[Muscular dystrophy due to mutations in anoctamin 5: clinical and molecular genetic findings]. / Muskeldystrophie infolge Anoctamin-5-Mutationen : Klinische und molekulargenetische Befunde.

Recessive mutations in the anoctamin 5 (ANO5) gene have been recently identified in families with limb girdle muscular dystrophy (LGMD2L) and distal non-dysferlin Miyoshi myopathy. Anoctamin 5 is supposed to be a putative calcium-activated chloride channel. We report five German patients (four index patients) with muscle dystrophy due to mutations in the ANO5 gene. Sequencing of the ANO5 exons 5, 13 and 20 was performed to screen for a common c.191dupA mutation and two other reported mutations (c.1295C>G and p.R758C). The whole coding region of the ANO5 gene was sequenced to identify new mutations. Phenotypically, three patients showed LGMD and one patient Miyoshi type distal myopathy. One sibling had asymptomatic hyperCKemia. The age at onset was 64, 38 and 40 years in patients with LGMD and 23 years in the patient with distal myopathy. The four symptomatic patients showed remarkable asymmetric muscle involvement. There was marked CK elevation (11 to 30 times). Electron microscopy showed multifocal gaps in the sarcolemmal membrane. All patients harboured the common c.191dupA mutation in at least one allele. Two patients with LGMD were homozygous and the third patient and his asymptomatic sister were compound heterozygous for the c.191dupA mutation and a novel p.T548I mutation. The patient with distal myopathy harboured the p.R758C mutation in the second allele. Mutations in the ANO5 gene seem to be a relatively common cause of muscular dystrophy in Germany. Cases with late onset or asymptomatic hyperCKemia can occur. Clinically, asymmetric manifestation is typical.

Biological treatment of waste gas containing mixture of monochlorobenzene (MCB) and benzene in a bench scale biofilter.

The paper outlines treatment of waste gas containing monochlorobenzene (MCB) and benzene in a mixture using biofilter packed with compost and woodchips seeded with Acinetobacter calcoaceticus. The biofilter could treat waste gas containing MCB and benzene effectively with an efficiency of (99+/-5%) and (97+/-6%) at optimal empty bed contact time (EBCT) of 3 min with a loading of 57 g/m(3)/h of MCB and 2g/m(3)/h of benzene. At optimum loading of MCB and benzene, the biofilter showed total bacterial count of 13 x 10(5)CFU/g of compost, while the MCB and benzene degrading bacterial count was 71 x 10(4)CFU/g and 5 x 10(4)CFU/g compost respectively. The experimental removal efficiency of MCB and benzene were in good agreement with the model predicted value.

Molecular diagnosis of German patients with late-onset glycogen storage disease type II.

UNLABELLED: In patients with late-onset glycogen storage disease type II, one mutation, c.-32-13T>G, in the α-glucosidase (GAA) gene is identified frequently in European populations from different regions along with many rarer mutations. We have performed molecular genetic investigations in 18 German index patients with late-onset disease. The c.-32-13T>G, c.525delT (p.Glu176fsX45), and c.2481+102_2646+31del mutations were detected by PCR/restriction enzyme digest. Other mutations were detected by sequencing. All patients were compound heterozygous and 17 patients harboured the c.-32-13T>G mutation. Seven other previously described mutations (including the c.-32-13T>G) were identified, of which the p.C103G (c.307T>G) and the c.2481+102_2646+31del mutations were present each in three unrelated patients. Sequencing revealed five novel mutations. CONCLUSIONS: Genetic testing was able to identify the genetic defects in all patients and screening of the c.-32-13T>G mutation identified 94% of the cases. This is important for quick and reliable diagnosis, especially in view of enzyme replacement. Among the rarer mutations, c.2481+102_2646+31del and p.C103G are rather frequent in Germany.

Analysis of spectrum and frequencies of mutations in McArdle disease. Identification of 13 novel mutations.

