A Simple RNA Target Capture NGS Strategy for Fusion Genes Assessment in the Diagnostics of Pediatric B-cell Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. Fusion genes are hallmarks of ALL, and they are used as biomarkers for risk stratification as well as targets for precision medicine. Hence, clinical diagnostics pursues broad and comprehensive strategies for accurate discovery of fusion genes. Currently, the gold standard methodologies for fusion gene detection are fluorescence in situ hybridization and polymerase chain reaction; these, however, lack sensitivity for the identification of new fusion genes and breakpoints. In this study, we implemented a simple operating procedure (OP) for detecting fusion genes. The OP employs RNA CaptureSeq, a versatile and effortless next-generation sequencing assay, and an in-house as well as a purpose-built bioinformatics pipeline for the subsequent data analysis. The OP was evaluated on a cohort of 89 B-cell precursor ALL (BCP-ALL) pediatric samples annotated as negative for fusion genes by the standard techniques. The OP confirmed 51 samples as negative for fusion genes, and, more importantly, it identified known (KMT2A rearrangements) as well as new fusion events (JAK2 rearrangements) in the remaining 38 investigated samples, of which 16 fusion genes had prognostic significance. Herein, we describe the OP and its deployment into routine ALL diagnostics, which will allow substantial improvements in both patient risk stratification and precision medicine.

Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABAAR modulation. [(3)H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[(3)H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([(3)H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1ß3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [(3)H]AziPm photoincorporation into GABAAR subunits maximally by ∼ 50%. When the amino acids photolabeled by [(3)H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the ß3-α1 subunit interface (ß3Met-286 in ß3M3 and α1Met-236 in α1M1), previously photolabeled by [(3)H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [(3)H]AziPm photolabeling of ß3Met-227 in ßM1, the amino acid in the α1-ß3 subunit interface photolabeled by R-[(3)H]mTFD-MPAB. The propofol-inhibitable [(3)H]AziPm photolabeling in the GABAAR ß3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the ß3-ß3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABAAR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.

Human α1ß3γ2L gamma-aminobutyric acid type A receptors: High-level production and purification in a functional state.

Gamma-aminobutyric acid type A receptors (GABA(A)Rs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA(A)Rs determine their function and pharmacological profile. GABAA Rs are heteropentamers of subunits, and (α1)2 (ß3)2 (γ2L)1 is a common subtype. Biochemical and biophysical studies of GABA(A)Rs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high-level production of active human α1ß3 GABA(A)R using tetracycline-inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline-inducible HEK293-TetR cell line expressing human (N)-FLAG-α1ß3γ2L-(C)-(GGS)3 GK-1D4 GABA(A)R. These cells achieved expression levels of 70-90 pmol [(3)H]muscimol binding sites/15-cm plate at a specific activity of 15-30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [(3)H]flunitrazepam to [(3)H]muscimol binding sites and sensitivity of GABA-induced currents to benzodiazepines and zinc. The α1ß3γ2L GABA(A)Rs were solubilized in dodecyl-D-maltoside, purified by anti-FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ∼ 30%. Typical purifications yielded 1.0-1.5 nmoles of [(3)H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [(3)H]muscimol binding were maintained in the purified state.

Mapping general anesthetic binding site(s) in human α1ß3 γ-aminobutyric acid type A receptors with [³H]TDBzl-etomidate, a photoreactive etomidate analogue.

The γ-aminobutyric acid type A receptor (GABA(A)R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA(A)Rs purified from bovine brain, [³H]azietomidate photolabeling of αMet-236 and ßMet-286 in the αM1 and ßM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the ß and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABA(A)R etomidate binding sites, we now use [³H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1ß3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [³H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner ß3Val-290, in the ß3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [³H]azietomidate, while no photolabeling of amino acids in the αM2 and ßM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA(A)R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [³H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of ß3Met-227 in ßM1 by [³H]TDBzl-etomidate and [³H]azietomidate also provides evidence of a homologous etomidate binding site at the ß3-ß3 subunit interface in the α1ß3 GABA(A)R.

p-(4-Azipentyl)propofol: a potent photoreactive general anesthetic derivative of propofol.

