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Detection of variants in dystroglycanopathy-associated genes through the application of targeted whole-exome sequencing analysis to a large cohort of patients with unexplained limb-girdle muscle weakness.
BACKGROUND: Dystroglycanopathies are a clinically and genetically heterogeneous group of disorders that are typically characterised by limb-girdle muscle weakness. Mutations in 18 different genes have been associated with dystroglycanopathies, the encoded proteins of which typically modulate the binding of α-dystroglycan to extracellular matrix ligands by altering its glycosylation. This results in a disruption of the structural integrity of the myocyte, ultimately leading to muscle degeneration. METHODS: Deep phenotypic information was gathered using the PhenoTips online software for 1001 patients with unexplained limb-girdle muscle weakness from 43 different centres across 21 European and Middle Eastern countries. Whole-exome sequencing with at least 250 ng DNA was completed using an Illumina exome capture and a 38 Mb baited target. Genes known to be associated with dystroglycanopathies were analysed for disease-causing variants. RESULTS: Suspected pathogenic variants were detected in DPM3, ISPD, POMT1 and FKTN in one patient each, in POMK in two patients, in GMPPB in three patients, in FKRP in eight patients and in POMT2 in ten patients. This indicated a frequency of 2.7% for the disease group within the cohort of 1001 patients with unexplained limb-girdle muscle weakness. The phenotypes of the 27 patients were highly variable, yet with a fundamental presentation of proximal muscle weakness and elevated serum creatine kinase. CONCLUSIONS: Overall, we have identified 27 patients with suspected pathogenic variants in dystroglycanopathy-associated genes. We present evidence for the genetic and phenotypic diversity of the dystroglycanopathies as a disease group, while also highlighting the advantage of incorporating next-generation sequencing into the diagnostic pathway of rare diseases.
Identification of GAA variants through whole exome sequencing targeted to a cohort of 606 patients with unexplained limb-girdle muscle weakness.
BACKGROUND: Late-onset Pompe disease is a rare genetic neuromuscular disorder caused by a primary deficiency of α-glucosidase and the associated accumulation of glycogen in lysosomal vacuoles. The deficiency of α-glucosidase can often be detected using an inexpensive and readily accessible dried blood spot test when Pompe disease is suspected. Like several neuromuscular disorders, Pompe disease typically presents with progressive weakness of limb-girdle muscles and respiratory insufficiency. Due to the phenotypic heterogeneity of these disorders, however, it is often difficult for clinicians to reach a diagnosis for patients with Pompe disease. Six hundred and six patients from a European population were recruited onto our study. Inclusion criteria stipulated that index cases must present with limb-girdle weakness or elevated serum creatine kinase activity. Whole exome sequencing with at least 250 ng DNA was completed using an Illumina exome capture and a 38 Mb baited target. A panel of 169 candidate genes for limb-girdle weakness was analysed for disease-causing variants. RESULTS: A total of 35 variants within GAA were detected. Ten distinct variants in eight unrelated index cases (and four siblings not sequenced in our study) were considered disease-causing, with the patients presenting with heterogeneous phenotypes. The eight unrelated individuals were compound heterozygotes for two variants. Six patients carried the intronic splice site c.-13 T > G transversion and two of the six patients also carried the exonic p.Glu176ArgfsTer45 frameshift. Four of the ten variants were novel in their association with Pompe disease. CONCLUSIONS: Here, we highlight the advantage of using whole exome sequencing as a tool for detecting, diagnosing and treating patients with rare, clinically variable genetic disorders.
Cyclic ADP-ribose and the regulation of calcium-induced calcium release in eggs and cardiac myocytes.
