Comprehensive genomic analysis of desmoplastic medulloblastomas: identification of novel amplified genes and separate evaluation of the different histological components.

Desmoplastic medulloblastoma (DMB) is a malignant cerebellar tumour composed of two distinct tissue components, pale islands and desmoplastic areas. Previous studies revealed mutations in genes encoding members of the sonic hedgehog pathway, including PTCH, SMOH and SUFUH in DMBs. However, little is known about other genomic aberrations. We performed comparative genomic hybridization (CGH) analysis of 22 sporadic DMBs and identified chromosomal imbalances in 20 tumours (91%; mean, 4.9 imbalances/tumour). Recurrent chromosomal gains were found on chromosomes 3, 9 (six tumours each), 20, 22 (five tumours each), 2, 6, 7, 17 (four tumours each) and 1 (three tumours). Recurrent losses involved chromosomes X (eight tumours), Y (six of eleven tumours from male patients), 9, 12 (four tumours each), as well as 10, 13 and 17 (three tumours each). Four tumours demonstrated high-level amplifications involving sequences from 1p22, 5p15, 9p, 12p13, 13q33-q34 and 17q22-q24, respectively. Further analysis of the 9p and 17q22-q24 amplicons by array-based CGH (matrix-CGH) and candidate gene analyses revealed amplification of JMJD2C at 9p24 in one DMB and amplification of RPS6KB1, APPBP2, PPM1D and BCAS3 from 17q23 in three DMBs. Among the 17q23 genes, RPS6KB1 showed markedly elevated transcript levels as compared to normal cerebellum in five of six DMBs and four of five classic medulloblastomas investigated. Finally, CGH analysis of microdissected pale islands and desmoplastic areas showed common chromosomal imbalances in five of six informative tumours. In summary, we have identified several novel genetic alterations in DMBs and provide genetic evidence for a monoclonal origin of their different tissue components.

Pbx4, a new Pbx family member on mouse chromosome 8, is expressed during spermatogenesis.

Members of the Pbx family are involved in a diverse range of developmental processes including axial patterning and organogenesis. Pbx functions are in part mediated by the interaction of Pbx proteins with members of the Hox and Meis/Prep families. We have identified a fourth mammalian Pbx family member. Pbx4 in the mouse and PBX4 in humans are located on chromosome 8 and chromosome 19, respectively. Pbx4 expression is confined to the testis, especially to spermatocytes in the pachytene stage of the first meiotic prophase.

Loss of the Y chromosome is a frequent chromosomal imbalance in pancreatic cancer and allows differentiation to chronic pancreatitis.

In a study for the identification of genomic alterations in pancreatic cancer, representational difference analysis was used and led to the isolation of 2 distinct fragments, deleted on the Y chromosome in the xenografted tumor DNA of a male patient with an adenocarcinoma of the pancreas. Loss of Y chromosomal material was further studied in 11 pancreatic cancer cell lines of male origin, using PCR amplification of 5 sequence tagged sites (STSs) distributed along the Y chromosome; 8/11 cell lines exhibited a complete loss of the Y chromosome and 3 had deletions. To examine the status of the Y chromosome in situ, interphase FISH analysis was performed on paraffin sections from pancreatic carcinoma (n=7) and chronic pancreatitis (n=7) tissues, and the loss of Y-chromosomal STS-markers was studied in 6 xenograft tumors obtained from male pancreatic cancer patients. This analysis revealed that a loss of the Y chromosome occurs in vivo in primary pancreatic tumor cells, whereas the Y chromosome was intact in chronic pancreatitis. Our data suggest that loss of Y is a frequent event occurring in male pancreatic tumors. Although there is no evidence for a functional implication of Y chromosome loss, it effectively differentiates between a malignant and a benign condition as e.g. chronic pancreatitis. Thus, this genetic alteration may be of diagnostic use.

Human homeodomain-interacting protein kinase-2 (HIPK2) is a member of the DYRK family of protein kinases and maps to chromosome 7q32-q34.

Here we identified the human serine/threonine kinase HIPK2 as a novel member of the DYRK kinase subfamily. Alignment of several DYRK family proteins including the kinases minibrain, MJAK, PKY, the Dictyostelium kinase YakA and Saccharomyces YAK1 allowed the identification of several evolutionary conserved DYRK consensus motifs within the kinase domain. A lysine residue conserved between all DYRK kinase family members was found to be essential for the kinase function of HIPK2. Human HIPK2 was mapped to chromosome 7q32-q34 and murine HIPK2 to chromosome 6B, the homologue to human chromosome 7.

Functional characterization of the gene encoding RLIM, the corepressor of LIM homeodomain factors.

