Ichthyosis bullosa of Siemens is caused by mutations in the keratin 2e gene.

Ichthyosis bullosa of Siemens is a blistering disorder with autosomal dominant inheritance. The disease resembles bullous congenital ichthyosiform erythroderma but is less severe. Keratins K1 and K10 have been implicated in bullous congenital ichthyosiform erythroderma. Linkage analysis pointed to the involvement of a keratin type II gene (12q11-13) in ichthyosis bullosa of Siemens. Mutations in the highly conserved regions of K1, a member of the type II gene cluster, were excluded. The gene coding for keratin 2e is also located in the type II gene cluster and the expression of the gene coincides with the occurrence of epidermolytic hyperkeratosis. Sequence analysis revealed the presence of mutations in the K2e gene in patients with ichthyosis bullosa of Siemens. Three different mutations were detected, one in the 1A domain and two in the 2B domain of the rod. Furthermore, histologic and ultrastructural examination of skin biopsies indicated that ichthyosis exfoliativa is identical to ichthyosis bullosa of Siemens. This was confirmed by the results of the molecular analysis. In the family diagnosed as ichthyosis exfoliativa, a mutation was detected that was identical to the mutation found in one of the families with ichthyosis bullosa of Siemens.

Measurement of totally activated pyruvate dehydrogenase complex activity in human muscle: evaluation of a useful assay.

A sensitive radiochemical method for the determination of the pyruvate dehydrogenase complex (PDHC) activity in skeletal muscle tissue, based on the decarboxylation of [1-14C]-pyruvate to 14CO2, is described. Measurements can be carried out either in muscle homogenate or in 600-g supernatant, both obtainable from a small muscle biopsy specimen (20 mg). In addition to NAD+, thiamine pyrophosphate and coenzyme A in the incubation mixture, a preparation of NADH:cytochrome c reductase (NADHCR) together with cytochrome c has a stimulating effect on the PDHC activity. NADHCR constitutes an oxidation system for NADH to prevent feedback inhibition. Addition of L-carnitine also results in stimulation of PDHC by trapping the produced acetyl-CoA as acetylcarnitine. Special care for radioactive pyruvate, with freeze drying and storage at -20 degrees C under nitrogen, and determination of the purity during every PDHC assay, is required. In the presented assay a Km value of 0.084 mmol/l was found for pyruvate. Nonsigmoidal kinetics was found with a Hill coefficient of 1.63. With the described method, a totally Mg2+,Ca(2+)-stimulated PDHC activity is measured. Addition of a purified specific pyruvate dehydrogenase phosphatase did not yield a higher PDHC activity. Finally, comparison of total PDHC activity with [1-14C]-pyruvate oxidation rates, both measured in the supernatant prepared from fresh muscle, shows an equimolar correlation, indicating that total PDHC activity is rate limiting in the assay for the pyruvate oxidation rate. Neonatal muscle exhibits five to ten times lower PDHC activities and pyruvate oxidation rates than controls (age > 3 years).

Deficiency of the alpha and beta subunits of pyruvate dehydrogenase in a patient with lactic acidosis and unexpected sudden death.

An infant with moderate muscular hypotonia and congenital lactic acidosis died suddenly at the age of 3 months. Autopsy revealed no abnormalities responsible for this unexpected death. Measurement of mitochondrial enzymes involved in energy production indicated a severely decreased total pyruvate dehydrogenase complex (PDHC) activity in muscle tissue (0.23 nmoles x min-1 x mg protein-1, control range 2.8-8.7) and moderately decreased PDHC activity in fibroblasts (0.27 nmoles x min-1 x mg protein-1, control range 0.37-2.32). The activity of the first component E1 (pyruvate dehydrogenase) in muscle tissue was 10 times lower than that of controls (0.008 nmoles x min-1 x mg protein-1, control range 0.10-0.25). The activities of dihydrolipoyl dehydrogenase (E3) and various other mitochondrial enzymes were normal. Immunochemical analysis in skeletal muscle tissue and fibroblasts demonstrated a decrease in the amount of the alpha and beta subunits of E1. The features of this patient are compared with those of other patients reported in the literature with immunochemically confirmed combined E1 alpha and beta deficiency.

Estimation of pyruvate dehydrogenase (E1) activity in human skeletal muscle; three cases with E1 deficiency.

Pyruvate dehydrogenase (E1) catalyzes the rate-limiting step of the pyruvate dehydrogenase complex. Since E1 activity of human muscle tissue is low, a sensitive method is needed for diagnostic purposes. Measurement of 14CO2 production from [1-14C]pyruvate provides a specific and sensitive assay for measuring E1 activity. We use as artificial electron acceptor dichlorophenolindophenol (DCPIP) instead of the often applied ferricyanide. The method can be applied to small muscle samples obtained by needle or open biopsy. We prefer to use total homogenate because E1 activities in homogenate are higher than in the corresponding 600-g supernatant of skeletal muscle tissue. Control values in homogenate are higher or of the same order as those reported by others.