Major sacrococcygeal teratoma in an extreme premature infant: a multidisciplinary approach.

Antenatally diagnosed, large sacrococcygeal teratomas in very premature infants are associated with a very poor outcome. We present an extreme premature infant with cardiac decompensation, diagnosed at 27 weeks and 1 day of gestational age. A positive outcome could be achieved with intensive multidisciplinary planning of the delivery, postnatal stabilization and surgical resection, as demonstrated in this case report.

Identification and characterization of interactions between the vertebrate polycomb-group protein BMI1 and human homologs of polyhomeotic.

In Drosophila melanogaster, the Polycomb-group (PcG) genes have been identified as repressors of gene expression. They are part of a cellular memory system that is responsible for the stable transmission of gene activity to progeny cells. PcG proteins form a large multimeric, chromatin-associated protein complex, but the identity of its components is largely unknown. Here, we identify two human proteins, HPH1 and HPH2, that are associated with the vertebrate PcG protein BMI1. HPH1 and HPH2 coimmunoprecipitate and cofractionate with each other and with BMI1. They also colocalize with BMI1 in interphase nuclei of U-2 OS human osteosarcoma and SW480 human colorectal adenocarcinoma cells. HPH1 and HPH2 have little sequence homology with each other, except in two highly conserved domains, designated homology domains I and II. They share these homology domains I and II with the Drosophila PcG protein Polyhomeotic (Ph), and we, therefore, have named the novel proteins HPH1 and HPH2. HPH1, HPH2, and BMI1 show distinct, although overlapping expression patterns in different tissues and cell lines. Two-hybrid analysis shows that homology domain II of HPH1 interacts with both homology domains I and II of HPH2. In contrast, homology domain I of HPH1 interacts only with homology domain II of HPH2, but not with homology domain I of HPH2. Furthermore, BMI1 does not interact with the individual homology domains. Instead, both intact homology domains I and II need to be present for interactions with BMI1. These data demonstrate the involvement of homology domains I and II in protein-protein interactions and indicate that HPH1 and HPH2 are able to heterodimerize.

Common origin of the human synovial sarcoma associated SS18 and SS18L1 gene loci.

The highly conserved synovial sarcoma associated protein SS18 is thought to act as a transcriptional co-activator through interactions with various proteins involved in (epigenetic) gene regulation. The SS18 SNH domain appears to act as a major interface for these protein-protein interactions. Previously, we used this SNH domain to identify SS18 paralogs (SS18L1 and SS18L2) and orthologs in various species. The functional significance of these SS18-like proteins is embodied by the observations that SS18L1 and SS18L2 can replace SS18 in its various protein-protein interactions, and that SS18L1 may act as a fusion partner of SSX in synovial sarcoma. In the current study, we performed a comprehensive comparison of SNH-containing loci in several distinct species. By doing so, we found that the vertebrate SS18 and SS18L1 genes map within co-linear DNA segments that may have evolved through a relatively recent genomic duplication event. An additional phylogenetic study indicated that the more divergent SS18L2 gene is most likely derived from an earlier gene duplication event, again in the vertebrate lineage.

Disruption of the gene Euchromatin Histone Methyl Transferase1 (Eu-HMTase1) is associated with the 9q34 subtelomeric deletion syndrome.

BACKGROUND: A new syndrome has been recognised following thorough analysis of patients with a terminal submicroscopic subtelomeric deletion of chromosome 9q. These have in common severe mental retardation, hypotonia, brachycephaly, flat face with hypertelorism, synophrys, anteverted nares, thickened lower lip, carp mouth with macroglossia, and conotruncal heart defects. The minimum critical region responsible for this 9q subtelomeric deletion syndrome (9q-) is approximately 1.2 Mb and encompasses at least 14 genes. OBJECTIVE: To characterise the breakpoints of a de novo balanced translocation t(X;9)(p11.23;q34.3) in a mentally retarded female patient with clinical features similar to the 9q- syndrome. RESULTS: Sequence analysis of the break points showed that the translocation was fully balanced and only one gene on chromosome 9 was disrupted--Euchromatin Histone Methyl Transferase1 (Eu-HMTase1)--encoding a histone H3 lysine 9 methyltransferase (H3-K9 HMTase). This indicates that haploinsufficiency of Eu-HMTase1 is responsible for the 9q submicroscopic subtelomeric deletion syndrome. This observation was further supported by the spatio-temporal expression of the gene. Using tissue in situ hybridisation studies in mouse embryos and adult brain, Eu-HMTase1 was shown to be expressed in the developing nervous system and in specific peripheral tissues. While expression is selectively downregulated in adult brain, substantial expression is retained in the olfactory bulb, anterior/ventral lateral ventricular wall, and hippocampus and weakly in the piriform cortex. CONCLUSIONS: The expression pattern of this gene suggests a role in the CNS development and function, which is in line with the severe mental retardation and behaviour problems in patients who lack one copy of the gene.

Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and cell lines.

The SSX genes, located on the X chromosome, encode a family of highly homologous nuclear proteins. The SSX1 and SSX2 genes were initially identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas. Recently, however, it was found that these two genes, as well as the highly homologous SSX4 and SSX5 genes, are aberrantly expressed in different types of cancers, including melanomas. Because normal SSX expression has been detected only in the testis and, at very low levels, the thyroid, these proteins are considered as new members of the still growing family of cancer/testis antigens. These antigens are presently considered as targets for the development of cancer immunotherapy protocols. In the present study, we developed a monoclonal antibody found to recognize SSX2, SSX3, and SSX4 proteins expressed in formaldehyde-fixed and paraffin-embedded tissues. This antibody was used to investigate SSX expression in normal testis and thyroid, benign melanocytic lesions, melanoma lesions, and melanoma cell lines. SSX nuclear expression in the testis was found to be restricted to spermatogenic cells, mainly spermatogonia. Of 18 melanoma cell lines analyzed, 9 showed SSX RNA and protein expression, although heterogeneously and at variable levels. Treatment of an SSX-negative cell line with 5-aza-2'-deoxycytidine, a demethylating agent, led to SSX RNA and protein expression, indicating a role for methylation in transcription regulation. Thirty-four of 101 primary and metastatic melanoma cases and 2 of 24 common nevocellular and atypical nevus cases showed SSX nuclear staining. Again, SSX expression was heterogeneous, ranging from widespread to scarce. Our findings stress the importance of assessing the a priori SSX expression status of melanoma cases that may be selected for immunotherapeutic trials.

Fluorinated steroids do not improve outcome of isolated atrioventricular block.

INTRODUCTION: Congenital atrioventricular block (CAVB) is a rare disorder with a significant morbidity and mortality. Consensus regarding the prescription and efficacy of prenatal corticosteroids is lacking. This nationwide study was initiated to evaluate the effects of prenatal treatment with corticosteroids on the outcome of CAVB in The Netherlands. METHODS: All fetuses identified with isolated congenital AVB-II° or AVB-III° in any of the eight academic fetal heart centers of The Netherlands between 2003 and 2013 were included and reviewed. RESULTS: Fifty-six fetuses were included. Fourteen (25%) fetuses were treated with dexamethasone. We found no differences between the steroid-treated and untreated cases regarding in utero progression of the AVB (63% vs 67% respectively), survival to birth (86% vs 84%), pacemaker implantations (74% vs 58%) or long-term dilated cardiomyopathy (13% vs 17%). Steroid treated fetuses demonstrated more in utero growth restriction (38% vs 11%). CONCLUSION: No benefit from prenatal corticosteroid treatment was demonstrated for fetuses with isolated CAVB in this study. However, we found negative side effects. Our data provide no evidence to support the routine administration of corticosteroids for the treatment of fetal CAVB.

Isolation and characterization of the mouse homolog of SYT, a gene implicated in the development of human synovial sarcomas.