Cyclic ADP-ribose (cADPR) is a cyclic metabolite of NAD+ synthesised in cells and tissues expressing ADP-ribosyl cyclases. Although it was first discovered in sea-urchin egg extracts as a potent calcium mobilizing agent, subsequent studies have indicated that it may have a widespread action in the activation of calcium-release channels in such diverse systems as mammalian neurones, myocytes, blood cells, eggs, and plant microsomes. In this review we focus on recent work suggesting that cADPR enhances the sensitivity of ryanodine-sensitive calcium-release channels (RyRs) to activation by calcium, a phenomenon termed calcium-induced calcium release (CICR). Two roles for cADPR in calcium signaling are discussed. The first is as a classical second messenger where its levels are controlled by extracellular stimuli, and the second mode of cellular regulation is that the levels of intracellular cADPR may set the sensitivity of RyRs to activation by an influx of calcium in excitable cells. These two possible actions of cADPR are illustrated by considering the signal transduction events during the fertilization of the sea-urchin egg and the modulation of CICR during excitation-coupling in isolated guinea-pig ventricular myocytes, respectively.
Lipoprotein (a) levels in end-stage renal failure and renal transplantation.
Some previous studies have documented an increase in lipoprotein (a) [Lp(a)] levels in renal diseases. Here, we report data in subjects with end-stage renal failure treated with hemodialysis (HD) or with continuous ambulatory peritoneal dialysis (CAPD) and in renal transplant recipients (RTR), compared with a group of normolipidemic controls (C). Lp(a) levels were significantly increased in HD and CAPD patients in comparison with C, while they were only slightly increased in RTR. Both HD and CAPD patients showed Lp(a) levels higher than in RTR, but no difference was found between the subjects of the two dialysis procedures. The prevalence of Lp(a) levels > 25 mg/dl was significantly higher in HD and CAPD patients, but not in RTR, in comparison with C. Moreover, Lp(a) levels did not change after HD. When patients were divided according to their fasting lipid levels in normolipidemics and hyperlipoproteinemics, no difference was found for Lp(a) levels in any group. Mechanisms underlying the increase in Lp(a) levels in these patients are not known. It is possible to suggest an active role of the kidney in the Lp(a) metabolism or that uremic plasma contains some factors affecting Lp(a) metabolism.
Ca2+ release induced by cyclic ADP-ribose.
Cyclic ADP-ribose (cADPR) is the most potent Ca(2+)-mobilizing agent known. It has been found in many different cell types, where it is synthesized from its precursor NAD(+) by ADP-ribosyl cyclases. cADPR binds to Ca(2+) channels in the endoplasmic reticulum membrane to activate a Ca(2+)-release mechanism. This release is itself potentiated by elevated cytoplasmic Ca(2+) concentrations. Thus, cADPR may function as an endogenous regulator of Ca(2+)-induced Ca(2+) release, and there is excitement that it may also function as a Ca(2+)-mobilizing second messenger.
Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.
The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.
Pancreatic enzymes in chronic renal failure and transplant patients.
The aim of the present study was to determine the frequency and degree of elevated serum levels of Total Amylase (TA), Pancreatic Amylase (PA), and Lipase (L) activity in patients with chronic renal failure (CRF) on conservative therapy; CRF on periodical hemodialysis (HD); in renal transplant (RT) and in a control Group (C). Mean values were significantly higher in all groups than Group C for TA (p < 0.005), PA (p < 0.0001) and L (p < 0.0001). A statistically significant correlation was found between TA and L vs creatininemia values in CRF patients, but only up to a certain level (creatininemia < 6 mg %) (p < 0.03 and p < 0.05), above which there was no correlation. The enzyme most frequently over the maximum normal limit was PA, both in the total CRF group (51%), in the hemodialysis patients (65%), and in the RT patients (55%); but only a few patients had values two times higher than the normal limits: 15% in the total CRF, 14% is the hemodialysis, and 10% in the RT groups, respectively. These results suggest that the increase in serum pancreatic enzyme during chronic renal pathology is slight but frequently occurs. It is possible that in these patients together with the renal excretion impairment there could also be some subclinical pancreatic damage; its genesis could also depend on the pharmacological treatment used (diuretics, immunosuppressive drugs) commonly adopted in these pathologies.
Cytosolic calcium oscillators.
