International trends in clozapine use: a study in 17 countries.

OBJECTIVE: There is some evidence that clozapine is significantly underutilised. Also, clozapine use is thought to vary by country, but so far no international study has assessed trends in clozapine prescribing. Therefore, this study aimed to assess clozapine use trends on an international scale, using standardised criteria for data analysis. METHOD: A repeated cross-sectional design was applied to data extracts (2005-2014) from 17 countries worldwide. RESULTS: In 2014, overall clozapine use prevalence was greatest in Finland (189.2/100 000 persons) and in New Zealand (116.3/100 000), and lowest in the Japanese cohort (0.6/100 000), and in the privately insured US cohort (14.0/100 000). From 2005 to 2014, clozapine use increased in almost all studied countries (relative increase: 7.8-197.2%). In most countries, clozapine use was highest in 40-59-year-olds (range: 0.6/100 000 (Japan) to 344.8/100 000 (Finland)). In youths (10-19 years), clozapine use was highest in Finland (24.7/100 000) and in the publicly insured US cohort (15.5/100 000). CONCLUSION: While clozapine use has increased in most studied countries over recent years, clozapine is still underutilised in many countries, with clozapine utilisation patterns differing significantly between countries. Future research should address the implementation of interventions designed to facilitate increased clozapine utilisation.

Serious adverse events reported for antiobesity medicines: postmarketing experiences from the EU adverse event reporting system EudraVigilance.

BACKGROUND: Use of antiobesity medicines has been linked with serious cardiac and psychiatric adverse events (AEs). Spontaneous reports can provide information about serious, rare and unknown AEs occurring after the time of marketing. In Europe, information about AEs reported for antiobesity medicines can be accessed in the EudraVigilance (EV) database. Therefore, we aimed to identify and characterise AEs associated with the use of antiobesity medicines in Europe. METHODS: AE reports submitted for antiobesity medicines (Anatomical Therapeutic Chemical (ATC) group A08A) from 2007 to 2014 and located in the EV database were analysed. AE data were categorised with respect to time, age and sex of patient/consumer, type of reporter, category and seriousness of reported AEs and medicines. Consumer AE reports were compared with reports from other types of reporters with respect to age and sex of consumer, seriousness, system organ class and medicine. The unit of analysis was one AE and one AE report, respectively. RESULTS: We located 4941 AE reports corresponding to 13 957 AEs for antiobesity medicines in the EV database. More than 90% of all AE cases were serious, including 159 deaths. The majority of AE cases were reported for female adults. The majority of serious AEs was reported for orlistat (37%) and rimonabant (22%). The largest share of serious AEs was of the type 'cardiac disorders' (19%) and 'psychiatric disorders' (18%). Consumer AEs reporting differed from other sources with respect to share and seriousness of AEs, type of AEs (system organ class) and medicines (ATC level 5). CONCLUSIONS: Many serious AEs were found for antiobesity medicines in EV, and consumers contributed with a relatively high share of reports. Although several products have been withdrawn from the market and new medicines are being marketed, the utilisation of antiobesity medicines is widespread, and therefore systematic monitoring of the safety of these medicines is necessary.

Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs.

Many microRNAs (miRNAs) are encoded within the introns of RNA Pol II transcripts, often as polycistronic precursors. Here, we demonstrate the optimization of an intron encoding three endogenous miRNAs for the ectopic expression of heterologous anti-HIV-1 small interfering RNAs (siRNAs) processed from a single RNA polymerase II primary miRNA. Our expression system, designated as MCM7, is engineered from the intron-embedded, tri-cistronic miR-106b cluster that endogenously expresses miR-106b, miR-93 and miR-25. Manipulation of the miR-106b cluster demonstrated a strict requirement for maintenance of the native flanking primary miRNA (pri-miRNA) sequences and key structural features of the native miRNAs for efficient siRNA processing. As a model for testing the efficacy of this approach, we have replaced the three endogenous miRNAs with siRNAs targeting the tat and rev transcripts of human immunodeficiency virus type 1 (HIV-1). This study has enabled us to establish guidelines for optimal processing of the engineered miRNA mimics into functional siRNAs. In addition, we demonstrate that the incorporation of a small nucleolar RNA TAR chimeric decoy (snoRNA) inserted within the MCM7 intron resulted in a substantial enhancement of HIV suppression in long-term acute infectious HIV-1 challenges.

