Autonomic neuropathy in Fabry disease: a prospective study using the Autonomic Symptom Profile and cardiovascular autonomic function tests

Background  

Fabry patients have symptoms and signs compatible with autonomic dysfunction. These symptoms and signs are considered to be due to impairment of the peripheral nervous system, but findings indicative of autonomic neuropathy in other diseases, such as orthostatic intolerance and male sexual dysfunction, are infrequently reported in Fabry disease. The aim of our study was to investigate autonomic symptoms and cardiovascular autonomic function in a large cohort of male and female Fabry patients.     

High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

Glycophorins are the most abundant sialoglycoproteins on the surface of human erythrocyte membranes. Genetic variation in glycophorin region of human chromosome 4 (containing GYPA, GYPB, and GYPE genes) is of interest because the gene products serve as receptors for pathogens of major public health interest, including Plasmodium sp., Babesia sp., Influenza virus, Vibrio cholerae El Tor Hemolysin, and Escherichia coli. A large structural rearrangement and hybrid glycophorin variant, known as Dantu, which was identified in East African populations, has been linked with a 40% reduction in risk for severe malaria. Apart from Dantu, other large structural variants exist, with the most common being deletion of the whole GYPB gene and its surrounding region, resulting in multiple different deletion forms. In West Africa particularly, these deletions are estimated to account for between 5 and 15% of the variation in different populations, mostly attributed to the forms known as DEL1 and DEL2. Due to the lack of specific variant assays, little is known of the distribution of these variants. Here, we report a modification of a previous GYPB DEL1 assay and the development of a novel GYPB DEL2 assay as high-throughput PCR-RFLP assays, as well as the identification of the crossover/breakpoint for GYPB DEL2. Using 393 samples from three study sites in Ghana as well as samples from HapMap and 1000 G projects for validation, we show that our assays are sensitive and reliable for genotyping GYPB DEL1 and DEL2. To the best of our knowledge, this is the first report of such high-throughput genotyping assays by PCR-RFLP for identifying specific GYPB deletion types in populations. These assays will enable better identification of GYPB deletions for large genetic association studies and functional experiments to understand the role of this gene cluster region in susceptibility to malaria and other diseases.     

Phosphorylation-dependent stimulation of prostanoid synthesis by nigericin in cerebral endothelial cells

Nigericin decreases intracellular pH(pH_i) and stimulates prostanoid(PG) synthesis in endothelial cells from cerebral microvessels ofnewborn pigs. Nigericin-induced PG production was abolished by proteintyrosine kinase (PTK) inhibitors and amplified by phorbol 12-myristate13-acetate (PMA) or protein tyrosine phosphatase (PTP) inhibitors.Nigericin-induced PG production in PMA-primed cells was potentiated byPTP inhibitors and abrogated by PTK inhibitors. PhospholipaseA₂(PLA₂) activity was stimulatedby nigericin in a phosphorylation-dependent manner. Nigericin'seffects on PG production and PLA₂activity were reproduced by ionomycin, which activates cytosolicPLA₂(cPLA₂).cPLA₂ was immunodetected in endothelial cell lysates. We found no evidence that nigericin's effects are mediated via mitogen-activated protein (MAP) kinase [extracellularly regulated kinase 1 (ERK1) and ERK2]activation: although nigericin stimulateddetergent-soluble MAP kinase, its effects were not amplified by PMA orPTP inhibitors. Phosphorylation-dependent stimulation of PG synthesiswas also observed when pH_i wasdecreased by sodium propionate or a high level ofCO₂. Altogether, our data indicatethat nigericin and decreased pH_istimulate PG synthesis by a protein phosphorylation-dependent mechanisminvolving cross talk between pathways mediated by PTK and PTP and byprotein kinase C; cPLA₂ appears tobe a key enzyme affected by nigericin and decreasedpH_i.