We synthesized 2,6-diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]phenol (p-(4-azipentyl)propofol), or p-4-AziC5-Pro, a novel photoactivable derivative of the general anesthetic propofol. p-4-AziC5-Pro has an anesthetic potency similar to that of propofol. Like propofol, the compound potentiates inhibitory GABA(A) receptor current responses and allosterically modulates binding to both agonist and benzodiazepine sites, assayed on heterologously expressed GABA(A) receptors. p-4-AziC5-Pro inhibits excitatory current responses of nACh receptors expressed in Xenopus oocytes and photoincorporates into native nACh receptor-enriched Torpedo membranes. Thus, p-4-AziC5-Pro is a functional general anesthetic that both modulates and photoincorporates into Cys-loop ligand-gated ion channels, making it an excellent candidate for use in identifying propofol binding sites.

A common variation in the cannabinoid 1 receptor (CNR1) gene is associated with pre-eclampsia in the Central European population.

OBJECTIVE: Recently it has been proposed that tightly regulated levels of endogenous cannabinoids play a fundamental role in early placental development. The aim of this study was to investigate associations of three single-nucleotide polymorphisms (SNPs) in the cannabinoid 1 receptor (CNR1) gene (rs1049353, rs12720071 and rs806368) and their inferred haplotypes with pre-eclampsia, a severe pregnancy-associated condition characterized by abnormal development and remodeling of spiral decidual arteries. STUDY DESIGN: The case-control study comprised a total of 115 pre-eclamptic women and 145 healthy pregnant controls, all originating from the Central-European Czech population. Using PCR-based methods, we tested rs1049353, rs12720071 and rs806368 in the CNR1 gene and haplotypes were constructed. RESULTS: Statistically significant difference in genotype distributions of rs806368 (p(g)<10(-3)) was observed when comparing the cases and the controls; the cases presenting with significantly lower proportion of CC homozygotes. In multivariate modeling, the rs806368 served as a predictor for pre-eclampsia development (ß=0.15; p=0.04). Haplotype analysis revealed presence of four common haplotypes; the CAA haplotype being less frequent in pre-eclamptic cases compared to the controls (p<0.008). Analysis of regression models confirmed the independent prediction role of AAC haplotype for pre-eclampsia onset (ß=-0.18; p=0.03). CONCLUSION: This is the first study focusing on the relationship between SNPs in the CNR1 gene and pre-eclampsia risk. Although limited by a relatively small sample size, the study indicates that rs806368 in the CNR1 gene may act as a susceptibility marker for pre-eclampsia in humans.

Circulating Levels of Agouti-Related Peptide in Endometrial Cancer Survivors.

BACKGROUND: Recently, it has been reported that central administration of agouti-related peptide (AgRP) might have protective effect against cachexia development in tumor-bearing mice. In this study, we determined whether the disease-free endometrial cancer survivors present with different plasma AgRP levels than controls and whether there was an association with the duration of the disease-free interval. METHODS: The total of 53 endometrial cancer survivors was enrolled in the study along with 93 healthy control women of similar age. Fasting blood samples were obtained and AgRP plasma levels determined using ELISA-based methodology. RESULTS: The AgRP plasma levels were significantly higher in the cases than in the controls; AgRP levels were the lowest in obese control women (77.4 ± 19.8 pg/ml); on the contrary, the AgRP plasma levels were highest in non-obese cancer survivors (100.5 ± 21.12 pg/ml). Moreover, we observed significant differences in AgRP levels between the endometrial cancer survivors and the control subjects [p (for comparison of the cases and the controls) = 0.002]. In the regression modeling, AgRP was significantly associated with the BMI as well as the case-control status, and the case-control differences in AgRP levels retained their statistical significance also after adjustment for BMI. CONCLUSIONS: Disease-free endometrial cancer survivors who did not develop cachexia during their treatment as well as post-treatment period present with significantly higher AgRP levels than the control population, independently on their BMI and menopausal status which could be indicative of the protective effect of circulating AgRP against cachexia development in endometrial cancer.

p-Trifluoromethyldiazirinyl-etomidate: a potent photoreactive general anesthetic derivative of etomidate that is selective for ligand-gated cationic ion channels.