In a previous study we reported the isolation of the human synovial sarcoma-associated t(X;18) breakpoint. As a result of this translocation, the SYT gene on chromosome 18 fuses to either the SSX1 or the SSX2 gene on the X chromosome, depending on the exact location of the breakpoint within band Xp11.2. As yet, little is known about the modes of action of the SYT and SSX genes and their respective (fusion) products. Here we report the isolation of the mouse homolog of SYT, its full length cDNA sequence, its chromosomal localization, and its spatio-temporal expression patterns in adult and embryonic tissues. The SYT gene was found to be well conserved during evolution and is part of a region of synteny between the human and mouse chromosomes 18. In early embryogenesis, Syt is ubiquitously expressed. In later stages, the expression becomes confined to cartilage tissues, specific neuronal cells and some epithelial derived tissues. In mature testis, expression was specifically observed in primary spermatocytes.

The synovial sarcoma associated protein SYT interacts with the acute leukemia associated protein AF10.

As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome. Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function. We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait. Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL. Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells. Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments. The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs. The C-terminal interaction domain of AF10 is located outside known functional domains. Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors. This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias.

Human growth-differentiation factor 3 (hGDF3): developmental regulation in human teratocarcinoma cell lines and expression in primary testicular germ cell tumours.

We describe the cloning and initial characterization of a novel cDNA from human embryonal carcinoma (EC) cells. This cDNA, which we named human growth differentiation factor 3 (hGDF3), encodes the homologue of mouse GDF3, a TGFbeta superfamily member belonging to the Growth/Differentiation Factors. We have analysed the expression of hGDF3 in human embryonal carcinoma cell lines and in primary testicular germ cell tumours of adolescents and adults (TGCTs). Expression of hGDF3 in human EC cell lines is stem cell-specific, is down-regulated upon RA-mediated differentiation and is increased upon culture of the cells in the presence of activin A. In TGCTs, hGDF3 expression is low in seminomas, while expression in non-seminomas is readily detectable and appears to be associated with the EC and yolk sac components in the tumours. We have also mapped the hGDF3 locus to the short arm of human chromosome 12, a region consistently overrepresented in human testicular germ cell tumours. Thus, hGDF3 represents an embryonal carcinoma stem cell-associated marker both in vitro and in vivo.

Towards the isolation of a human malignant extragonadal germ cell tumour-associated breakpoint in chromosome 11q13.

In a previous study we have defined a subgroup of human malignant extragonadal germ cell tumours that is characterized by complex translocations involving chromosomes 6 and 11 (Echten et al. 1995). Here we report (i) the use of fluorescent in situ hybridization, pulsed field gel electrophoresis and direct visual hybridization techniques to localize the tumour-associated breakpoint within band 11q13, and (ii) the construction of a phage library enriched for this region to facilitate genomic walks towards the breakpoint. Extensive breakpoint-flanking contigs were generated and within these contigs six candidate genes could be identified.

Simultaneous bilateral fluorescein angiography.

Two fundus cameras were used in the development of a setup for bilateral fluorescein angiography. The indications for this method are listed and examples of simultaneous fluoroangiograms are given. This technique allows the simultaneous study of hemodynamics in the carotid artery, the study of intraocular and anterior eye segment circulatory disturbances, and it is time-saving in bilateral pathology.

Double chambered right ventricle: delineation by multiplane transoesophageal echocardiography.

Three consecutive patients (median age 5 years; median weight 15 kg) with double chambered right ventricle (DCRV) were studied by multiplane transoesophageal echocardiography (TOE), using a specially designed paediatric probe. Using a 30 degrees angulation from the longitudinal plane, the body and outflow tract of the right ventricle could be imaged with minimal foreshortening, allowing detailed description of the level and nature of obstruction. In all cases, angiography and subsequent surgical inspection confirmed the TOE findings. Multiplane TOE should avoid the necessity for angiography in the preoperative assessment of this unusual lesion.

Severe Progressive Autism Associated with Two de novo Changes: A 2.6-Mb 2q31.1 Deletion and a Balanced t(14;21)(q21.1;p11.2) Translocation with Long-Range Epigenetic Silencing of LRFN5 Expression.

