D.R.H. de Bruijn

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.

Mapping and characterization of the mouse and human SS18 genes, two human SS18-like genes and a mouse Ss18 pseudogene.

We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus explaining the high and widespread expression of the SS18 genes.

Masked t(X;18)(p11;q11) in a biphasic synovial sarcoma revealed by FISH and RT‐PCR

The initial cytogenetic analysis of a biphasic synovial sarcoma showed an apparently normal karyotype. After FISH using chromosome X‐ and 18‐specific probes and RT‐PCR using SYT‐ and SSX‐specific primer sets, a cryptic synovial sarcoma‐associated t(X;18)(p11;q11) could be revealed. The “masked” nature of the translocation may best be explained by a two‐step scenario in which a genuine t(X;18)(p11;q11) has occurred as a first step and a reverse reciprocal X;18 translocation as a second step, leaving the synovial sarcoma‐associated SYT–SSX1 fusion intact. The findings further underline our previous suggestion that SYT–SSX1 fusions may correlate with a biphasic nature of the tumor. In addition, our findings indicate that, in analogy to, e.g., the Philadelphia translocation in chronic myeloid leukemia, “masked” translocations may occur in soft tissue tumors and that, as a standard, RT‐PCR and/or FISH analyses should be carried out in order to provide karyotypic information that may be relevant to tumor diagnosis and/or prognosis. Genes Chromosomes Cancer 23:198–201, 1998.

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.

Ebstein's anomaly with severe hypoplastic and stenotic pulmonary venous connections: an unusual cause of congenital chylothorax

this condition are increasing skin thickness, particularly over the forehead and scalp, where thick creases may be evident, and polyhydramnios, with increasing echodensity of the amniotic fluid. This appears to be a fairly late feature and the sediment appears to develop quite rapidly once the hydramnios is detected. Congenital ichthyosis has been reported as a feature of various genodermatoses1–6 (Table 1). However, these do not appear to have the same histopathological features as the condition described here. As neither of the parents in this case shows any signs of being affected by the condition, autosomal recessive inheritance is assumed. Advances in molecular genetics and the use of invasive interventions, such as fetal skin biopsy, have enabled prenatal diagnosis7. Recent advances in threedimensional ultrasonography have enabled non-invasive prenatal diagnosis of congenital ichthyosis8,9. In conclusion, ichthyosis should be considered in cases of increased echogenicity of the amniotic fluid and fetal skin thickening. A positive diagnosis is not always associated with a lethal outcome.

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.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.

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.