Structure-function analysis of SUV39H1 reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression.

SUV39H1, a human homologue of the Drosophila position effect variegation modifier Su(var)3-9 and of the Schizosaccharomyces pombe silencing factor clr4, encodes a novel heterochromatic protein that transiently accumulates at centromeric positions during mitosis. Using a detailed structure-function analysis of SUV39H1 mutant proteins in transfected cells, we now show that deregulated SUV39H1 interferes at multiple levels with mammalian higher-order chromatin organization. First, forced expression of full-length SUV39H1 (412 amino acids) redistributes endogenous M31 (HP1beta) and induces abundant associations with inter- and metaphase chromatin. These properties depend on the C-terminal SET domain, although the major portion of the SUV39H1 protein (amino acids 89 to 412) does not display affinity for nuclear chromatin. By contrast, the M31 interaction surface, which was mapped to the first 44 N-terminal amino acids, together with the immediately adjacent chromo domain, directs specific accumulation at heterochromatin. Second, cells overexpressing full-length SUV39H1 display severe defects in mitotic progression and chromosome segregation. Surprisingly, whereas localization of centromere proteins is unaltered, the focal, G(2)-specific distribution of phosphorylated histone H3 at serine 10 (phosH3) is dispersed in these cells. This phosH3 shift is not observed with C-terminally truncated mutant SUV39H1 proteins or with deregulated M31. Together, our data reveal a dominant role(s) for the SET domain of SUV39H1 in the distribution of prominent heterochromatic proteins and suggest a possible link between a chromosomal SU(VAR) protein and histone H3.

Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres.

Centromeres of eukaryotes are frequently associated with constitutive heterochromatin and their activity appears to be coregulated by epigenetic modification of higher order chromatin. Recently, we isolated murine (Suv39h1) and human (SUV39H1) homologues of the dominant Drosophila suppressor of position effect variegation Su(var)3-9, which is also related to the S. pombe silencing factor Clr4. We have shown that mammalian Su(var)3-9 homologues encode novel centromeric proteins on metaphase-arrested chromosomes. Here, we describe a detailed analysis of the chromatin distribution of human SUV39H1 during the cell cycle. Although there is significant heterochromatic overlap between SUV39H1 and M31 (HP1(beta)) during interphase, mitotic SUV39H1 displays a more restricted spatial and temporal association pattern with metaphase chromosomes than M31 (HP1(beta)), or the related HP1(&agr;) gene product. SUV39H1 specifically accumulates at the centromere during prometaphase but dissociates from centromeric positions at the meta- to anaphase transition. In addition, SUV39H1 selectively associates with the active centromere of a dicentric chromosome and also with a neocentromere. Interestingly, SUV39H1 is shown to be a phosphoprotein with modifications at serine and, to a lesser degree, also at threonine residues. Whereas SUV39H1 steady-state protein levels appear constant during the cell cycle, two additional phosphorylated isoforms are detected in mitotic extracts. This intriguing localisation and modification pattern would be consistent with a regulatory role(s) for SUV39H1 in participating in higher order chromatin organisation at mammalian centromeres.

Chromatin-association of the Polycomb group protein BMI1 is cell cycle-regulated and correlates with its phosphorylation status.

The human proto-oncogene Bmi1 is a member of the mammalian Polycomb Group (Pc-G) genes. The subnuclear distribution of the BMI1 protein was studied in several primary human and tumor-derived cell lines using immunohistochemical and biochemical methods. In primary and tumor cells, nuclear BMI1 shows a fine-grain distribution over chromatin, usually dense in interphase nuclei and significantly weaker along mitotic chromosomes. In addition, BMI1 preferentially associates with several distinct heterochromatic domains in tumor cell lines. In both primary and tumor cell lines a marked cell cycle-regulation of Pc-G-chromatin interaction is observed: nuclear BMI1-staining dissipates in late S phase and is re-established early in G(1)-phase. Chromatin-association of BMI1 inversely correlates with its phosphorylation status in a cell cycle-dependent fashion: at G(1)/S, hypophosphorylated BMI1 is specifically retained in the chromatin-associated nuclear protein fraction, whereas during G(2)/M, phosphorylated BMI1 is not chromatin-bound. Our findings indicate a strict cell cycle-controlled regulation of Pc-G complex-chromatin association and provide molecular tools for improving our understanding of Pc-G complex regulation and function in mammalian cells.

Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31.

The chromo and SET domains are conserved sequence motifs present in chromosomal proteins that function in epigenetic control of gene expression, presumably by modulating higher order chromatin. Based on sequence information from the SET domain, we have isolated human (SUV39H1) and mouse (Suv39h1) homologues of the dominant Drosophila modifier of position-effect-variegation (PEV) Su(var)3-9. Mammalian homologues contain, in addition to the SET domain, the characteristic chromo domain, a combination that is also preserved in the Schizosaccharyomyces pombe silencing factor clr4. Chromatin-dependent gene regulation is demonstrated by the potential of human SUV39H1 to increase repression of the pericentromeric white marker gene in transgenic flies. Immunodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere-specific localization during metaphase. In addition, Suv39h1/SUV39H1 proteins associate with M31, currently the only other characterized mammalian SU(VAR) homologue. These data indicate the existence of a mammalian SU(VAR) complex and define Suv39h1/SUV39H1 as novel components of mammalian higher order chromatin.

Oncogenic aberrations of p16INK4/CDKN2 and cyclin D1 cooperate to deregulate G1 control.

The p16INK4/CDKN2, D-type cyclins, their partner cyclin-dependent kinases, and retinoblastoma protein constitute a G1 regulatory pathway commonly targeted in oncogenesis. We show that, unexpectedly, abnormalities of p16INK4/CDKN2 occur concomitantly in two-thirds of cancer cell lines harboring aberrations of cyclin D1. Gene and protein transfer experiments demonstrated that concurrent alterations of cyclin D1 and p16 levels cooperate to (de)regulate G1 control in diploid fibroblasts, and that both events influence growth of retinoblastoma (RB)-positive, but not RB-deficient cancer cells. These results show that biological consequences of deregulating individual components along the pathway are unequal, reflecting their hierarchical roles in the G1 checkpoint control. Whereas RB defects eliminate the checkpoint completely, aberrations of the upstream components, such as cyclin D1 and p16INK4/CDKN2, can cooperate in multistep tumorigenesis.

Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins.

We report the characterization of three novel members of the KRAB-domain containing C2-H2 zinc finger family (ZNF133, 136 and 140). KRAB (Krüppel-associated box) is an evolutionarily conserved protein domain found N-terminally with respect to the zinc finger repeats that encodes the DNA binding domain. ZNF133 and ZNF140 have both the KRAB A- and KRAB B-boxes present at their N-terminus, whereas ZNF136 contains only the KRAB A-box. We have previously demonstrated that the KRAB domains derived from ZNF133 and ZNF140 are potent transcriptional repression domains [Margolin et al. (1994) Proc. Natl. Acad. Sci. USA 91, 4509-4513]. The KRAB domain from ZNF136, containing only subdomain A, is a considerable weaker suppression domain; however, when fused to the heterologous KRAB B subdomain of ZNF10 (KOX1) the two subdomains from a KRAB domain which induces repression as potently as previously reported KRAB domains.

Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16.

D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, promote progression through the G1 phase of the cell cycle by phosphorylating the retinoblastoma protein (RB). The activities of Cdk4 and Cdk6 are constrained by inhibitors such as p16, the product of the CDKN2 gene on human chromosome 9p21 (refs 12-14). The frequent deletion or mutation of CDKN2 in tumour cells suggests that p16 acts as a tumour suppressor. We show that wild-type p16 arrests normal diploid cells in late G1, whereas a tumour-associated mutant of p16 does not. Significantly, the ability of p16 to induce cell-cycle arrest is lost in cells lacking functional RB, including primary fibroblasts from Rb-/- mouse embryos. Thus, loss of p16, overexpression of D-cyclins and loss of RB have similar effects on G1 progression, and may represent a common pathway to tumorigenesis.

Aberrations of p16Ink4 and retinoblastoma tumour-suppressor genes occur in distinct sub-sets of human cancer cell lines.

