Prospective evaluation of NGS-based sequencing in epilepsy patients: results of seven NASGE-associated diagnostic laboratories.

Background: Epilepsy is one of the most common and disabling neurological disorders. It is highly prevalent in children with neurodevelopmental delay and syndromic diseases. However, epilepsy can also be the only disease-determining symptom. The exact molecular diagnosis is essential to determine prognosis, comorbidity, and probability of recurrence, and to inform therapeutic decisions. Methods and materials: Here, we describe a prospective cohort study of patients with epilepsy evaluated in seven diagnostic outpatient centers in Germany. Over a period of 2 months, 07/2022 through 08/2022, 304 patients (317 returned result) with seizure-related human phenotype ontology (HPO) were analyzed. Evaluated data included molecular results, phenotype (syndromic and non-syndromic), and sequencing methods. Results: Single exome sequencing (SE) was applied in half of all patients, followed by panel (P) testing (36%) and trio exome sequencing (TE) (14%). Overall, a pathogenic variant (PV) (ACMG cl. 4/5) was identified in 22%; furthermore, a significant number of patients (12%) carried a reported clinically meaningful variant of unknown significance (VUS). The average diagnostic yield in patients ≤ 12 y was higher compared to patients >12 y cf. Figure 2B vs. Figure 3B. This effect was more pronounced in cases, where TE was applied in patients ≤ 12 vs. >12 y [PV (PV + VUS): patients ≤ 12 y: 35% (47%), patients > 12 y: 20% (40%)]. The highest diagnostic yield was achieved by TE in syndromic patients within the age group ≤ 12 y (ACMG classes 4/5 40%). In addition, TE vs. SE had a tendency to result in less VUS in patients ≤ 12 y [SE: 19% (22/117) VUS; TE: 17% (6/36) VUS] but not in patients >12 y [SE: 19% (8/42) VUS; TE: 20% (2/10) VUS]. Finally, diagnostic findings in patients with syndromic vs. non-syndromic symptoms revealed a significant overlap of frequent causes of monogenic epilepsies, including SCN1A, CACNA1A, and SETD1B, confirming the heterogeneity of the associated conditions. Conclusion: In patients with seizures-regardless of the detailed phenotype-a monogenic cause can be frequently identified, often implying a possible change in therapeutic action (36.7% (37/109) of PV/VUS variants); this justifies early and broad application of genetic testing. Our data suggest that the diagnostic yield is highest in exome or trio-exome-based testing, resulting in a molecular diagnosis within 3 weeks, with profound implications for therapeutic strategies and for counseling families and patients regarding prognosis and recurrence risk.

An intronic splice site alteration in combination with a large deletion affecting VPS13B (COH1) causes Cohen syndrome.

Cohen syndrome (CS) is a rare, autosomal recessive disorder characterized by intellectual disability, postnatal microcephaly, facial abnormalities, abnormal truncal fat distribution, myopia, and pigmentary retinopathy. It is often considered an underdiagnosed condition, especially in children with developmental delay and intellectual disability. Here we report on four individuals from a large Jordanian family clinically diagnosed with CS. Using Trio Exome Sequencing (Trio-WES) and MLPA analyses we identified a maternally inherited novel intronic nucleotide substitution c.3446-23T>G leading to the activation of a cryptic splice site and a paternally inherited multi-exon deletion in VPS13B (previously termed COH1) in the index patient. Expression analysis showed a strong decrease of VPS13B mRNA levels and direct sequencing of cDNA confirmed splicing at a cryptic upstream splice acceptor site, resulting in the inclusion of 22 intronic bases. This extension results in a frameshift and a premature stop of translation (p.Gly1149Valfs*9). Segregation analysis revealed that three affected maternal cousins were homozygous for the intronic splice site variant. Our data show causality of both alterations and strongly suggest the expansion of the diagnostic strategy to search for intronic splice variants in molecularly unconfirmed patients affected by CS.

Novel MECP2 Mutation c.1162_1172del; p.Pro388* in Two Patients with Symptoms of Atypical Rett Syndrome.

We report 2 cases of girls with MECP2 gene variants who do not have typical clinical features of Rett syndrome except for intellectual disability and seizures. Both patients present with adipositas, macrocephalia, precocious puberty, and seizures. They have prominent eyebrows and a short neck as well as short and plump fingers. Sequencing by NGS revealed a novel variant c.1162_1172del; p.Pro388* in both patients.