In a 19-year-old severely autistic and mentally retarded girl, a balanced de novo t(14;21)(q21.1;p11.2) translocation was found in addition to a de novo 2.6-Mb 2q31.1 deletion containing 15 protein-encoding genes. To investigate if the translocation might contribute to developmental stagnation at the age of 2 years with later regression of skills, i.e. a more severe phenotype than expected from the 2q31.1 deletion, the epigenetic status and expression of genes proximal and distal to the 14q21.1 breakpoint were investigated in Ebstein Barr Virus-transformed lymphoblast and primary skin fibroblast cells. The 14q21.1 breakpoint was found to be located between a cluster of 7 genes 0.1 Mb upstream, starting with FBXO33, and the single and isolated LRFN5 gene 2.1 Mb downstream. Only expression of LRFN5 appeared to be affected by its novel genomic context. In patient fibroblasts, LRFN5 expression was 10-fold reduced compared to LRFN5 expressed in control fibroblasts. In addition, a relative increase in trimethylated histone H3 lysine 9 (H3K9M3)-associated DNA starting exactly at the translocation breakpoint and going 2.5 Mb beyond the LRFN5 gene was found. At the LRFN5 promoter, there was a distinct peak of trimethylated histone H3 lysine 27 (H3K27M3)-associated DNA in addition to a diminished trimethylated histone H3 lysine 4 (H3K4M3) level. We speculate that dysregulation of LRFN5, a postsynaptic density-associated gene, may contribute to the patient's autism, even though 2 other patients with 14q13.2q21.3 deletions that included LRFN5 were not autistic. More significantly, we have shown that translocations may influence gene expression more than 2 Mb away from the translocation breakpoint.

The C terminus of the synovial sarcoma-associated SSX proteins interacts with the LIM homeobox protein LHX4.

As a result of the synovial sarcoma-associated t(X;18) translocation, the SS18 gene on chromosome 18 is fused to either one of the three closely related SSX genes on the X chromosome. The SS18 protein is thought to act as a transcriptional co-activator, whereas the SSX proteins are thought to act as transcriptional corepressors. The main SSX-repression domain is located in its C terminus, a domain that is retained in the respective SS18-SSX fusion proteins. Both the SS18 and SSX proteins lack DNA-binding domains. Previously, we found that the SS18 and SS18-SSX fusion proteins may be tethered to DNA targets via the SS18-interacting protein AF10. Here, we set out to isolate proteins that interact with the SSX C-terminal repression domain using a yeast two-hybrid interaction trap. Of the positive clones isolated, two corresponded to the LIM homeobox protein LHX4, a DNA-binding protein that is involved in transcription regulation. An endogenous interaction was subsequently established in mammalian cells via colocalization and coimmunoprecipitation of the respective proteins. Interestingly, the LHX4 gene was previously found to be deregulated in various human leukemias. In addition, it was previously found that LIM homeobox proteins may bind to and activate the glycoprotein-alpha (CGA) promoter. Using LHX4 chromatin immunoprecipitation and CGA-promoter assays, we found that endogenous LHX4 binds to the CGA promoter and that LHX4-mediated CGA activation is enhanced by the SS18-SSX protein, but not by the SSX protein. Taken together, we conclude that this novel protein - protein interaction may have direct consequences for the (de)regulation of SSX and/or SS18-SSX target genes and, thus, for the development of human synovial sarcomas.

The effect of computer reminders on GPs' prescribing behaviour: a cluster-randomised trial.

