Genome-wide analysis implicates microRNAs and their target genes in the development of bipolar disorder.

Bipolar disorder (BD) is a severe and highly heritable neuropsychiatric disorder with a lifetime prevalence of 1%. Molecular genetic studies have identified the first BD susceptibility genes. However, the disease pathways remain largely unknown. Accumulating evidence suggests that microRNAs, a class of small noncoding RNAs, contribute to basic mechanisms underlying brain development and plasticity, suggesting their possible involvement in the pathogenesis of several psychiatric disorders, including BD. In the present study, gene-based analyses were performed for all known autosomal microRNAs using the largest genome-wide association data set of BD to date (9747 patients and 14 278 controls). Associated and brain-expressed microRNAs were then investigated in target gene and pathway analyses. Functional analyses of miR-499 and miR-708 were performed in rat hippocampal neurons. Ninety-eight of the six hundred nine investigated microRNAs showed nominally significant P-values, suggesting that BD-associated microRNAs might be enriched within known microRNA loci. After correction for multiple testing, nine microRNAs showed a significant association with BD. The most promising were miR-499, miR-708 and miR-1908. Target gene and pathway analyses revealed 18 significant canonical pathways, including brain development and neuron projection. For miR-499, four Bonferroni-corrected significant target genes were identified, including the genome-wide risk gene for psychiatric disorder CACNB2. First results of functional analyses in rat hippocampal neurons neither revealed nor excluded a major contribution of miR-499 or miR-708 to dendritic spine morphogenesis. The present results suggest that research is warranted to elucidate the precise involvement of microRNAs and their downstream pathways in BD.

The KMO allele encoding Arg452 is associated with psychotic features in bipolar disorder type 1, and with increased CSF KYNA level and reduced KMO expression.

The kynurenine pathway metabolite kynurenic acid (KYNA), modulating glutamatergic and cholinergic neurotransmission, is increased in cerebrospinal fluid (CSF) of patients with schizophrenia or bipolar disorder type 1 with psychotic features. KYNA production is critically dependent on kynurenine 3-monooxygenase (KMO). KMO mRNA levels and activity in prefrontal cortex (PFC) are reduced in schizophrenia. We hypothesized that KMO expression in PFC would be reduced in bipolar disorder with psychotic features and that a functional genetic variant of KMO would associate with this disease, CSF KYNA level and KMO expression. KMO mRNA levels were reduced in PFC of bipolar disorder patients with lifetime psychotic features (P=0.005, n=19) or schizophrenia (P=0.02, n=36) compared with nonpsychotic patients and controls. KMO genetic association to psychotic features in bipolar disorder type 1 was studied in 493 patients and 1044 controls from Sweden. The KMO Arg(452) allele was associated with psychotic features during manic episodes (P=0.003). KMO Arg(452) was studied for association to CSF KYNA levels in an independent sample of 55 Swedish patients, and to KMO expression in 717 lymphoblastoid cell lines and 138 hippocampal biopsies. KMO Arg(452) associated with increased levels of CSF KYNA (P=0.03) and reduced lymphoblastoid and hippocampal KMO expression (P≤0.05). Thus, findings from five independent cohorts suggest that genetic variation in KMO influences the risk for psychotic features in mania of bipolar disorder patients. This provides a possible mechanism for the previous findings of elevated CSF KYNA levels in those bipolar patients with lifetime psychotic features and positive association between KYNA levels and number of manic episodes.

Duplications in RB1CC1 are associated with schizophrenia; identification in large European sample sets.

