Identification of candidate tumour suppressor gene loci for Hodgkin and Reed-Sternberg cells by characterisation of homozygous deletions in classical Hodgkin lymphoma cell lines.

Several tumour suppressor genes (TSG) have been identified as a result of mapping homozygous deletions in cancer cells. To identify putative TSG involved in the pathogenesis of classical Hodgkin lymphoma (cHL), we investigated four cHL cell lines (L428, HDLM2, KMH2, L1236) using four different array-Comparative Genomic Hybridisation (array-CGH) platforms and focused on high resolution identification of homozygous deletions. Out of 79 candidate regions of bi-allelic loss identified by array-CGH, besides previously described regions, 28 novel regions of homozygous deletions could be verified by polymerase chain reaction. These regions ranged from 13 kb to 619 kb in size. Eleven of the 28 novel bi-allelic losses were putative copy number polymorphisms. This left 17 regions that might harbour novel tumour suppressors involved in Hodgkin lymphoma. Expression profiling with two different platforms confirmed lack of expression of the majority of the genes located in the homozygous deletions. Furthermore, analysis of ontology annotations of genes located in the homozygously deleted regions indicated an enrichment of genes involved in apoptosis and cell death. In summary, through the mapping of homozygous deletions in cell lines this study identified a series of genes, such as SEPT9, GNG7 and CYBB, which might encode candidate tumour suppressors involved in the pathogenesis of cHL.

T-/H-cadherin (CDH13): a new marker for differentiating podocytes.

Cadherin molecules are known to be involved in various biological processes other than cell adhesion such as morphogenesis, cell-cell communication, cell recognition or cell signalling. While the classical cadherin molecule is characterized by an extracellular moiety, a transmembrane region and a variable cytoplasmic domain, T-/H-cadherin differs from this pattern due to the absence of a transmembrane region and a cytoplasmic domain, respectively. Its extracellular moiety is bound to the apical cell membrane by a glycosyl-phosphatidyl-inositol anchor and localized to lipid raft domains. As its molecular function and expression pattern is still not fully understood, we used a newly generated anti-T-/H-cadherin antiserum to study immunohistochemically the expression of T-/H-cadherin during the differentiation of foetal human glomeruli. At the early capillary loop stage a strong apical signal comes up for visceral epithelial cells of Bowman's capsule, which begin to differentiate towards podocytes. At the advanced capillary loop stage, when podocytes have become part of the glomerular filtration barrier, the expression pattern, however, becomes more distinct and most likely restricted to the foot processes of the podocytes. We thus postulate a functional role of T-/H-cadherin for the differentiation of the podocytes and the formation of the glomerular capillary network.

Expression, alternative splicing and haplotype analysis of transcribed testis specific protein (TSPY) genes.

Testis specific protein (TSPY) is a human Y-chromosome derived gene with numerous functional and non-functional copies. Specific expression patterns in testis and testicular tumors, as in prostate cancer samples and cell lines led to the postulation of a potential role in cell proliferation, supported by the presence of a suppressor of variegation, enhancer of zeste and Trithorax/nucleosome assembling protein (nucleosome assembly protein) domain in the mature protein. Expression studies have now identified two transcripts of variable length, termed TSPY-S and -L, which differ in their 3'-translated region due to alternative splicing, and in the quantitative level of transcripts, with TSPY-S being at least 3-4-fold more abundant. In immunoblot experiments on human testis and LNCaP protein extracts using an anti-peptide-antiserum against the TSPY-L specific C-terminus TSPY-L was characterized as a functional variant on the protein level. As there are at least three intragenic positions differing between various TSPY genes and thus defining certain haplotypes, the alternatively spliced TSPY transcripts were analysed for their haplotypes in order to link them to well defined TSPY loci. Surprisingly, no evidence of a G-G-18 haplotype was found for the TSPY-L transcript, while this haplotype makes up almost 50% of all TSPY-S transcripts. This excludes the corresponding TSPY-1 locus from alternative splicing. The only significant differences between the TSPY-1 locus and eight other loci were identified in the promotor region as revealed by detailed sequence comparisons. Thus one might speculate that the alternative splicing could be influenced by elements binding to the promotor region.

Quantitative morphometric analysis of the submucous plexus in age-related control groups.

An increased number and density of the so-called "giant ganglia" (seven or greater ganglion cells per ganglion) serve as histopathological criteria for a bowel motility disorder called intestinal neuronal dysplasia of the submucous plexus (IND B). However, because these morphological criteria have been defined based upon observations in constipated patients, the diagnostic value of previous studies is open to controversy. Moreover, no age-related reference data from unaffected controls are available. This study reports on data from unaffected controls on the variability of size and distribution of ganglia in the submucous plexus during development. Therefore, for the first time, the normal status has been defined. Four age groups have been defined: (a) premature births, gestational age less than 35 weeks; (b) 1-365 days; (c) 1-14 years and (d) 15 years to greater than 70 years). All of these groups revealed giant ganglia in the submucous plexus. With advancing age, there was a decrease in the number of giant ganglia (from 32.7% in group a to 11.2% in group d) accompanied by an inverse increase in the mean distance between all ganglia (from 0.52 mm in group a to 1.17 mm in group d). The data presented permit the conclusion that the criteria mentioned above are not apt to define IND B as an entity, since they do not allow a sufficient demarcation from the age-correlated normal values presented here.

CK2-dependent C-terminal phosphorylation at T300 directs the nuclear transport of TSPY protein.

TSPY (testis-specific protein, Y-encoded) is a member of the greater SET/NAP family of molecules with various functions, e.g., in chromatin remodeling, regulation of gene expression, and has been implicated to play a role in the malignant development of gonadoblastoma, testicular and prostate cancer. Here we demonstrate that the C-terminus has a functional role for the nucleo-cytoplasmatic shuttling of the TSPY protein. Using various combinations of in vitro mutagenesis and enhanced green fluorescent protein reporter gene-expression experiments we were able to show that while the deletion of C-terminus leads to a decreased stability and enhanced degradation of the protein, the selective mutation of a C-terminal CK2 phosphorylation site (T300) prevents the TSPY protein from entering the nucleus. We conclude that phosphorylation of the (T300) residue is a necessary and functional prerequisite for TSPY's transport into the nucleus reminding of comparable data from a related Drosophila molecule, NAP1.

Alport syndrome with diffuse leiomyomatosis.

Alport syndrome (AS) is a hereditary nephropathy with hematuria progressing to end-stage renal failure (ESRF), sensorineural deafness, and specific eye signs (lenticonus, macular flecks, and congenital cataracts). Inheritance is X-linked in about 85% of the cases, caused by different mutations in the COL4A5 gene. Rarely AS is seen in combination with diffuse leiomyomatosis (DL). DL is a tumorous process involving smooth muscle cells, mostly of the esophagus, but also of the tracheobronchial tree and the female genital tract. Characteristically, the patients have deletions of the 5'-end of both the COL4A5 and the COL4A6 genes, respectively. We here present a 9-year-old boy who was admitted because of a newly diagnosed sensorineural deafness. He was born with cataracts and presented symptoms of dysphagia and bronchial irritation in the first year of life. Macroscopic hematuria was first noticed at 2 years during a febrile infection. Since early childhood the boy suffered from severe constipation. Taking together these symptoms, the diagnosis of Alport syndrome with diffuse leiomyomatosis (AS-DL) has to be considered. Genetic analysis demonstrated the predicted deletion of the COL4A5/COL4A6 genes.