The p16Ink4/MTS1/CDKN2 is a cell-cycle regulatory inhibitor of cyclin-dependent kinase 4 (cdk4), and a candidate tumour suppressor whose gene on chromosome band 9p21 is frequently deleted or mutated in diverse types of cancer. Cdk4 in association with its D-type cyclin partners, together with p16Ink4, and the product of the retinoblastoma tumour-suppressor gene (pRB), appear to constitute a G1-phase-controlling pathway which can become de-regulated through oncogenic aberrations of any of the components. In an attempt to elucidate the underlying molecular mechanisms, we have now surveyed expression of p16Ink4, at the protein and the mRNA levels, in 21 human cell types expressing normal pRB, as compared with another series of 21 cell lines whose pRB is mutant and/or inactivated through sequestration by DNA tumour virus onco-proteins. In contrast to aberrant lack of p16 expression in the majority of RB-positive cell types, expression of apparently normal (as shown by electrophoretic mobility and/or the ability to form protein-protein complexes with cdk4 in vivo) p16 was uniformly preserved in the cancer cell lines whose RB function was compromised. These data indicate that p16 operates upstream of pRB along the same pathway in G1. The results are discussed in view of the nature of a selective growth advantage potentially gained by cells through de-regulation of this key cell-cycle control mechanism.

A zinc-finger gene ZNF141 mapping at 4p16.3/D4S90 is a candidate gene for the Wolf-Hirschhorn (4p-) syndrome.

Chromosomal aneusomy is a major cause of reproductive wastage and congenital malformations in man. Zinc finger encoding genes would be good candidates for being involved in the multiple developmental defects associated with chromosomal aneusomy--by virtue of their role as transcriptional regulators, their abundance in the genome and their known association with specific developmental disorders. We have isolated and mapped a zinc finger encoding cDNA (ZNF141) of the C2-H2/KRAB subfamily to the 4p- (Wolf-Hirschhorn) syndrome (WHS) chromosome region. ZNF141 mapped to the distal end of the 2.2 Mb smallest region of deletion overlap of WHS, 300 kb from the 4p telomere on cosmid CD1 defining the anonymous locus D4S90. ZNF141 was expressed ubiquitously at low levels in the analysed tissue. The identification of a candidate gene for a chromosomal aneusomy syndrome belonging to a class of evolutionary conserved genes will provide options for studying its normal and abnormal expression during mammalian embryogenesis.

Pancreatic beta cells express two autoantigenic forms of glutamic acid decarboxylase, a 65-kDa hydrophilic form and a 64-kDa amphiphilic form which can be both membrane-bound and soluble.

The 64-kDa pancreatic beta-cell autoantigen, which is a target of autoantibodies associated with early as well as progressive stages of beta-cell destruction, resulting in insulin-dependent diabetes (IDDM) in humans, has been identified as the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase. We have identified two autoantigenic forms of this protein in rat pancreatic beta-cells, a Mr 65,000 (GAD65) hydrophilic and soluble form of pI 6.9-7.1 and a Mr 64,000 (GAD64) component of pI 6.7. GAD64 is more abundant than GAD65 and has three distinct forms with regard to cellular compartment and hydrophobicity. A major portion of GAD64 is hydrophobic and firmly membrane-anchored and can only be released from membrane fractions by detergent. A second portion is hydrophobic but soluble or of a low membrane avidity, and a third minor portion is soluble and hydrophilic. All the GAD64 forms have identical pI and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results of pulse-chase labeling with [35S]methionine are consistent with GAD64 being synthesized as a soluble protein that is processed into a firmly membrane-anchored form in a process which involves increases in hydrophobicity but no detectable changes in size or charge. All the GAD64 forms can be resolved into two isoforms, alpha and beta, which differ by approximately 1 kDa in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but are identical with regard to all other parameters analyzed in this study. GAD65 has a shorter half-life than the GAD64 forms, remains hydrophilic and soluble, and does not resolve into isomers. Comparative analysis of the brain and beta-cell forms of GAD show that GAD65 and GAD64 in pancreatic beta-cells correspond to the larger and smaller forms of GAD in brain, respectively. The expression of different forms and the flexibility in subcellular localization of the GAD autoantigen in beta-cells may have implications for both its function and autoantigenicity.

Analysis of a 1963-bp polymorphic region flanking the human insulin gene.

The nucleotide sequence of a long polymorphic region located 365 bp upstream from the human insulin gene is reported. The region is composed of 139 repeating sequences whose consensus structure is related to ACAGGGGTGTGGGG. Expansion in the number of repeating sequences appears to have taken place through duplication and triplication of 112-141-bp regions. However, ancestral polymorphic regions containing additions or deletions of 50 bp or more were not detected in two previous generations.