Schizophrenia (SCZ) is a severe and debilitating neuropsychiatric disorder with an estimated heritability of ~80%. Recently, de novo mutations, identified by next-generation sequencing (NGS) technology, have been suggested to contribute to the risk of developing SCZ. Although these studies show an overall excess of de novo mutations among patients compared with controls, it is not easy to pinpoint specific genes hit by de novo mutations as actually involved in the disease process. Importantly, support for a specific gene can be provided by the identification of additional alterations in several independent patients. We took advantage of existing genome-wide single-nucleotide polymorphism data sets to screen for deletions or duplications (copy number variations, CNVs) in genes previously implicated by NGS studies. Our approach was based on the observation that CNVs constitute part of the mutational spectrum in many human disease-associated genes. In a discovery step, we investigated whether CNVs in 55 candidate genes, suggested from NGS studies, were more frequent among 1637 patients compared with 1627 controls. Duplications in RB1CC1 were overrepresented among patients. This finding was followed-up in large, independent European sample sets. In the combined analysis, totaling 8461 patients and 112 871 controls, duplications in RB1CC1 were found to be associated with SCZ (P=1.29 × 10(-5); odds ratio=8.58). Our study provides evidence for rare duplications in RB1CC1 as a risk factor for SCZ.

Association between genetic variation in a region on chromosome 11 and schizophrenia in large samples from Europe.

Recent molecular studies have implicated common alleles of small to moderate effect and rare alleles with larger effect sizes in the genetic architecture of schizophrenia (SCZ). It is expected that the reliable detection of risk variants with very small effect sizes can only be achieved through the recruitment of very large samples of patients and controls (that is tens of thousands), or large, potentially more homogeneous samples that have been recruited from confined geographical areas using identical diagnostic criteria. Applying the latter strategy, we performed a genome-wide association study (GWAS) of 1169 clinically well characterized and ethnically homogeneous SCZ patients from a confined area of Western Europe (464 from Germany, 705 from The Netherlands) and 3714 ethnically matched controls (1272 and 2442, respectively). In a subsequent follow-up study of our top GWAS results, we included an additional 2569 SCZ patients and 4088 controls (from Germany, The Netherlands and Denmark). Genetic variation in a region on chromosome 11 that contains the candidate genes AMBRA1, DGKZ, CHRM4 and MDK was significantly associated with SCZ in the combined sample (n=11 540; P=3.89 × 10(-9), odds ratio (OR)=1.25). This finding was replicated in 23 206 independent samples of European ancestry (P=0.0029, OR=1.11). In a subsequent imaging genetics study, healthy carriers of the risk allele exhibited altered activation in the cingulate cortex during a cognitive control task. The area of interest is a critical interface between emotion regulation and cognition that is structurally and functionally abnormal in SCZ and bipolar disorder.

Genome-wide survey implicates the influence of copy number variants (CNVs) in the development of early-onset bipolar disorder.

We used genome-wide single nucleotide polymorphism (SNP) data to search for the presence of copy number variants (CNVs) in 882 patients with bipolar disorder (BD) and 872 population-based controls. A total of 291 (33%) patients had an early age-at-onset < or =21 years (AO < or =21 years). We systematically filtered for CNVs that cover at least 30 consecutive SNPs and which directly affect at least one RefSeq gene. We tested whether (a) the genome-wide burden of these filtered CNVs differed between patients and controls and whether (b) the frequency of specific CNVs differed between patients and controls. Genome-wide burden analyses revealed that the frequency and size of CNVs did not differ substantially between the total samples of BD patients and controls. However, separate analysis of patients with AO < or =21 years and AO>21 years showed that the frequency of microduplications was significantly higher (P=0.0004) and the average size of singleton microdeletions was significantly larger (P=0.0056) in patients with AO < or =21 years compared with controls. A search for specific BD-associated CNVs identified two common CNVs: (a) a 160 kb microduplication on 10q11 was overrepresented in AO < or = 21 years patients (9.62%) compared with controls (3.67%, P=0.0005) and (b) a 248 kb microduplication on 6q27 was overrepresented in the AO< or = 21 years subgroup (5.84%) compared with controls (2.52%, P=0.0039). These data suggest that CNVs have an influence on the development of early-onset, but not later-onset BD. Our study provides further support for previous hypotheses of an etiological difference between early-onset and later-onset BD.

Susceptibility variants on chromosome 7p21.1 suggest HDAC9 as a new candidate gene for male-pattern baldness.

