate heterozygosity for a de novo CGT-CAT (Arg167His) mutation in a constitutively expressed exon (exon 5) of slow alpha-tropomyosin (TPM3). This is the first mutation identified in a constitutively expressed exon of TPM3 in a nemaline myopathy patient, but is similar to recently described mutations in beta-tropomyosin (TPM2) associated with nemaline myopathy and mutations in fast alpha-tropomyosin (TPM1) which cause hypertrophic cardiomyopathy.

Fluorescence videomicroscopic assessment of xenogeneic microcirculation and impact of antibody removal by immunoadsorption.

BACKGROUND: Alterations in microcirculation are considered central to the pathogenesis of hyperacute xenogeneic rejection (HXR) of vascularized xenografts, but currently there exist no data describing these microhemodynamic alterations. METHODS: Rat livers were perfused in situ with either isogeneic rat blood or xenogeneic human blood. The microcirculation of these xenoperfused livers was investigated directly using intravital fluorescence microscopy, and compared with that of isogeneic hemoperfused livers. In addition, the impact of antibody depletion by immunoadsorption was investigated. RESULTS: Although a homogenous microcirculation was found during isogeneic liver perfusion (index of acinar perfusion 90.4%/sinusoidal perfusion rate 93.6%), xenoperfusion resulted in a rapid breakdown of the microcirculation (47.5%/67.1%, respectively). Perfusion deficits were found predominantly in the periportal areas. Immunoadsorption reduced the total amount of IgM and IgG by 75.2% and 96.2%, respectively, and caused a significantly improved liver perfusion (80.2%/84.4%) and liver function, as indicated by bile production. In contrast, the massive hepatic leukocyte and platelet accumulation observed during perfusion with untreated xenogeneic blood was not altered by antibody depletion. CONCLUSIONS: Thus, the combination of isolated rat liver perfusion and intravital fluorescence microscopy enables the observation and quantification of the early phase of HXR. This is an important step forward for sensitive characterization of the rejection process and will enable the mechanisms involved in HXR to be elucidated. Antibody depletion was shown to improve liver function and perfusion, but did not reconstitute liver viability to the level of the isogeneic perfusion. These findings highlight the need for additional therapeutic regimens in xenografting.

IG-therasorb immunoapheresis in orthotopic xenotransplantation of baboons with landrace pig hearts.

BACKGROUND: The major problem of xenotransplantation is, that hyperacute xenograft rejection (HXR) causes graft failure within minutes or a few hours because of natural antibodies and activation of the complement system. As a preclinical model we transplanted pig hearts orthotopically into baboons. To prevent HXR after orthotopic xenotransplantation (oXHTx), the immunoglobulins (Ig) and natural antibodies were adsorbed to reusable Ig-Therasorb immunoadsorption (IA) columns. METHODS: We performed three oXHTx of landrace pig hearts into baboons (19+/-6.8 kg), using extracorporeal circulation (ECC) connected to the IA unit. After separating the recipient's blood into plasma and cellular fraction by a plasma filter, plasma flow was directed to the Ig-Therasorb column coated with polyclonal sheep-antibodies against human IgG, IgM, and IgA. Intraoperative treatment consisted of 4 cycles of IA. For a control, we transplanted one pig heart into a baboon (16.9 kg) without applying IA. Perioperatively, serum concentrations of Ig, anti-pig-antibodies, complement and cardiac enzymes were determined. Tissue samples of myocardium were collected at the end of the study for immunohistochemical examinations, light microscopic examination (LM) and electron microscopic examination (EM). For cardiac monitoring after oXHTx, we used ECG, echocardiography, and invasive measurement of cardiac output. To prevent a mismatch of donor and recipient heart size, the donor pig had a 30-40% lower body weight than the recipient baboon. RESULTS: Four cycles of IA removed >80% of IgG, IgM, and IgA from plasma. The graft of the control animal failed after 29 min. The first oXHTx with IA was intentionally terminated after 100 min, the second oXHTx after 11 hr and the third oXHTx after 21 hr. All xenografts showed no histological signs of HXR. After weaning off ECC, these donor hearts worked in sinus rhythm without electrocardiographic ST-segment elevation. An excellent cardiac output was measured by echocardiography and thermodilution (2 L/min). Serological parameters indicating cardiac damage were significantly lower after IA if compared with the control experiment. Macroscopically, the xenograft of the control animal showed massive hemorrhage in comparison with the almost inconspicuous grafts after IA. The myocardium of the IA group demonstrated fewer deposits of Ig and complement components compared with the control animal. CONCLUSION: Baboons do not hyperacutely reject a porcine xenograft after antibody depletion by the Ig-Therasorb column. In our experiment only 4 cycles of immunoapheresis effectively prevented HXR after oXHTx of baboons. The Ig-Therasorb column is a reusable device, which can be handled easily in combination with the ECC. IA must be tested in oXHTx longterm survival experiments, especially in combination with transgenic pig organs, which could be a reliable preclinical approach for future clinical xenotransplantation.

