Biomedical subjects

Steven Petrou

Publications and source records attributed to Steven Petrou.

At least 19 recordsLinked to original sources

PubMed2026

Individualized antisense oligonucleotides for SCN2A-related developmental epileptic encephalopathy.

SCN2A variants are among the most common genetic causes of developmental and epileptic encephalopathies (DEEs), which can present with uncontrolled seizures at birth and account for 1-2% of all epileptic encephalopathies. A substantial fraction of causal variants are gain-of-function or mixed-function variants associated with increased channel open probability or greater sodium current flux. Here two parallel n = 1 clinical studies were conducted in two patients (9-year-old and 14-year-old boys) with SCN2A-related DEE. Individualized allele-selective antisense oligonucleotides (ASOs) were designed to target heterozygous intronic single-nucleotide polymorphisms (SNPs) for decreased expression of mutant SCN2A transcript while preserving the wild-type copy. Primary endpoints included quantitative change from baseline in seizure frequency and neurodevelopment, including motor scores. Efficacy measures were also individualized to each patient's phenotype, including refractory seizures, developmental delay, autism spectrum disorder, choreoathetosis and gastrointestinal dysfunction. Patients experienced a reduction in seizure frequency (26% and 90% in the two patients, respectively), decreased use of concomitant medications and improvement in neurodevelopmental skills. Both ASOs were well tolerated, with no ASO-related serious adverse events. Continued long-term follow-up of these preliminary positive safety and efficacy findings is needed to confirm the disease-modifying potential of these ASOs. Haplotype phasing in a separate cohort of infants with SCN2A-related disorder (SCN2A-RD), diagnosed by rapid whole-genome sequencing, identified 16% of patients with compatible SNPs. These data provide a pathway from n = 1 to n of more patients with SCN2A-RD and other monogenic disorders. ClinicalTrials.gov registration: NCT06314490 .

Adolescent↗

PubMed2006

Temporal lobe epilepsy and GEFS+ phenotypes associated with SCN1B mutations.

SCN1B, the gene encoding the sodium channel beta 1 subunit, was the first gene identified for generalized epilepsy with febrile seizures plus (GEFS+). Only three families have been published with SCN1B mutations. Here, we present four new families with SCN1B mutations and characterize the associated phenotypes. Analysis of SCN1B was performed on 402 individuals with various epilepsy syndromes. Four probands with missense mutations were identified. Detailed electroclinical phenotyping was performed on all available affected family members including quantitative MR imaging in those with temporal lobe epilepsy (TLE). Two new families with the original C121W SCN1B mutation were identified; novel mutations R85C and R85H were each found in one family. The following phenotypes occurred in the six families with SCN1B missense mutations: 22 febrile seizures, 20 febrile seizures plus, five TLE, three other GEFS+ phenotypes, two unclassified and ten unaffected individuals. All individuals with confirmed TLE had the C121W mutation; two underwent temporal lobectomy (one with hippocampal sclerosis and one without) and both are seizure free. We confirm the role of SCN1B in GEFS+ and show that the GEFS+ spectrum may include TLE alone. TLE with an SCN1B mutation is not a contraindication to epilepsy surgery.

Adolescent↗

PubMed2006

The mechanism of carbamazepine aggravation of absence seizures.

Carbamazepine (CBZ) aggravates many generalized seizures types, particularly absence seizures, but the mechanisms underlying this are poorly understood. GABA signaling within the reticular nucleus (Rt) and the ventrobasal complex (VB) of the thalamus is critical to the neurophysiology of absence seizures. The hypothesis that CBZ aggravates absence seizures by acting at the VB thalamus via a GABA(A) receptor-mediated mechanism was investigated in a genetic rat model, generalized absence epilepsy rats from Strasbourg (GAERS). Seizure activity was quantified by a 90-min electroencephalogram recording postdrug injection. Intracerebroventricular injections of CBZ (15 microg in 4 microl) resulted in seizure aggravation versus vehicle treatment, with a mean increase in seizure time of 40%. This indicates that CBZ acts directly, rather than via a metabolite, on the brain to aggravate seizures. Seizure aggravation also occurred following bilateral microinjection of CBZ (0.75 microg in 0.2 microl) into the VB (53%) but not following injection into the Rt (-9%). However, seizure aggravation was blocked when the GABA(A) receptor antagonist, bicuculline (BIC, 0.04 microg in 0.2 microl), was coinjected with CBZ into the VB. Injection of BIC alone (versus vehicle) into the VB also blocked seizure aggravation following systemic administration of CBZ (15 mg/kg i.p.). In vitro studies in Xenopus oocytes expressing recombinant GABA(A) receptors demonstrated that CBZ produced a dose-dependent potentiation of the GABA current at a physiological relevant concentration range (1-100 microM). These data demonstrate that CBZ acts at the VB thalamus to aggravate absence seizures in GAERS and that activation of GABA(A) receptors is critical to this effect.