BACKGROUND: Male-pattern baldness (androgenetic alopecia, AGA) is the most common form of hair loss among humans. Research has shown that it is caused by genetic factors. Numerous studies have unequivocally identified two major genetic risk loci for AGA: the X-chromosomal AR/EDA2R locus, and the PAX1/FOXA2 locus on chromosome 20. OBJECTIVES: To identify further candidate genes for AGA, and thus gain further insights into this phenotype. METHODS: A German sample of 581 severely affected cases and 617 controls was used to perform a genome-wide association study. The identified associated locus was further analysed by fine-mapping, and then independently replicated in an Australian sample. Expression and pathway analyses were performed to characterize the susceptibility gene identified. RESULTS: The most significant association signal was obtained for rs756853 (P = 1·64 × 10(-7) ), which is located intronically in the histone deacetylase 9 (HDAC9) gene. Fine-mapping and a family-based analysis revealed that rs756853 and the 6-kb distal rs2249817 were the most highly associated single nucleotide polymorphisms. The association finding was replicated in an independent Australian sample, when the analysis was restricted to severely affected cases and unaffected controls (P = 0·026). Analysis of rs2249817 in a combined sample of severely affected German and Australian cases and unaffected controls revealed a strong association signal (P = 9·09 × 10(-8) ). Tissue expression studies demonstrated HDAC9 expression in various tissues, including tissues of relevance to AGA. No strong genotypic effects were observed in genotype-specific expression or splice studies. Pathway analyses supported the hypothesis that HDAC9 plays a functional role in AGA via interaction with the AR gene. CONCLUSIONS: The present study suggests that HDAC9 is the third AGA susceptibility gene.

The ultrarapid and the transient outward K(+) current in human atrial fibrillation. Their possible role in postoperative atrial fibrillation.

Atrial fibrillation (AF) causes distinct changes in atrial conduction, characterized as electrical remodeling. Experimental data on the possible significance of alterations of specific K(+)outward currents in this process are still limited in human AF. The ultra-rapid delayed rectifier current (I(Kur)) has not been studied in AF with respect to its sensitivity to 4-Aminopyridine (4-AP). To clarify the role of (1) the 4-AP sensitive I(Kur)current, compared to recordings without using 4-AP (I(Kur*)), and (2) the transient outward current (I(to)) in changes of atrial repolarization associated with AF, whole cell voltage-clamp recordings were obtained from atrial myocytes of patients undergoing elective cardiac surgery, with and without a history of atrial fibrillation (AF/non-AF). Further, a possible relation between experimental data and postoperative AF was studied. In AF patients, I(Kur*)was reduced by 40% [5.00+/-0.32 pA/pF (non-AF) and 2.91+/-0. 45 pA/pF (AF) at +50 mV, P<0.0001, n=22/11], I(Kur)by 55% [3.81+/-0. 30 pA/pF (non-AF) and 1.71+/-0.20 pA/pF (AF) at +50 mV, P<0.0001, n=22/11]. The mean amplitude of I(Kur)was significantly smaller than I(Kur*). Consistently, I(to)was reduced by 44% [11.57+/-0.77 pA/pF (non-AF) and 6.51+/-1.31 pA/pF (AF), P<0.01, n=25/11]. In 48% of non-AF patients, postoperative AF was detected. The corresponding voltage-clamp recordings showed a trend to reduced I(Kur*)and I(Kur)currents, although it did not reach statistical significance. The consistent reduction of all three K(+)currents investigated due to the presence of AF indicates an important association of abnormalities in cellular repolarization with the onset and the self-sustaining nature of human AF.

[Chylothorax as a postoperative complication of aortocoronary bypass operation]. / Der Chylothorax als postoperative Komplikation einer aortokoronaren Bypassoperation.