We synthesized the R- and S-enantiomers of ethyl 1-(1-(4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazole-5-carboxylate (trifluoromethyldiazirinyl-etomidate), or TFD-etomidate, a novel photoactivable derivative of the stereoselective general anesthetic etomidate (R-(2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate)). Anesthetic potency was similar to etomidate's, but stereoselectivity was reversed and attenuated. Relative to etomidate, TFD-etomidate was a more potent inhibitor of the excitatory receptors, nAChR (nicotinic acetylcholine receptor) ((alpha1)(2)beta1delta1gamma1) and 5-HT(3A)R (serotonin type 3A receptor), causing significant inhibition at anesthetic concentrations. S- but not R-TFD-etomidate enhanced currents elicited from inhibitory alpha1beta2gamma2L GABA(A)Rs by low concentrations of GABA, but with a lower efficacy than R-etomidate, and site-directed mutagenesis suggests they act at different sites. [(3)H]TFD-etomidate photolabeled the alpha-subunit of the nAChR in a manner allosterically regulated by agonists and noncompetitive inhibitors. TFD-etomidate's novel pharmacology is unlike that of etomidate derivatives with photoactivable groups in the ester position, which behave like etomidate, suggesting that it will further enhance our understanding of anesthetic mechanisms.

High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors.

The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABA(A) receptors containing α1 and ß3 subunits could be expressed with specific activities of 29-34 pmol/mg corresponding to 140-170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT(3A)) receptors were 49-63 pmol/mg and 245-315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3-3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one step after solubilization using ANTI-FLAG affinity chromatography with yields of 30-40%. Purified receptors were functional. Binding of the agonist [(3)H]muscimol to the purified GABA(A)R was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles.

Comparison of agouti-related peptide levels in peripheral blood of postpartum pre-eclamptic and non pre-eclamptic women and in umbilical cord blood from their pregnancies.

Plasma levels of agouti-related peptide (AgRP) were reported to continuously rise during ongoing pregnancy in rats. The aim of the study was to investigate the maternal pre-partum peripheral plasma levels of AgRP and levels in umbilical cord blood in pre-eclamptic and physiological pregnancies in humans.

Is there any link between severe pre-eclampsia and defined polymorphisms in leptin and adiponectin genes?

AIM: The pathophysiology of pre-eclampsia, one of the leading causes of maternal mortality worldwide, still remains unclear. Recently, it has been suggested that impaired regulation of complex interactions among various adipokines plays an important role in the development of pre-eclampsia. The aim of this study was to investigate whether the two common polymorphisms of the leptin (LEP) and adiponectin (APM1) genes are associated with the development of pre-eclampsia and its related traits (gestational hypertension, proteinuria and various measures of reduced fetal growth ) in the Czech pre-eclamptic population. METHODS: The case-control study comprised a total of 123 pre-eclamptic women and 150 healthy controls of similar age and parity distribution. They were genotyped for the LEP -2548G/A (5'-untranslated region) and APM1 T94G (exon 2) polymorphisms using polymerase chain reaction. RESULTS: The allele frequency of the LEP -2548G polymorphism was 0.541 in the pre-eclamptic group versus 0.583 in the control group (P=0.578); the frequency of the APM1 94G polymorphism was 0.073 and 0.079 (P=0.628), respectively. No significant associations were detected between either of the two single nucleotide polymorphisms or any of the parameter biomarkers related to pre-eclampsia, such as gestational hypertension or proteinuria. However, the APM1 T94G polymorphism was significantly associated with a low birth weight in pre-eclamptic pregnancies, with mothers carrying the T-allele having an almost three-fold increase in the likelihood of giving birth to a child with a low birth weight for its gestational age (odds ratio, 2.7; 95% confidence interval, 0.18-5.9; P=0.004). CONCLUSIONS: The APM1 T94G and LEP -2548G/A polymorphisms do not seem to be major genetic determinants of susceptibility to pre-eclampsia in the Czech Caucasian population. However, evidence has been provided for possible APM1 T94G involvement in controlling the birth weight of children from pre-eclamptic pregnancies, thus supporting the hypothesis of T94G involvement in controlling the birth weight of newborns.