Mucosa of human detrusor impairs contraction and ß-adrenoceptor-mediated relaxation.

OBJECTIVES: To elucidate the impact of the mucosa on detrusor muscle function by investigating force of contraction under various stimulatory conditions and during subsequent relaxation using catecholamines. PATIENTS AND METHODS: Detrusor tissue was obtained from patients who had undergone cystectomy for bladder cancer and strips of intact or mucosa-denuded muscle were set up for force measurement. Preparations were precontracted with KCl, carbachol or electric-field stimulation (EFS). Precontracted strips were relaxed using increasing concentrations of catecholamines in the absence and presence of the subtype-selective ß-adrenoceptor (AR) blockers CGP 20712A (ß1-ARs), ICI 118,551 (ß2-ARs), and L-748,337 (ß3-ARs). RESULTS: Force development in response to KCl (40 mM), carbachol (1 µM) or EFS was larger in the absence of mucosa than in intact muscle strips. The force of contraction of mucosa-denuded strips with detached urothelium incubated in the same chamber was as low as in intact strips. Noradrenaline relaxed precontracted detrusor strips to a significantly larger extent and at lower concentrations in denuded than in intact strips. CGP 20712A did not affect noradrenaline-induced relaxation of denuded and intact strips, and ICI 118,551 did not influence noradrenaline-induced relaxation in denuded strips, but abolished the difference between denuded and intact strips. The antagonism of the relaxant effects of noradrenaline by L-748,337 was slightly smaller in intact than denuded strips. CONCLUSIONS: The mucosa of human detrusor strips impairs force development when stimulated with KCl, carbachol or EFS. The mucosa also blunts the relaxing effects of catecholamines. The latter effect does not involve the activation of ß1-ARs but only of ß2-ARs, whereas ß3-ARs mediate the relaxation of human detrusor.

Impaired vascular function in small resistance arteries of LOX-1 overexpressing mice on high-fat diet.

AIMS: LOX-1 is a major vascular receptor for oxidized low-density lipoprotein (oxLDL). In this study, we analysed the impact of LOX-1 overexpression and high dietary fat intake on vascular function in small resistance arteries. METHODS AND RESULTS: Relaxation of mesenteric arteries was measured using a wire myograph. Compared with the control group, mice overexpressing LOX-1 on a high-fat diet (FD) had preserved vascular smooth muscle relaxation, but impaired endothelium-dependent relaxation via NO. Vascular NO availability was decreased by exaggerated formation of reactive oxygen species and decreased endothelial NO synthase expression. Endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation via cytochrome P450 metabolites was increased in LOX-1 + FD animals, but did not completely compensate for the loss of NO. Currents of calcium-activated potassium channels with large conductance (BKCa channels) were measured by the voltage-clamp method. The BKCa current amplitudes were not altered in endothelial cells, but highly increased in vascular smooth muscle cells from resistance arteries of LOX-1-overexpressing mice on FD. BK(Ca) currents were activated by low-dose H2O2 and cytochrome P450 metabolites 11,12-EET and 14,15-EET as EDHF in control mice. CONCLUSION: LOX-1 overexpression and FD caused functional changes in endothelial and vascular smooth muscle cells of small resistance arteries.

Catecholamines relax detrusor through beta 2-adrenoceptors in mouse and beta 3-adrenoceptors in man.