HISTORY AND CLINICAL FINDINGS: One month after a coronary bypass grafting operation, pericardial- und pleural effusions were found in a 75-year-old woman. Dressler-syndrome was assumed and an antiphlogistic and cortisone were prescribed. Under this therapy, the pericardial effusion disappeared, but the pleural effusion increased in size. After thoracocentesis, the diagnosis of a chylothorax could be confirmed. Three days later, the thoracic radiography was inconspicuous and the patient was discharged without changes in medication. As dyspnea occurred two weeks later, the patient was admitted to our hospital. INVESTIGATIONS: The thoracic radiography showed a reappearance of the pleural effusion. DIAGNOSIS, THERAPY AND COURSE: The laboratory test confirmed a relapse of the chylothorax. After a pleurodesis by drainage and a prescription of a high-caloric, fat-reduced diet the patient could be discharged two weeks later. The diet could be terminated two months later after a recurrence of the chylothorax had been excluded in the thoracic radiography. In the follow-up examinations, there was no evidence for a relapse of the chylothorax. CONCLUSIONS: The chylothorax is an uncommon postoperative complication of the aortocoronary bypass surgery. A relapse is likely and it can result in life-threatening cachexia. Therefore, a early and adequate therapy is important.

Cell swelling causes the action potential duration to shorten in guinea-pig ventricular myocytes by activating IKATP.

In guinea-pig ventricular myocytes, cell swelling by incubation in hypotonic solution caused a pronounced shortening of the action potential duration (APD90: 15.5+/-14.6% compared to control; mean +/- SD) after a latency of 12 min when the intracellular ATP concentration was 2 mM. This shortening was partially reversible within 10 min after reperfusion with isotonic solution (APD90: 80. 5+/-12.1% compared to control). With 5 mM intracellular ATP in the pipette electrode, the effect of cell swelling on the action potential was significantly reduced. Incubation with 1 microM glibenclamide, a blocker of the ATP-dependent K+ current (IKATP), abolished the swelling-induced shortening of the action potential duration, whereas incubation with 0.5 mM 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), a blocker of the swelling-induced Cl- current (ICl,swell), had no effect on the action potential duration in hypotonic solution. Simultaneous measurements of membrane currents substantiate that IKATP is the current that underlies this effect. These results suggest that in the ischaemic myocardium IKATP may be partially activated by cell swelling, resulting in a shortening of the action potential duration before the intracellular ATP concentration has fallen below 2 mM.

Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle.

BACKGROUND: The role of the L-type calcium channel in human heart failure is unclear, on the basis of previous whole-cell recordings. METHODS AND RESULTS: We investigated the properties of L-type calcium channels in left ventricular myocytes isolated from nonfailing donor hearts (n= 16 cells) or failing hearts of transplant recipients with dilated (n=9) or ischemic (n=7) cardiomyopathy. The single-channel recording technique was used (70 mmol/L Ba2+). Peak average currents were significantly enhanced in heart failure (38.2+/-9.3 fA) versus nonfailing control hearts (13.2+/-4.5 fA, P=0.02) because of an elevation of channel availability (55.9+/-6.7% versus 26.4+/-5.3%, P=0.001) and open probability within active sweeps (7.36+/-1.51% versus 3.18+/-1.33%, P=0.04). These differences closely resembled the effects of a cAMP-dependent stimulation with 8-Br-cAMP (n= 11). Kinetic analysis of the slow gating shows that channels from failing hearts remain available for a longer time, suggesting a defect in the dephosphorylation. Indeed, the phosphatase inhibitor okadaic acid was unable to stimulate channel activity in myocytes from failing hearts (n=5). Expression of calcium channel subunits was measured by Northern blot analysis. Expression of alpha1c- and beta-subunits was unaltered. Whole-cell current measurements did not reveal an increase of current density in heart failure. CONCLUSIONS: Individual L-type calcium channels are fundamentally affected in severe human heart failure. This is probably important for the impairment of cardiac excitation-contraction coupling.

Simulation study of cellular electric properties in heart failure.