Animals↗

PubMed2006

Febrile seizures: traffic slows in the heat.

Febrile seizures, which occur in young children, have long been known to have a major inherited component. Mutations in some genes that encode sodium channel and GABA(A) receptor subunits have been found in a few families affected by febrile seizures. These mutations account only for a minority of cases, and much remains to be learnt about the molecular architecture of febrile seizures. A rare inherited cause--a mutation in the GABA(A) receptor subunit GABRG2 gene--has been recently shown to cause a temperature-dependent intracellular trafficking defect. This is an important step in unravelling the molecular pathogenesis of this common childhood disorder.

Biological Transport↗

PubMed2006

Human epilepsies: interaction of genetic and acquired factors.

Epilepsies, once regarded as due to demoniacal possession, can have both genetic and acquired causes, with interaction of these factors in many cases. To date, nearly all the genes discovered to be involved in human epilepsies encode subunits of ion channels, both voltage-gated and ligand-gated. Established acquired causes include serious brain trauma, stroke, tumours and infective lesions. Thus, in terms of exploring the neurobiology of "nature and nurture" in disease, the epilepsies are an excellent paradigm. Here, we review the evidence and discuss the possibility that ion channels are a common biological substrate for both genetic and acquired epilepsies. This review is part of the INMED/TINS special issue "Nature and nurture in brain development and neurological disorders", based on presentations at the annual INMED/TINS symposium (http://inmednet.com/).

Brain Neoplasms↗

PubMed2005

A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs ATP-induced mycobacterial killing by macrophages.

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.

Adenosine Triphosphate↗

PubMed2005

SCN1A mutations and epilepsy.

SCN1A is part of the SCN1A-SCN2A-SCN3A gene cluster on chromosome 2q24 that encodes for alpha pore forming subunits of sodium channels. The 26 exons of SCN1A are spread over 100 kb of genomic DNA. Genetic defects in the coding sequence lead to generalized epilepsy with febrile seizures plus (GEFS+) and a range of childhood epileptic encephalopathies of varied severity (e.g., SMEI). All published mutations are collated. More than 100 novel mutations are spread throughout the gene with the more debilitating usually de novo. Some clustering of mutations is observed in the C-terminus and the loops between segments 5 and 6 of the first three domains of the protein. Functional studies so far show no consistent relationship between changes to channel properties and clinical phenotype. Of all the known epilepsy genes SCN1A is currently the most clinically relevant, with the largest number of epilepsy related mutations so far characterized.

Alternative Splicing↗

PubMed2005

Ion transport blockers inhibit human rhinovirus 2 release.

Picornavirus replication causes leakage of cytoplasmic K+ and an influx of Na+ and Ca2+. In this study, we have explored the possibility that a blockade of Ca2+ and Na+ influx would reduce rhinovirus production and/or release. The Ca2+-channel blockers, verapamil and diltiazem, as well as the blocker of Na+/H+ exchange and the epithelial Na+ channel, EIPA, inhibited both virus production and release. The effect on virus release was more pronounced than the effect on production, thus raising the possibility that rhinovirus release may serve as a target for antiviral agents. Unexpectedly, our results also showed that the antiviral activity of the Ca2+-channel blockers was not due to the block of Ca2+ influx. Similarly, the antiviral activity of EIPA appeared to be unrelated to the blockade of cellular Na+/H+ exchanger or the epithelial Na+ channel. Potential alternative mechanisms of the antiviral activity of these compounds are discussed.

Calcium↗

PubMed2004

GABRD encoding a protein for extra- or peri-synaptic GABAA receptors is a susceptibility locus for generalized epilepsies.

A major challenge in understanding complex idiopathic generalized epilepsies has been the characterization of their underlying molecular genetic basis. Here, we report that genetic variation within the GABRD gene, which encodes the GABAA receptor delta subunit, affects GABA current amplitude consistent with a model of polygenic susceptibility to epilepsy in humans. We have found a GABRD Glu177Ala variant which is heterozygously associated with generalized epilepsy with febrile seizures plus. We also report an Arg220His allele in GABRD which is present in the general population. Compared with wild-type receptors, alpha1beta2Sdelta GABAA receptors containing delta Glu177Ala or Arg220His have decreased GABAA receptor current amplitudes. As GABAA receptors mediate neuronal inhibition, the reduced receptor current associated with both variants is likely to be associated with increased neuronal excitability. Since delta subunit-containing receptors localize to extra- or peri-synaptic membranes and are thought to be involved in tonic inhibition, our results suggest that alteration of this process may contribute to the common generalized epilepsies.