BACKGROUND: McArdle disease, a common metabolic myopathy with autosomal recessive inheritance, is caused by a frequent R50X mutation and many rare mutations in the myophosphorylase gene. OBJECTIVES: To identify spectrum and frequencies of myophosphorylase gene mutations in a large cohort of patients with McArdle disease, to discuss diagnostic implications, and to analyse genotype-phenotype relationship. METHODS: Molecular genetic analysis of 56 index patients with muscle biopsy-proven myophosphorylase deficiency from Germany (n = 35), UK (n = 13), and several other countries (n = 8) was performed using direct sequencing. RESULTS: Allele frequency of the R50X mutation was 58%, and 71% of the patients carried this mutation at least on one allele. We detected 26 other less common mutations, 13 of which are novel: G157V, R161C, Q337R, E384K, S450L, G486D, R570W, K575E, IVS6-2A>T, IVS10+1G>A, R650X, c.1354insC, c.1155_1156delGG. There was no genotype-phenotype correlation with respect to age of onset and severity. R270X was the most frequent mutation among the less common mutations reaching an allele frequency of 5% followed by R94W and G686R representing a frequency of 4% each. CONCLUSIONS: The study further extends the genetic heterogeneity of myophosphorylase gene mutations showing no mutational hotspot and no genotype-phenotype correlation. Most novel missense mutations were located in secondary structures or active sites of the enzyme. Some of the less common mutations are recurrent with different frequencies within Europe. Ethnic origin and frequency of less common mutations must be considered to establish efficient strategies in molecular genetic testing. Performing molecular testing can avoid muscle biopsy.

A vertical flow chamber for Xenopus oocyte electrophysiology and automated drug screening.

Xenopus laevis oocytes are used extensively in the study of ion channel coupled receptors. Efficient use of oocytes for ion channel characterization requires a system that is inherently stable, reproducible, minimizes drug volumes, and maximizes oocyte longevity. We have constructed a vertical flow oocyte recording chamber to address the aforesaid issues, where the oocyte is placed in a funnel-shaped chamber and perfused from the bottom of the funnel. The vertical rather than horizontal flow of perfusate results in an unusually stable environment for oocyte recording. Two-electrode voltage clamp recordings from a single oocyte are acquired easily and routinely over several hours while maintaining stable baseline currents and reproducible response profiles. Chamber characteristics were tested using a serotonin ligand-gated ion channel receptor (5-HT3R). Data obtained from this system corresponds well with published data. To further test the stability and reliability of this perfusion chamber, we constructed an automated oocyte perfusion system utilizing a commonly available HPLC autosampler. We were able to obtain dose-response curves for various 5-HT3AR ligands using the automated perfusion system with minimal user intervention. Such a system can easily satisfy need for automated oocyte electrophysiology in academic settings, especially small to medium sized laboratories.

Prevalence of diphtheria and tetanus antibodies in young adults in Delhi.

A total of 574 blood samples collected mainly from adult males, on a random basis, were tested for diphtheria and tetanus antibodies by Indirect Haemagglutination (IHA) test to find out the percentage of individuals with protective titres (> or = 0.015 IU/ml). A total of 502 (87.5 per cent) and 437 (76.2 per cent) of these had protective titres against diphtheria and tetanus respectively. The vaccination status of these subjects against diphtheria and tetanus was not ascertainable. The relevance of these findings is discussed.

Nasopharyngeal carriage of Neisseria meningitidis in general population and meningococcal disease.

Nasopharyngeal carriage of Neisseria meningitidis was determined in the normal healthy population in Delhi at monthly intervals for a period of 2 years from January, 1986 to December, 1987. Of a total of 6513 individuals screened only 107 (1.64 per cent) were found to carry Neisseria meningitidis serogroup A. There was no age and sex difference in carriage. During the same period, data of laboratory confirmed cases of meningitis due to N. meningitidis serogroup A was obtained from 6 hospitals of Delhi which acted as sentinel centres. Of the total 11,870 pyogenic C.S.F. samples processed, only 557 (4.69 per cent) were due to N. meningitidis serogroup A. There was no correlation observed between the nasopharyngeal meningococcal carriage in the healthy population with the disease prevalence. There was no seasonal variation in nasopharyngeal carriage though upsurge in the number of meningococcal meningitis cases was noticed from January to April.