Patients with severe heart failure are at high risk of sudden cardiac death. In the majority of these patients, sudden cardiac death is thought to be due to ventricular tachyarrhythmias. Alterations of the electric properties of single myocytes in heart failure may favor the occurrence of ventricular arrhythmias in these patients by inducing early or delayed afterdepolarizations. Mathematical models of the cellular action potential and its underlying ionic currents could help to elucidate possible arrhythmogenic mechanisms on a cellular level. In the present study, selected ionic currents based on human data are incorporated into a model of the ventricular action potential for the purpose of studying the cellular electrophysiological consequences of heart failure. Ionic currents that are not yet sufficiently characterized in human ventricular myocytes are adopted from the action potential model developed by Luo and Rudy (LR model). The main results obtained from this model are as follows: The action potential in ventricular myocytes from failing hearts is longer than in nonfailing control hearts. The major underlying mechanisms for this prolongation are the enhanced activity of the Na+-Ca2+ exchanger, the slowed diastolic decay of the [Ca2+]i transient, and the reduction of the inwardly rectifying K+ current and the Na+-K+ pump current in myocytes of failing hearts. Furthermore, the fast and slow components of the delayed rectifier K+ current (I(Kr) and I(Ks), respectively) are of utmost importance in determining repolarization of the human ventricular action potential. In contrast, the influence of the transient outward K+ current on APD is only small in both cell groups. Inhibition of I(Kr) promotes the development of early afterdepolarizations in failing, but not nonfailing, myocytes. Furthermore, spontaneous Ca2+ release from the sarcoplasmic reticulum triggers a premature action potential only in failing myocytes. This model of the ventricular action potential and its alterations in heart failure is intended to serve as a tool for investigating the effects of therapeutic interventions on the electric excitability of the human ventricular myocardium.

An overview of Medicaid managed care litigation.

Since the enactment of Medicaid in 1965, states have had the option of offering beneficiaries enrollment in managed care arrangements. With the advent of mandatory managed care reaching millions of beneficiaries (including a growing proportion of disabled recipients), the amount and scope of litigation involving Medicaid managed care plans can be expected to grow. A review of the current litigation regarding Medicaid managed care reveals two basic types of lawsuits: (1) those that challenge the practices of managed care companies under various federal and state laws that safeguard consumer rights, protect health care quality, and prohibit discrimination; and (2) suits that assert claims arising directly under the Medicaid statute and implementing regulations, as well as claims related to Constitutional safeguards that undergird the program. Lawsuits asserting claims arising under Medicaid tend to raise two basic questions: (1) the extent to which enrollment in a Medicaid managed care plan alters existing Medicaid beneficiary rights and state agency duties under federal or state Medicaid law; and (2) the extent to which managed care companies, as agents of the state, act under "color of law" (i.e., undertaking to perform official duties or acting with the imprimatur of state authority). Additionally, states might see an increase in litigation brought by prospective and current contractors who assert that they have been wrongfully denied contracts or improperly penalized for poor performance. These assertions may involve claims that are grounded in federal and state law, the Medicaid statute, and the Constitution. Moreover, in light of the consumer protection elements of the managed care reforms contained in the Balanced Budget Act, future managed care litigation may focus on the manner in which companies carry out states' obligations toward managed care enrollees. Resolution of Medicaid managed care cases involves the application of general principles of administrative and regulatory law. Thus, Medicaid managed care cases have implications for other public purchasers of managed care arrangements, including state mental health and alcohol and substance abuse agencies.

Functional interaction between K(ATP) channels and the Na(+)-K(+) pump in metabolically inhibited heart cells of the guinea-pig.

1. Transmembrane current through ATP-regulated K(+) channels (IK(ATP)) was measured in ventricular heart cells of the guinea-pig in the whole-cell and cell-attached patch configurations under conditions of metabolic poisoning with the mitochondrial uncoupler 2,4-dinitrophenol (DNP). 2. Maintained exposure of the cells to DNP resulted in a transient appearance of whole-cell IK(ATP) When IK(ATP) had reached several nanoamps, blocking the forward-running Na(+)-K(+) pump with 0.5 mM strophanthidin decreased IK(ATP) after a delay. The time course of this decrease could be described by a single exponential function, which yielded a time constant(T)of 4.51+/- 1.89 s (n=8). 3. Hyperpolarization from 0 mV to -100 or -150 mV for 2 s caused IK(ATP) (measured at 0 mV) to decrease by 34.2 +/- 14.1 % (n = 8) and 37.6 +/- 9.4% (n = 8), respectively. After the hyperpolarizing pulse, IK(ATP) returned to its higher initial level within a couple of seconds. 4. Driving the pump backwards by removing the extracellular K(+) ions caused the permanent disappearance of DNP-induced IK(ATP). 5. Application of 0.5 mM strophanthidin in the absence of external K(+) ions induced a transient increase in IK(ATP), as did washing out the glycoside (n = 5). 6. When pump action was inhibited by using Na(+), K(+)-free Tyrode solution (see Methods) in the bath, strophanthidin did not have a comparable direct effect on IK(ATP). 7. In cell-attached patches, strophanthidin applied via the bath caused a reduction in IK(ATP) with a similar time course to that in whole-cell experiments. This suggests that the interaction between the pump molecules and the K(ATP) channels is not restricted to closely neighbouring molecules. 8. The data support the hypothesis that [ATP] at the cytosolic face of the membrane may drop to practically zero, thereby passing an 'ATP window' in which the channels first open and then close, and that the submembrane [ATP] is readily controlled by the cytosolic [ATP].