OBJECTIVE: It is difficult to control drug-prescribing behaviour in general practice, despite the development and distribution of guidelines. The purpose of this study was to assess the effect on drug-prescribing behaviour of implementing prescribing guidelines by means of a reactive computer reminder system (CRS). DESIGN: Cluster-randomised controlled trial with an incomplete block design in the south of the Netherlands: 25 GPs (7 GP practices) received reminders about antibiotics and asthma/COPD prescriptions, 28 GPs (7 GP practices) received reminders about cholesterol prescriptions. Prescription guidelines were integrated into the computerised GP information system. MEASUREMENTS: Both performance indicators and prescription volumes were calculated as the main outcome measures. Next to individual volume measure, sum scores were constructed on the volume measures per drug group (antibiotics, asthma/COPD and cholesterol). RESULTS: Variation between GPs turned out to be larger and more skewed than expected. No differences between groups were found for indicators and volumes related to recommendations advocating certain drugs. Although there was a tendency towards clinically relevant results for prescription volumes that were supposed to drop, the difference in sum score between the groups was not significant. For antibiotic prescriptions that were supposed to drop, the sum score for the intervention group was 28.2 (95% CI: 20.8-44.5) prescriptions per 1000 patients per GP, while this was 39.7 (95% CI: 29.7-64.1) for the control group (p 0.2). For prescriptions asthma/COPD that were supposed to drop, the sum score for the intervention group was 1.1 (95% CI: 0.6-2.6) prescriptions per 1000 patients per GP, while this was 2.2 (95% CI: 1.4-4.3) for the control group (p 0.1). On three specific recommendations (on quinolones for cystitis, corticosteroids for CPOD, and antibiotics for acute sore throat) significant differences were found. CONCLUSIONS: This study turned out to be underpowered due to high inter doctor variation in prescribing behaviour. Nevertheless, computerised reminders sometimes have a favourable effect on restricting certain drugs that are not or no longer indicated in general practice.

Targeted disruption of the synovial sarcoma-associated SS18 gene causes early embryonic lethality and affects PPARBP expression.

The synovial sarcoma-associated protein SS18 (also known as SYT or SSXT) is thought to act as a transcriptional co-activator. This activity appears to be mediated through the SWI/SNF proteins BRG1 and INI1 and the histone acetyl transferase p300. Here, we report that disruption of the mouse Ss18 gene results in a recessive embryonic lethal phenotype, due to placental failure caused by impairment of placental vascularization and/or chorio-allantoic fusion. This phenotype resembles the p300 knockout phenotype, but is distinct from the Brg1 and Ini1 knockout phenotypes. Through expression profiling of knockout embryos, we observed altered expression of genes known to affect placental development, including the peroxisome proliferator-activated receptor-binding protein (Pparbp). Since Pparbp null mutant embryos display a similar, lethal phenotype with placental failure, we suggest that the functional and phenotypic co-linearities between Ss18 and p300 may also include the transcriptional co-activator Pparbp. Additional interbreeding of Ss18 and Ss18l1 (Crest) mutant mice indicates that these two functionally and structurally related genes may act synergistically during critical stages of embryonic development.

Symposium overview. The Shell Conference on Computer-Aided Molecular Modelling.

The 'Shell Conference on ...' series began in 1985 and meetings are held approximately twice a year. The idea behind the conferences is to bring together invited scientists from both universities and industry, and representatives from different Shell Research laboratories, to create a forum to discuss the future directions of the chosen research area. These meetings have embraced a wide range of topics of interest to Shell Research as a whole. This particular conference, organized by the Analytical Department of the Koninklijke/shell-Laboratorium, Amsterdam (KSLA), was held on 4-6 October, 1987 at Hoenderloo in the Netherlands. The aim was to review the state-of-the-art and to discuss the future of molecular modeling and design. The programme itself consisted of a series of presentations on prescribed topics, panel discussions, and software and hardware demonstrations. Many of the presentations given consisted of overviews, experiences, advice and predictions for the future. The panel sessions, which involved the speakers within that session and a discussion leader who summarised some of the points made in an introduction, encouraged even further discussion and speculation. This overview attempts to catch the flavour of the meeting and convey some personal views that were expressed and conclusions drawn.

Simultaneous bilateral fluorescein angiography of the retina and the iris in central retinal vein occlusion.

Twenty-two patients with a recently developed C.R.V.O. (central retinal vein occlusion) (symptoms: less than four weeks) are discussed. A delay in A.R.C.T. (arm to retina circulation time) in the affected eye was found in two patients with an ischaemic type of C.R.V.O. and in 3 patients with a non-ischaemic type of C.R.V.O. No side difference could be demonstrated in A.I.C.T. (arm to iris circulation time). Leakage of radial iris vessels on fluorescein angiography was found in 6 out of 7 patients with an ischaemic type of C.R.V.O. and in 2 out of 15 patients with a non-ischaemic type of C.R.V.O. Radial iris vessel leakage may be a helpful prognostic sign in C.R.V.O.