Many cells display oscillations in intracellular calcium resulting from the periodic release of calcium from intracellular reservoirs. Frequencies are varied, but most oscillations have periods ranging from 5 to 60 s. For any given cell, frequency can vary depending on external conditions, particularly the concentration of natural stimuli or calcium. This cytosolic calcium oscillator is particularly sensitive to those stimuli (neurotransmitters, hormones, growth factors) that hydrolyze phosphoinositides to give diacylglycerol and inositol 1,4,5-trisphosphate (Ins1,4,5P3). The ability of Ins1,4,5P3 to mobilize intracellular calcium is a significant feature of many of the proposed models that are used to explain oscillatory activity. Receptor-controlled oscillator models propose that there are complex feedback mechanisms that generate oscillations in the level of Ins1,4,5P3. Second messenger-controlled oscillator models demonstrate that the oscillator is a component of the calcium reservoir, which is induced to release calcium by a constant input of either Ins1,4,5P3 or calcium itself. In the latter case, the process of calcium-induced calcium release might be the basis of oscillatory activity in many cell types. The function of calcium oscillations is still unknown. Because oscillator frequency can vary with agonist concentration, calcium transients might be part of a frequency-encoded signaling system. When an external stimulus arrives at the cell surface the information is translated into a train of calcium spikes, i.e., the signal is digitized. Certain cells may then convey information by varying the frequency of this digital signal.
ADP-ribose gates the fertilization channel in ascidian oocytes.
We report an ion channel in the plasma membrane of unfertilized oocytes of the ascidian Ciona intestinalis that is directly gated by the second messenger ADP-ribose. The ion channel is permeable to Ca2+ and Na+ and is characterized by a reversal potential between 0 and +20 mV and a unitary conductance of 140 pS. Preinjection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or antagonists of intracellular Ca2+ release channels into oocytes did not inhibit the ADP-ribose current, demonstrating that the channel is activated in a Ca2+-independent manner. Both the fertilization current and the current induced by the injection of nicotinamide nucleotides are blocked by nicotinamide, suggesting that the ADP-ribose channel is activated at fertilization in a nicotinamide-sensitive manner. These data suggest that ascidian sperm trigger the hydrolysis of nicotinamide nucleotides in the oocyte to ADP-ribose and that this mechanism is responsible for the production of the fertilization current.
Actions of cADP-ribose and its antagonists on contraction in guinea pig isolated ventricular myocytes. Influence of temperature.
Although it is becoming widely accepted that cADP-ribose (cADPR) can regulate calcium release from the endoplasmic reticulum in sea urchin eggs and in a variety of mammalian cell types, it remains controversial whether this substance might influence calcium release during excitation-contraction coupling in cardiac muscle. We have investigated possible actions of cADPR in intact cells isolated from guinea pig ventricle, paying particular attention to the possible influence of temperature. At 36 degrees C, myocyte contraction was influenced by cytosolic application of cADPR in a concentration-dependent manner (showing an approximately 30% increase in contraction with 5 mumol/L cADPR applied via a patch pipette in myocytes stimulated to fire action potentials at 1 Hz). Calcium transients measured with fura 2 were also increased by 5 mumol/L cADPR. Antagonists of cADPR reduced contraction at 36 degrees C (by approximately 35% with either 50 mumol/L 8-Br-cADPR or 5 mumol/L 8-amino-cADPR applied via the patch pipette). At room temperature (approximately 20 degrees C to 24 degrees C), no significant effects on contraction were detected with either cADPR or its antagonists. At 36 degrees C, treatment of the cells with a mixture of 2 mumol/L ryanodine and 1 mumol/L thapsigargin to suppress function of the sarcoplasmic reticulum stores of calcium prevented the action of 5 mumol/L cADPR applied via a patch pipette. These observations are consistent with an action of cytosolic cADPR to enhance calcium-induced calcium release from the sarcoplasmic reticulum in guinea pig ventricular myocytes at 36 degrees C. The observed influence of temperature under the conditions of our experiments is one factor that might help to account for failure to detect actions of cADPR and its analogues in some previous studies.
Mechanisms of calcium release and sequestration in eggs of Chaetopterus pergamentaceus.