RLIM is a RING H2 zinc finger protein that acts as a negative coregulator for LIM homeodomain transcription factors. We have isolated genomic lambda clones that cover the entire mouse RLIM-encoding Rnf12 gene. The Rnf12 gene encompasses 20 kb and consists of at least five exons and four introns. Several transcriptional start sites within a 24-bp region were mapped around 300 nt upstream of the translational start site. Rnf12-specific mRNA can be detected in many tissues as revealed by Northern blot analysis. Transient cotransfections reveal that the proximal Rnf12 promoter can be activated in vitro by ubiquitously and more restrictively expressed transcription factors, some of which are known mediators of signal transduction pathways, e.g., mammalian Krüppel-like transcription factors, Sox and ets-related proteins, and RBP-J. We isolated a cDNA encoding human RLIM, which is highly conserved with mouse and chick RLIM. By fluorescence in situ hybridization and interspecific backcross analysis, we have localized the Rnf12 gene to the central regions of mouse and human chromosome X.

Children with ocular motor apraxia type Cogan carry deletions in the gene (NPHP1) for juvenile nephronophthisis.

Congenital ocular motor apraxia type Cogan is characterized by impairment of horizontal voluntary eye movements, ocular attraction movements, and optokinetic nystagmus. Two patients with congenital ocular motor apraxia type Cogan exhibited a newly recognized association with nephronophthisis type 1, an autosomal recessive kidney disease. Both patients possess large deletions of the NPHP1 gene. The deletion occurred on both chromosomes 2q13 in one patient and heterozygously in combination with a point mutation of the NPHP1 gene in the other. The findings will help to elucidate the pathogenetic processes involved.

Adrenocortical carcinoma is characterized by a high frequency of chromosomal gains and high-level amplifications.

Distinction of adrenocortical carcinoma from benign adrenocortical lesions by standard criteria is often difficult. In order to search for additional diagnostic parameters, a series of 25 adrenocortical tumors, 8 adenomas, 14 primary carcinomas, 1 metastasis, and the 2 adrenocortical carcinoma cell lines SW13 and NCI-H295 were analyzed by the approach of comparative genomic hybridization (CGH). Except for the two smallest adenomas, all tumors showed chromosomal imbalances with a high incidence of chromosomal gains, most frequently involving chromosomes or chromosome arms 5, 7, 8, 9q, 11q, 12q, 14q, 16, 17q, 19, 20, and 22q. The only significant loss of material concerned the distal part of 9p. Furthermore, 21 high-level amplifications were identified in 15 different regions of the genome. The consensus regions of recurrent gains and the focal high-level amplifications allowed identification of a series of chromosomal subregions containing candidate proto-oncogenes of potential pathogenic function in adrenocortical tumors: 1p34.3-pter, 1q22-q25, 3p24-pter, 3q29, 7p11.2-p14, 9q34, 11q12-11q13, 12q13, 12q24.3, 13q34, 14q11.2-q12, 14q32, 16p, 17q24-q25, 19p13.3, 19q13.4, and 22q11.2-q12. A subset of the CGH data was independently confirmed by interphase cytogenetics. Interestingly, the adenomas larger than 4 cm contained gained material of regions also overrepresented in carcinomas. In addition, several chromosomal gains, in particular the high-level amplifications, were exclusive for the malignant status of the tumors. These data indicate that the larger adrenal lesions need to be carefully considered in the diagnosis of adrenocortical tumors, and that genetic aberrations might provide useful markers for a better diagnostic differentiation.

Primary structure, chromosomal mapping, expression and transcriptional activity of murine hepatocyte nuclear factor 4gamma.

We demonstrate the presence of a new member of the orphan nuclear receptor hepatocyte nuclear factor 4 (HNF4) subfamily in mouse which is genetically distinct from the previously characterized mouse HNF4alpha gene. The new member of the HNF4 subfamily shows highest amino acid identity, similar tissue distribution and syntenous chromosomal localization to the recently described human HNF4gamma (NR2A2), we therefore classify it as mouse HNF4gamma (mHNF4gamma). A combination of RT-PCR and immunohistochemical analysis showed expression of mHNF4gamma mRNA and protein in the endocrine pancreas, testes, kidney and gut. By co-transfection experiments, we show that mHNF4gamma is able to activate transcription, acting through binding sites that have been previously characterized as HNF4alpha binding sites. The presence of HNFgamma in human and mouse implies that a complex transcriptional network exists in higher vertebrates involving a number of HNF4 members with overlapping yet distinct function and tissue distribution.

Predetermined chromosomal deletion encompassing the Nf-1 gene.