Amino Acid Substitution↗

PubMed2004

An Arg307 to Gln polymorphism within the ATP-binding site causes loss of function of the human P2X7 receptor.

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by (7)loss-of-function polymorphisms that change Glu(496) to Ala (E496A) and Ile(568) to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg(307) (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X(7) carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X(7) and blocks P2X(7) function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X(7) function in cells heterozygous for the R307Q to a value 10-40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X(7), and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X(7) receptor, and since the affected Arg(307) is homologous to those amino acids essential for ATP binding to P2X(1) and P2X(2), it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X(7).

Adenosine Triphosphate↗

PubMed2003

Glucose transporter GLUT12-functional characterization in Xenopus laevis oocytes.

We have recently identified and cloned the cDNA of a new member of the glucose transporter family that has been designated GLUT12. GLUT12 possesses the structural features critical to facilitative transport of glucose but the key to understanding the possible physiological roles of this novel protein requires analysis of functional glucose transport. In the current study, we have utilized the Xenopus laevis oocyte expression system to assay transport of the glucose analog 2-deoxy-D-glucose and characterize the glucose transport properties and hexose affinities of GLUT12. Our results demonstrate that GLUT12 facilitates transport of glucose with an apparent preferential substrate affinity for glucose over other hexoses assayed. The results are significant to understanding the potential role and importance of GLUT12 in insulin-sensitive tissues and also cells with high glucose utilization such as cancer cells.

Animals↗

PubMed2003

An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X7 receptor.

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X(7) receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into various lymphocyte subsets and was decreased to values of approximately 25% of normal. The expression of the P2X(7) receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X(7) carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-gamma up-regulated P2X(7) function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X(7) receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.

Asparagine↗

PubMed2003

Channelopathies as a genetic cause of epilepsy.

PURPOSE OF REVIEW: This review describes the significant number of new gene associations with epilepsy syndromes that have emerged during the past year, together with additional mutations and new electrophysiological data relating to previously known gene associations. RECENT FINDINGS: Autosomal dominant juvenile myoclonic epilepsy was demonstrated to be a channelopathy associated with a GABA(A) receptor, alpha1 subunit mutation. Benign familial neonatal infantile seizures were delineated as another channelopathy of infancy, by molecular characterization of sodium channel, alpha2 subunit defects. A sodium channel, alpha2 subunit defect was previously found to be associated with generalized epilepsy with febrile seizures plus. Similarly, the clinical spectrum associated with potassium channel, KQT-like mutations was extended to include the channelopathy myokymia and neonatal epilepsy. Mutations in the non-ion channel genes, leucine-rich, glioma inactivated 1 gene and Aristaless related homeobox gene, have emerged as important causes of their specific syndromes, with mutations in the latter gene frequently underlying X-linked mental retardation with epilepsy. SUMMARY: All but one of the idiopathic epilepsies with a known molecular basis are channelopathies. Where the ion channel defects have been identified, however, they generally account for a minority of families and sporadic cases with the syndrome in question. The data suggest that ion channel mutations of large effect are a common cause of rare monogenic idiopathic epilepsies, but are rare causes of common epilepsies. Additive effects of genetic variation, perhaps within the same ion channel gene families, are likely to underlie the common idiopathic generalized epilepsies with complex inheritance. The genetics of epilepsy is progressing rapidly toward a more detailed molecular dissection and definition of syndromes.

Calcium Channels↗

PubMed2002

P2X7 receptor cell surface expression and cytolytic pore formation are regulated by a distal C-terminal region.

The importance of the cytosolic C-terminal region of the P2X7 receptor (P2X7R) is unquestioned, yet little is known about the functional domains of this region and how they may contribute to the numerous properties ascribed to this receptor. A structure-function analysis of truncated and single-residue-mutated P2X7 receptors was performed in HEK-293 cells and Xenopus oocytes. Cells expressing receptors truncated at residue 581 (of 595) have negligible ethidium ion uptake, whereas those expressing the P2X7R truncated at position 582 give wild type ethidium ion uptake suggesting that pore formation requires over 95% of the C-terminal tail. Channel function was evident even in receptors that were truncated at position 380 indicating that only a small portion of the cytosolic region is required for channel activity. Surprisingly, truncations in the region between residues 551 and 581 resulted in non-functional receptors with no detectable cell surface expression in HEK-293 cells. A more detailed analysis revealed that mutations of single residues within this region could also abolish receptor function and cell surface expression, suggesting that this region may participate in regulating the surface expression of the pore-forming P2X7R.