Increases in the intracellular free calcium concentration are of great importance to the initiation of development in deuterostomes. Their involvement has not yet been clearly defined in protostomes. We used endogenous ligands (IP3, cADPR, ryanodine and NAADP) and pharmacological agents (thapsigargin [Tg], thimerosal, caffeine and heparin) to study smooth endoplasmic reticulum Ca2+ pump and release mechanisms in eggs of an annelid, Chaetopterus. Oocyte homogenates effectively sequestered Ca2+ and released it in response to IP3 in a concentration-dependent manner. Repeated additions of IP3 were unable to cause further release. Heparin inhibited Ca2+ release in response to IP3. The homogenates also released Ca2+ in response to thimerosal, and this release was sensitive to heparin. Two antibodies to IP3 receptors recognized an appropriate band in Chaetopterus egg lysates. These results indicate that the oocytes possess type-1 IP3-gated Ca2+ channels. Neither calcium itself, nor strontium, cADPR, ryanodine, caffeine nor NAADP released appreciable Ca2+. At low concentrations, Tg caused a slow release of Ca2+; at higher concentrations, it elicited a rapid release. Release of Ca2+ by Tg activated development. Since one theory of fertilization invokes the introduction of a Ca2+ releasing soluble protein into the egg upon sperm-egg fusion, we also tested whether soluble extracts of Chaetopterus sperm could stimulate Ca2+ release in Chaetopterus egg homogenates. There was no Ca2+ release when the sperm extract was added to the homogenate; however, homogenates exposed to sperm extract became refractory to IP3. Thus, Ca2+ release at fertilization in these oocytes occurs through IP3-gated channels.
Effects of glyburide-cyclosporin A interaction on interleukin-2 production in rats.
The effects of simultaneous administrations of Cyclosporin A (CsA) and Glyburide on the immune system of rats has been evaluated in terms of Interleukin-2 (IL-2) production by Concanavalin A (ConA) stimulated splenocytes and exogenous IL-2 binding capacity. The inhibitory effect of Cyclosporin A on IL-2 production of lymphoid cells is well known. Spleen cells from rats receiving CsA had reduced levels of IL-2 when compared to untreated controls or rats receiving Glyburide only. Splenocytes from rats receiving both drugs had reduced levels of IL-2 when they were sacrificed 24 hours after one or three CsA administrations; instead when the animals were sacrificed 6 days after three CsA administrations, their ability of producing IL-2 is increased as well as increasing exogenous IL-2 binding capacity. These findings let us hypothesize that when there are lower concentrations of CsA in lymphocytes there is an increase of cellular metabolism induced by Glyburide that leads to an increase in IL-2 secretion and in IL-2 receptor expression on cellular surface restoring these levels to normal or slightly above normal levels.
Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg.
1. The sea urchin egg homogenate is an ideal model to characterize Ca2+-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP3- and ryanodine-sensitive Ca2+-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist. 2. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca2+-releasing agent, NAADP, compared to inositol trisphosphate (InsP3) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors. 3. NAADP-induced Ca2+-release was blocked by L-type Ca2+-channel blockers and by Bay K 8644, while InsP3- and cyclic ADPR-induced Ca2+-release were insensitive to these agents. L-type Ca2+-channel blockers did not displace [32P]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion. 4. Similarly, a number of K+-channel antagonists blocked NAADP-induced Ca2+-release selectively over InsP3- and cyclic ADPR-induced Ca2+-release. Radioligand studies showed that these agents were not competitive antagonists. 5. As has been shown for InsP3 and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca2+-release mechanisms. 6. The presence of K+ was not essential for NAADP-induced Ca2+-release, since substitution of K+ with other monovalent cations in the experimental media did not significantly alter Ca2+ release by NAADP. On the contrary, cyclic ADPR and InsP3-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K+. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca2+-release. While the alkaline condition permitted InsP3 and cyclic ADPR-induced Ca2+-release, the acidic condition completely hampered both Ca2+-release mechanisms. 7. The present results characterize pharmacologically and biochemically the novel Ca2+-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.