Complex chromosomal rearrangements (deletions, inversions, translocations) are a hallmark of human tumour cells. Yet, the generation of animal models for gross chromosomal abnormalities still presents a formidable challenge. Here, we describe a versatile procedure for chromosomal engineering that was used to generate an ES cell line with a megabase deletion encompassing the tumour suppressor gene neurofibromatosis-1 (Nf-1) on mouse chromosome 11, which is often deleted in tumours of neural crest origin. Homologous recombination into sites flanking Nf-1 was used to introduce artificial sequences (triple-helix, loxP, vector backbone) that can be employed for in vitro recovery of intervening sequences or the generation of in vivo deletions. This strategy may be developed into a scheme by which large chromosomal regions with precisely defined end points may be excised from mammalian cells and reintroduced after suitable in vitro modification.

Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis.

Rhythmic activity of neurons and heart cells is endowed by pacemaker channels that are activated by hyperpolarization and directly regulated by cyclic nucleotides (termed HCN channels). These channels constitute a multigene family, and it is assumed that the properties of each member are adjusted to fit its particular function in the cell in which it resides. Here we report the molecular and functional characterization of a human subtype hHCN4. hHCN4 transcripts are expressed in heart, brain, and testis. Within the brain, the thalamus is the predominant area of hHCN4 expression. Heterologous expression of hHCN4 produces channels of unusually slow kinetics of activation and inactivation. The mean potential of half-maximal activation (V(1/2)) was -75.2 mV. cAMP shifted V(1/2) by 11 mV to more positive values. The hHCN4 gene was mapped to chromosome band 15q24-q25. The characteristic expression pattern and the sluggish gating suggest that hHCN4 controls the rhythmic activity in both thalamocortical neurons and pacemaker cells of the heart.

Spleen-specific expression of the malaria-inducible intronless mouse gene imap38.

We characterize the mouse gene imap38 and its inducibility by Plasmodium chabaudi malaria among different lymphoid tissues and mouse strains of different H-2 complex and non-H-2 background. Imap38 is a single copy gene assigned to chromosome 6B. It consists of only one exon of 1900 base pairs encoding a highly basic 25.8-kDa protein. Confocal laser scanning microscopy localizes differently tagged IMAP38 proteins in nuclei of transfected cells. Reporter gene assays reveal that the 1730-base pair 5'-flanking region, containing an RSINE1 repeat immediately adjacent to initiation site +1, exhibits promoter activity in nonmurine cells, while it is largely repressed in diverse mouse cell lines, which corresponds to the situation in mouse tissues. P. chabaudi malaria induces imap38 expression almost exclusively in the spleen but not in other lymphoid organs. Parasite lysates are able to induce imap38 in the spleen, but not in spleen cells ex vivo. Activation of spleen cells by LPS and other stimuli is not sufficient to induce imap38. Inducibility of imap38 requires signals from both parasites and the intact spleen, and it is controlled by genes of that non-H-2 background, which also controls development of protective immunity against P. chabaudi malaria.

Genomic organization of mouse gene zfp162.

We report the cloning and characterization of the alternatively spliced mouse gene zfp162, formerly termed mzfm, the homolog of the human ZFM1 gene encoding the splicing factor SF1 and a putative signal transduction and activation of RNA (STAR) protein. The zfp162 gene is about 14 kb long and consists of 14 exons and 13 introns. Comparison of zfp162 with the genomic sequences of ZFM1/SF1 revealed that the exon-intron structure and exon sequences are well conserved between the genes, whereas the introns differ in length and sequence composition. Using fluorescent in situ hybridization, the zfp162 gene was assigned to chromosome 19, region B. Screening of a genomic library integrated in lambda DASH II resulted in the identification of the 5'-flanking region of zfp162. Sequence analysis of this region showed that zfp162 is a TATA-less gene containing an initiator control element and two CCAAT boxes. The promoter exhibits the following motifs: AP-2, CRE, Ets, GRE, HNF5, MRE, SP-1, TRE, TCF1, and PU.1. The core promoter, from position -331 to -157, contains the motifs CRE, SP-1, MRE, and AP-2, as determined in transfected CHO-K1 cells and IC-21 cells by reporter gene assay using a secreted form of human placental alkaline phosphatase. The occurrence of PU.1/GRE supports the view that the zfp162 gene encodes a protein involved not only in nuclear RNA metabolism, as the human ZFM1/SF1, but also in as yet unknown macrophage-inherent functions.

Transcriptional and translational downregulation of H-REV107, a class II tumour suppressor gene located on human chromosome 11q11-12.