Identification of 2.3-Mb gene locus for congenital aural atresia in 18q22.3 deletion: a case report analyzed by comparative genomic hybridization.

OBJECTIVE: 18q deletion syndrome is a multiple-anomaly mental retardation syndrome associated with congenital aural atresia. The purpose of this study was to determine the frequency of the congenital aural atresia phenotype in 18q deletion syndrome patients and to delineate a potential critical region for congenital aural atresia at the 18q22.3-18q23 region. STUDY DESIGN AND PATIENTS: The study describes one 18q deletion syndrome clinical report (Patient 15) with an overview of 19 other selected 18q deletion syndrome patients presenting congenital aural atresia from 18 published articles and one presented poster on 18q deletion syndrome. RESULTS: Our investigation, together with the results of published 18q deletion syndrome reports, shows that the average frequency of congenital aural atresia is approximately 52%. A combination of three 18q deletion syndrome probands defines a chromosomal deletion site for congenital aural atresia at 18q22.3-18q23 in the region between markers D18S489 and D18S554. These polymorphic markers outline a putative critical interval of approximately 2.3 Mb, including the genes ZNF407, ZADH2, SDCCAG33, ZNF516, FLJ44881, ZNF236, MBP-Golli, and GALR1. The haploinsufficiency of these genes is suggested to be a primary cause of congenital aural atresia phenotype in 18q deletion syndrome individuals. CONCLUSION: Congenital aural atresia is a relevant diagnostic clue and a major recognizable feature of 18q deletion syndrome. Early diagnosis of 18q deletion syndrome may enable application of hearing aids. Knockout studies on the congenital aural atresia mouse gene homolog may add further insight into the genes responsible for this condition.

Association of leptin genetic polymorphism -2548 G/A with gestational diabetes mellitus.

The aim of this study was to investigate possible associations of -2548 G/A polymorphism in leptin gene promoter and pregnancy-associated diseases with abnormal fetal growth such as preeclampsia and gestational diabetes. The study was also focused on whether it is rather maternal or fetal variants that determines the pathological growth status. Peripheral or cord blood samples obtained from 49 preeclamptic women and their 39 newborns, 53 healthy controls and their 53 healthy newborns and 48 patients with gestational diabetes mellitus were evaluated for leptin gene (LEP) locus -2548 genotypes. The significantly higher risk for gestational diabetes mellitus was observed in the presence of an allele (AA and AG genotypes) against carriers of GGgenotype(OR=2.84, 95%CI1.14-7.07,p=0.02). Thereisa significant risk of diabetes mellitus associated to A allele (OR=1.79, 95%CI 1.02-3.14, p=0.03). Furthermore, evaluations of preeclamptic patients' data revealed a significant association of genotype distribution and delivery and spontaneous abortion rate, where the GG carriers performed the highest pregnancy rate while the AG carriers performed the lowest spontaneous abortion rate. Our results support the hypothesis for -2548 G/A leptin gene polymorphism involvement in ethiopathogenesis of pregnancy-associated diseases with abnormal fetal growth, especially gestational diabetes mellitus.