Amino Acid Sequence↗

PubMed2002

Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit mutation [gamma 2(R43Q)] found in human epilepsy.

The gamma-aminobutyric acid type A (GABA(A)) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA(A) receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABA(A) gamma(2)-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABA(A) receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from alpha(1)beta(2)gamma(2) or alpha(1)beta(2)gamma(2)(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imidazopyridine zolpidem. These results provide evidence of impaired GABA(A) receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Amino Acid Substitution↗

PubMed2002

Site of action of fatty acids and other charged lipids on BKCa channels from arterial smooth muscle cells.

Fatty acids and other negatively charged single-chain lipids increase large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel activity, whereas sphingosine and other positively charged single-chain lipids suppress activity. Because these molecules are effective on both inside-out and outside-out patches and because they can flip across the bilayer, the location of their site of action is unclear. To identify the site of action of charged lipids on this channel, we used two compounds that are unlikely to flip across the lipid bilayer. Palmitoyl coenzyme A (PCoA) was used to identify the site of action of negatively charged lipids, and a positively charged myristoylated pentapeptide (myr-KPRPK) was used to investigate the site of action of positively charged lipids. The effect of these compounds on channel activity was studied in excised patches using patch-clamp techniques. In "normal" ionic strength solutions and in experiments where high-ionic strength solutions were used to shield membrane surface charge, PCoA increased channel activity only when applied to outside-out patches, suggesting that the site of action of negatively charged lipids is located on the outer surface of the membrane. A decrease in activity, similar to that of other positively charged lipids, was observed only when myr-KPRPK was applied to outside-out patches, suggesting that positively charged lipids suppress activity by also acting on the outer membrane surface. Some channel blockade effects of myr-KPRPK and KPRPK are also described. The sidedness of action suggests that modulation of channel activity by single-chain lipids can occur by their interaction with the channel protein.

Amines↗

PubMed2002

Fatty acid augmentation of the cardiac slowly activating delayed rectifier current (IKs) is conferred by hminK.

The mechanism by which dietary fatty acids confer protection against cardiac arrhythmias and sudden cardiac death is not resolved. Here, we study the effects of several known cardio-protective and arrhythmogenic fatty acids on the slowly activating delayed rectifier potassium current (IKs), which is responsible for the repolarization phase of the cardiac action potential. cRNAs encoding either or both of the two subunits, KvLQT1 and hminK, that together produce IKs, were injected into Xenopus oocytes, and the effects of various fatty acids were determined. Docosahexaenoic acid (DHA) significantly augmented IKs as did the short-chained fully saturated lauric acid, and to a lesser extent the cis-unsaturated oleic acid. Eicosapentaenoic acid (EPA) was without significant effect on current magnitude, although it reduced the rate of activation. These results suggest that not all "antiarrhythmic" fatty acids target the same channel. To examine the role of hminK in this response, KvLQT1 was expressed alone. In this case, DHA, lauric acid, and oleic acid did not augment current, suggesting that hminK confers fatty acid sensitivity to IKs.

Animals↗

PubMed2002

Release of mitochondrial Ca2+ via the permeability transition activates endoplasmic reticulum Ca2+ uptake.

Regulatory interactions between the endoplasmic reticulum (ER) and the mitochondria in the control of intracellular free Ca2+ concentration ([Ca2+]I), may be of importance in the control of many cell functions, and particularly those involved in initiating cell death. We used targeted Ca2+ sensors (cameleons) to investigate the movement of Ca2+ between the ER and mitochondria of intact cells and focused on the role of the mitochondrial permeability transition (MPT) in this interaction. We hypothesized that release of Ca2+ from mitochondria in response to a known MPT agonist (atractyloside) would cause release of ER Ca2+, perpetuating cellular Ca2+ overload, and cell death. Targeted cameleons (mitochondria and ER) were imaged with confocal microscopy 2-3 days following transient transfection of human embryonic kidney 293 cells. Opening of the MPT resulted in specific loss of mitochondrial Ca2+ (blocked by cyclosporin A), which was sequestered initially by ER. The ER subsequently released this Ca2+ load, leading to a global Ca2+ elevation, a response that was not observed when ER Ca2+-ATPases were blocked with cyclopiazonic acid. Thus, ER plays an important role in moderating changes in intracellular Ca2+ following MPT and may play a key role in cell death initiated by mitochondrial mechanisms.

Atractyloside↗