(-)-Isoproterenol [4-[1-hydroxy-2-[(1-methylethyl)amino]ethyl]-1,2-benzene diol hydrochloride] relaxes murine detrusor through beta-adrenoceptors (ARs); however, the beta-AR subtypes involved are unknown. beta(2)-ARs have been associated with caveolae, plasma-lemmal scaffolding domains that are absent in caveolin-1 (cav-1) knockout (KO) mice. Here, we studied detrusor responses in the absence and presence of beta-AR subtype-selective antagonists in wild-type (WT) and cav-1 KO mice. To inquire whether the murine detrusor model is relevant to man, beta-AR subtypes that mediate (-)-isoproterenol-evoked human detrusor relaxation were investigated. In WT mice, (-)-isoproterenol concentration-dependently relaxed the KCl (40 mM)-precontracted detrusor (-logEC(50)M = 8.04, E(max) = 62%). The effects of (-)-isoproterenol were surmountably antagonized by the beta(2)-AR-selective antagonist ICI 118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] (pK(B) = 9.28) but not affected by the beta(1)-AR-selective antagonist CGP 20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol] and beta(3)-AR-selective L-748,337 [(S)-M-[4-[2-[3-[3-[acetamidomethyl)phenoxy)-2-hydroxypropyl]-amino]-ethyl]-phenylbenzsulfonamide)], suggesting involvement of beta(2)-AR only. The cav-1 KO detrusor displayed significant contractile dysfunction. (-)-Isoproterenol was less potent and efficient in relaxing detrusor from cav-1 KO (-logEC(50)M, 7.76; E(max) = 44%), but ICI 118,551 caused similar antagonism (pK(B) = 9.15), suggesting that beta(2)-AR function persisted in cav-1 KO. The beta(3)-AR-selective antagonist L-748,337 in the presence of ICI 118,551 and CGP 20712 caused additional blockade of (-)-isoproterenol effects in cav-1 KO, consistent with a beta(3)-AR involvement during relaxation and suppression of this effect in WT. (-)-Isoproterenol relaxed human detrusor muscle precontracted with carbachol (-logEC(50)M = 6.39, E(max) = 52%). However, the effects of (-)-isoproterenol in human detrusor were not blocked by CGP 20712 or ICI 118,551 but antagonized by L-748,337 (pK(B) = 7.65). We conclude that murine detrusor relaxation occurs via beta(2)-AR, and loss of caveolae does not perturb beta(2)-AR function but unmasks an additional activation of beta(3)-AR. In contrast, detrusor relaxation in man is mediated exclusively via beta(3)-AR.

Muscarinic receptor expression and receptor-mediated detrusor contraction: comparison of juvenile and adult porcine tissue.

Urinary bladder function is known to mature during fetal and postnatal development, including changes in neurotransmitter regulation of detrusor contraction. However, only few experimental data are available about muscarinic receptor antagonist function in the urinary bladder from young animals. In the present study, we compare the muscarinic receptor-mediated contractions in juvenile and adult porcine detrusor and the effects of antimuscarinic compounds. Urinary bladders from young (8-12 weeks; 12- to 35-kg body weight) and mature pigs (>40 weeks; >100 kg) were compared. Muscarinic receptor expression was assessed by real time polymerase chain reaction and radioligand binding. Muscle contraction was measured with a force transducer; L-type Ca2+ currents (ICa,L) of isolated detrusor myocytes were recorded with standard voltage clamp technique. Juvenile and adult detrusor expressed similar quantities of the messenger RNA of M2 and M3 receptors. The number of [3H]QNB-binding sites and their affinity for the radioligand were also similar between juvenile and adult detrusor. In contrast, maximum contractile responses to the muscarinic receptor agonist carbachol were slightly larger in juvenile than adult bladders. On the other hand, carbachol was slightly less potent in juvenile than in adult tissue. The M3 antagonist DAU 5884 and the spasmolytic drug propiverine inhibited contractile responses with comparable efficacies and potencies in juvenile and adult tissue. ICa,L was somewhat smaller in juvenile than in adult cells. Taken together, these data suggest that expression and function of M2 and M3 receptors are similar in the detrusor of juvenile and mature pigs. Therefore, similar responses to antimuscarinic compounds could be expected in young and adult patients.

Vascular large conductance calcium-activated potassium channels: functional role and therapeutic potential.

Large-conductance Ca2+-activated K+ channels (BK Ca or maxiK channels) are expressed in different cell types. They play an essential role in the regulation of various cell functions. In particular, BK Ca channels have been extensively studied in vascular smooth muscle cells, where they contribute to the control of vascular tone. They facilitate the feedback regulation against the rise of intracellular Ca2+, membrane depolarization and vasoconstriction. BK Ca channels promote a K+ outward current and lead to membrane hyperpolarization. In endothelial cells expression and function of BK Ca channels play an important role in the regulation of the vascular smooth muscle activity. Endothelial BK Ca channels modulate the biosyntheses and release of various vasoactive modulators and regulate the membrane potential. Because of their regulatory role in vascular tone, endothelial BK Ca channels have been suggested as therapeutic targets for the treatment of cardiovascular diseases. Hypertension, atherosclerosis, and diabetes are associated with altered current amplitude, open probability, and Ca2+-sensing of BK Ca channels. The properties of BK Ca channels and their role in endothelial and vascular smooth muscle cells would address them as potential therapeutic targets. Further studies are necessary to identify the detailed molecular mechanisms of action and to investigate selective BK Ca channels openers as possible therapeutic agents for clinical use.