Prevention of hyperacute rejection by human decay accelerating factor in xenogeneic perfused working hearts.

As a potential source of organs for xenotransplantation, pigs that are transgenic for human decay accelerating factor (DAF) have been bred in order to overcome hyperacute rejection. We investigated the protective effect of human DAF in a porcine working heart model perfused by human blood. Hearts of normal landrace pits served as controls. The following parameters were measured: stroke work index, coronary flow and arteriovenous oxygen consumption, 6-keto prostaglandin F1alpha and prostaglandin E2 as markers of endothelial cell activation; creatine phosphokinase and lactate dehydrogenase for evaluation of the extent of myocardial damage; TNFalpha and IL-6 as markers of mononuclear cell activation. Histological and ultrastructural investigations from myocardial tissue sections were done at the end of perfusion. Human (h) DAF appeared to inhibit complement-mediated endothelial cell activation of transgenic pig hearts successfully. This was in contrast to landrace pig hearts, which had a sixfold increase of prostaglandin levels during perfusion with human blood. The cardiac weight increase during perfusion time due to interstitial edema tended to be less in the hDAF group. Myocardial damage was minimal in transgenic hearts, whereas normal pig hearts produced a threefold increase of creatine phosphokinase and lactate dehydrogenase levels. In these hearts, electron microscopy revealed single cell necrosis of myocytes and vacuolization of mitochondria with cristae rupture. According to the results obtained in the working heart model, the breeding of pigs that are transgenic for hDAF represents a promising step to making heart xenotransplantation a clinical reality in the future.

Immunoreactivity of p53, Ki-67, and Bcl-2 in oncocytic adenomas and carcinomas of the thyroid gland.

To analyze relevant factors of neoplastic transformation in oncocytic neoplasms of the thyroid, expression of p53, Ki-67, and bcl-2 has been studied in oncocytic carcinomas (n = 17) and compared with results obtained in oncocytic adenomas (n = 20). P53 protein accumulation was found immunohistochemically in 75% of the oncocytic adenomas (15 of 20) and 88% of the oncocytic carcinomas (15 of 17). Eight of 17 of the carcinomas (47%), but only 3 of the 20 adenomas (15%), showed nuclear p53 accumulation in more than 10% of the cells, mostly in a focal pattern. Ki-67 expression also differed significantly between adenomas and carcinomas. The median of Ki-67-positive cells was 12/10 high-power fields (HPF) for adenomas and 76/10 HPF for carcinomas (P < .001). Furthermore, metastatic carcinomas had a significantly higher Ki-67 positivity than nonmetastasized carcinomas (164/10 HPF v 42/10 HPF, P < .05). Bcl-2 immunohistochemistry showed a constantly positive reaction in normal thyroid tissue. In contrast, bcl-2 protein was not detected in most of the adenomas (70%) and carcinomas (76%). In conclusion, p53 protein and Ki-67 is more prevalent in oncocytic carcinomas than in oncocytic adenomas of the thyroid, indicating that these factors may be involved in the progression of oncocytic neoplasms in the thyroid. In contrast, loss of bcl-2 appears to be an early event in the formation of oncocytic neoplasms of the thyroid. Its importance for malignant transformation is, however, unclear.

Fatal mitochondrial myopathy with cytochrome-c-oxidase deficiency and subunit-restricted reduction of enzyme protein in two siblings: an autopsy-immunocytochemical study.

Lack of cytochrome-c oxidase activity and of cytochromes aa3 + b has been reported previously in the skeletal muscle of one of two siblings (Müller-Höcker et al, 1983). The present study reports a deficiency of immunoreactive enzyme protein in the skeletal muscle of both siblings, who had an identical fatal clinical course. In all specimens the defect did not involve the whole enzyme protein, but was selectively expressed in the mitochondrially derived subunits II/III and nuclear coded subunits VIIbc. Neither the specific fibers of the muscle spindles nor the mitochondria of the heart, liver, kidneys, vessel walls and/or gastrointestinal tract were affected. These results are most consistent with a primary nuclear defect being responsible for the organ specific and subunit selective expression of the enzyme defect.

P53 gene product and EGF-receptor are highly expressed in placental site trophoblastic tumor.

Immunohistochemical analysis of curettage material from a placental site trophoblastic tumor (PSTT) revealed a high expression of p53 gene products, of epidermal growth factor receptor (EGF-R) and of Ki-67 (MIB-1) proliferation associated antigen. bcl-2 was not expressed. These results show that in PSTT inactivation/dysregulation of p53 and upregulation of EGF-R and MiB-1 occurs, indicating that these factors are probably involved in tumor genesis and propagation of PSTT. The prognostic significance of the molecular genetic data, however, remains to be established.