The H-rev107 tumour suppressor was isolated as a gene specifically expressed in rat fibroblasts resistant toward malignant transformation by the activated HRAS gene (Sers et al., 1997; Hajnal et al., 1994). Here we describe the human homologue of the rat H-rev107 gene. The predicted rat and human proteins are highly conserved exhibiting an overall amino acid identity of 83%. The H-REV107-1 gene is ubiquitously expressed with the exception of haematopoetic cells and tissues. In contrast, H-REV107-1 mRNA was found only in eight of 27 cell lines derived from mammary carcinoma, lung carcinoma, gastric carcinoma, kidney carcinoma, melanoma, neuroblastoma and other tumours. The H-REV107-1 protein was not detectable in any of these tumour cells. Loss of H-REV107-1 expression was not restricted to cultured human tumour cell lines, but also found in primary squamous cell carcinomas. Gross structural aberrations of the H-REV107-1 gene were absent in tumorigenic cell lines. Thus, the block to H-REV107-1 expression is achieved both at the level of transcription and translation. By fluorescence in situ hybridisation the human H-REV107-1 gene was localised to chromosome 11q11-12.

Construction of a gene map of the nephronophthisis type 1 (NPHP1) region on human chromosome 2q12-q13.

A gene for the autosomal recessive kidney disorder juvenile nephronophthisis (NPH) is located on chromosome 2q between markers D2S1893 and D2S1888. Recently, the presence of large homozygous deletions was described in the majority of NPH patients. We constructed an integrated YAC/PAC contig of 54 markers and 30 PAC clones that encompasses this deletion and the flanking inverted duplication. Thirty-six novel sequence-tagged site markers were generated for this region of 2-3 Mb, 22 of which represent PAC ends. Ten of 18 multiplex NPH families show a homozygous deletion for 8 consecutive markers. BlastN database search and expressed sequence tag (EST) mapping led to the localization of 18 EST clones to the integrated contig, representing 11 putative transcribed sequences. Seven EST clones were localized to the NPHP1 region between D2S1893 and D2S1888. Two EST clones, zc07a11 from a human parathyroid tumor library and yy63e10 from a multiple sclerosis lesion library, are located in the deletion region. PCR amplification experiments indicate that zc07a11 represents a chimeric cDNA. Through FISH analysis the NPHP1 deletion region was localized to 2q12-q13. In summary, our study provides a high-resolution physical map of the NPHP1 region with 7 precisely localized expressed sequences, 2 of which have recently been shown to be part of a gene for NPH. These data will alleviate the identification of further genes of a homozygous gene deletion syndrome in patients with NPH and oculomotor apraxia and will be instrumental in the characterization of the molecular mechanism leading to the large homozygous deletion in this region. The data furthermore provide an important step toward the construction of a sequence-ready PAC contig of this region.

The mouse filensin gene: structure and evolutionary relation to other intermediate filament genes.

Filensin and phakinin are two lens-specific members of the intermediate filament (IF) superfamily of proteins. They coassemble to form a beaded submembraneous filamentous network, the beaded filaments (BFs). The low sequence homology and differences in assembly compared to other IF proteins do not allow their classification in any of the five IF subgroups. The organization of the phakinin gene exon/intron boundaries provides evidence that this partner may be sharing a common origin with type I cytokeratin genes. Here we report the molecular cloning, sequence and characterization of the mouse filensin gene. The filensin gene consists of 8 exons and 7 introns, with 6 introns interrupting its rod domain in a highly conserved manner characteristic of type III IF genes, like vimentin, desmin, or peripherin. Of the two tail domain exons the one adjacent to the rod domain, compares to exon 7 of the non-neuronal cytoplasmic IF gene of helix aspersa and to the lamin region bridging the end of the rod domain to the nuclear localization signal. Altogether, these observations indicate that the lens beaded filaments form an independent class of IF.

Repetin (Rptn), a new member of the "fused gene" subgroup within the S100 gene family encoding a murine epidermal differentiation protein.

We report the cloning and characterization of a murine epidermal differentiation gene, repetin (Rptn), exhibiting striking similarity to the genes of the intermediate filament-associated proteins profilaggrin and trichohyalin. The repetin gene consists of three exons and two introns. The first exon is short and untranslated. The deduced amino acid sequence distributed between exons II and III contains 1130 amino acids with a calculated molecular mass of 130 kDa and pI of 7.7. The amino terminus exhibits significant homology to the S100 proteins containing two calcium-binding motifs of the EF-hand type. The remainder coding sequence contains a central segment consisting of 49 tandem repeats of a 12-amino-acid sequence rich in glutamines. By fluorescence in situ hybridization the repetin gene was localized to chromosome band 3 F1-2. Expression of repetin mRNA is detectable in the stratified internal epithelia of forestomach and tongue and to a lesser degree in normal skin epidermis, where it is restricted to the differentiated suprabasal cell layers. Based on its chromosomal localization, its genomic organization, and its stage-specific expression during late epidermal differentiation, as well as on the structural features of the encoded protein, we conclude that the repetin gene represents a novel member of the "fused gene" subgroup of the S100 gene family encoding multifunctional epidermal matrix proteins.