Divergence between genotype and phenotype in relatives of patients with the intron 2 mutation of steroid-21-hydroxylase.

We studied 95 patients and their relatives with the classical salt wasting (SW) and simple virilizing (SV) form of CAH. SSCP/heteroduplex analysis allowed fast and efficient screening for the most common 21-hydroxylase mutations (e.g. deletions, splice site mutation in intron 2 (bp 656), Ile172Asn mutation in exon 4) and determination of the relative intensities of CYP21A and CYP21B genes. The splice site mutation in intron 2 was found as the most frequent cause of 21-hydroxylase deficiency (35% of our patients). There is a strong genetic association between the mutation in intron 2 and the SW form of CAH. On the other hand, about 20% of our patients with the intron 2 mutation have the SV phenotype. Interestingly, homozygous splice site mutations in intron 2 were also detected in some parents or other relatives with no phenotypic changes typical for CAH (clinical evaluation, steroid hormone levels). In those patients with SV-CAH and especially in the relatives with the homozygous intron 2 mutation and an unaffected phenotype, the splice site mutation could be "leaky". mRNA-splicing in the adrenal cortex should result in a high degree of normal mRNA species. This is in contrast to in vitro expression studies of CYP21B genes containing the intron 2 mutation, performed by other groups. However, the results of in vitro expression studies are not always reflecting the in vivo conditions in the adrenal cortex. This situation is in good agreement with the variable degree of normal spliced mRNA and different phenotypic severity in intron mutations found in thalassemia.

[Semiquantitative determination of the cutaneous blood flow]. / Semiquantitative Bestimmung der Hautdurchblutung.

A semiquantitative determination of the cutaneous blood flow is possible by a simple thermic method using a heat conduction probe. There is a quantitative uncertainty because of the possible existence of relatively big vessels which do not participate in the blood supply of the examined tissular region but in the determination of the convection within the temperature field of the thermal conduction probe. The values of blood circulation derived from the stationary temperature field will therefore always be higher than the values of the real blood supply. This procedure necessitates a determination of the tissular heat conductivity. This will not be possible in all cutaneous regions. A relative method can be useful here inserting a binding lambda G value which lies below the thermal transport value lambda for normal blood circulation.

[Optimal measurement technic for the vasoconstrictor effect of corticosteroids. Comparative studies with reflection photometry and flow measurement]. / Uber die optimale messtechnische Erfassung des gefässverengenden Effektes der Kortikosteroide. Vergleichende Untersuchungen mit der Reflexionsphotometrie und der Fluvographie.

In 20 healthy male test subjects, a non-specific ointment base was applied to the back of the hand over 16 hours. Two days later, the same ointment base with 0.25% triamcinolone acetonide was once more applied to the back of the hand over 16 hours. The light reflection of the skin was measured by reflection photometry and the heat transport coefficient was measured fluvographically under the condition of the tourniquet test. The results show that the vasoconstrictor effect of triamcinolone acetonide is best detected by the reflection photometric measurements. In fluvographic measurement, the heat transport coefficient is most affected in maximal reactive hyperemia when the tourniquet has been removed. Application of the base led to a reduction of the blood flow and the water content of the skin after 16 hours, whereas the water content of the skin was unaffected in the presence of triamcinolone acetonide.