Search

Breast cancer is a heterogeneous genetic disease driven by the accumulation of individual mutations per tumor. Whole-genome sequencing approaches have identified numerous genes with recurrent mutations in primary tumors. Although mutations in well characterized tumor suppressors and oncogenes are overrepresented in these sets, the majority of the genetically altered genes have so far unknown roles in breast cancer progression. To improve the basic understanding of the complex disease breast cancer and to potentially identify novel drug targets or regulators of known cancer-driving pathways, we analyzed 86 wild-type genes and 94 mutated variants for their effect on cell growth using a serially constructed panel of MCF7 cell lines. We demonstrate in subsequent experiments that the metal cation transporter CNNM4 regulates growth by induction of apoptosis and identified a tumor suppressive role of complement factor properdin (CFP) in vitro and in vivo. CFP appears to induce the intracellular upregulation of the pro-apoptotic transcription factor DDIT3 which is associated with endoplasmic reticulum-stress response.

The fragile X syndrome (FraX), the most common inherited form of mental retardation, has been located to Xq27.3. As a step in the molecular analysis of this mutation, we have cloned a contiguous 1.8 Mb region containing the entire fragile X region in YAC and cosmid clones. The cloned area defines a region of 50 kb containing a CpG island, found to be selectively methylated in patients expressing the fragile X phenotype. In this 50kb area we have localised the breakpoints of four somatic cell hybrids selected to break at the position of the fragile site. Fluorescence in-situ hybridisation of cosmids flanking this area shows that the breakpoints, the CpG island and the fragile site coincide.

Deleted in malignant brain tumors 1 (DMBT1) is a secreted glycoprotein displaying a broad bacterial-binding spectrum. Recent functional and genetic studies linked DMBT1 to the suppression of LPS-induced TLR4-mediated NF-kappaB activation and to the pathogenesis of Crohn's disease. Here, we aimed at unraveling the molecular basis of its function in mucosal protection and of its broad pathogen-binding specificity. We report that DMBT1 directly interacts with dextran sulfate sodium (DSS) and carrageenan, a structurally similar sulfated polysaccharide, which is used as a texturizer and thickener in human dietary products. However, binding of DMBT1 does not reduce the cytotoxic effects of these agents to intestinal epithelial cells in vitro. DSS and carrageenan compete for DMBT1-mediated bacterial aggregation via interaction with its bacterial-recognition motif. Competition and ELISA studies identify poly-sulfated and poly-phosphorylated structures as ligands for this recognition motif, such as heparansulfate, LPS, and lipoteichoic acid. Dose-response studies in Dmbt1(-/-) and Dmbt1(+/+) mice utilizing the DSS-induced colitis model demonstrate a differential response only to low but not to high DSS doses. We propose that DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands providing a molecular basis for its broad bacterial-binding specificity and its inhibitory effects on LPS-induced TLR4-mediated NF-kappaB activation. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-α, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-κB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn’s disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-κB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn’s disease.

Udgivelsesdato: 2007-Nov BACKGROUND & AIMS: Impaired mucosal defense plays an important role in the pathogenesis of Crohn's disease (CD), one of the main subtypes of inflammatory bowel disease (IBD). Deleted in malignant brain tumors 1 (DMBT1) is a secreted scavenger receptor cysteine-rich protein with predominant expression in the intestine and has been proposed to exert possible functions in regenerative processes and pathogen defense. Here, we aimed at analyzing the role of DMBT1 in IBD. METHODS: We studied DMBT1 expression in IBD and normal tissues by quantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and mRNA in situ hybridization. Genetic polymorphisms within DMBT1 were analyzed in an Italian IBD case-control sample. Dmbt1(-/-) mice were generated, characterized, and analyzed for their susceptibility to dextran sulfate sodium-induced colitis. RESULTS: DMBT1 levels correlate with disease activity in inflamed IBD tissues. A highly significant fraction of the patients with IBD displayed up-regulation of DMBT1 specifically in the intestinal epithelial surface cells and Paneth cells. A deletion allele of DMBT1 with a reduced number of scavenger receptor cysteine-rich domain coding exons is associated with an increased risk of CD (P = .00056; odds ratio, 1.75) but not for ulcerative colitis. Dmbt1(-/-) mice display enhanced susceptibility to dextran sulfate sodium-induced colitis and elevated Tnf, Il6, and Nod2 expression levels during inflammation. CONCLUSIONS: DMBT1 may play a role in intestinal mucosal protection and prevention of inflammation. Impaired DMBT1 function may contribute to the pathogenesis of CD

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

Udgivelsesdato: 2004-Nov-12 The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight for group B. The protein DMBT1 (deleted in malignant brain tumors 1), which is identical to salivary agglutinin and lung gp-340, belongs to the group B SRCR proteins and is considered to be involved in tumor suppression and host defense by pathogen binding. In a previous study we used nonoverlapping synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteria-binding protein loop (peptide SRCRP2; QGRVEVLYRGSWGTVC) within the SRCR domains. In this study, using overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an 11-amino acid motif (DMBT1 pathogen-binding site 1 or DMBT1pbs1; GRVEVLYRGSW). An alanine substitution scan revealed that VEVL and Trp are critical residues in this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical. In addition, the homologous consensus sequences of a number of SRCR proteins were synthesized and tested for bacteria binding. Only consensus sequences of DMBT1 orthologues bound bacteria by this motif.

Mutations in the DKC1 gene are responsible for causing X-linked recessive dyskeratosis congenita (DKC) and a more severe allelic variant of the disease, Hoyeraal–Hreidarsson syndrome. Both diseases are characterized by progressive and fatal bone marrow failure. The nucleolar protein dyskerin is the pseudouridine synthase component of the box H+ACA snoRNAs and also interacts with the RNA component (human telomerase, hTR) of the telomerase complex. Dyskerin is therefore thought to function in the processing of pre-rRNA and of the hTR, strengthening the notion that the underlying mechanism of DKC is a premature senescence of cells, especially of the rapidly dividing epithelial and hemopoietic cells. To examine the functions of dyskerin in vivo, it will be necessary to generate mouse models. As a first step, we here provide the genomic structure of the mouse Dkc1 gene and expression analysis of the transcript. Northern hybridizations revealed the tissue-specific expression of an alternative 4.5-kb transcript, in addition to the ubiquitous 2.6-kb transcript. RNA in situ hybridizations on day 10.5–18.5 postconception embryos showed a ubiquitous expression of Dkc1 with a notably higher level of expression confined to the epithelial tissues. In addition, higher level Dkc1 expression was confined to embryonic neural tissues as well as to specific neurons in the cerebellum (Purkinje cells) and the olfactory bulb (mitral cells) of the adult brain. In adult testis, elevated expression was limited to the Leydig cells. The results indicate that some of the pertinent functions of dyskerin may be more tissue-specific than previously thought and are not limited to rapidly dividing cells.

Familial incontinentia pigmenti (IP; MIM 308310) is a genodermatosis that segregates as an X-linked dominant disorder and is usually lethal prenatally in males. In affected females it causes highly variable abnormalities of the skin, hair, nails, teeth, eyes and central nervous system. The prominent skin signs occur in four classic cutaneous stages: perinatal inflammatory vesicles, verrucous patches, a distinctive pattern of hyperpigmentation and dermal scarring. Cells expressing the mutated X chromosome are eliminated selectively around the time of birth, so females with IP exhibit extremely skewed X-inactivation. The reasons for cell death in females and in utero lethality in males are unknown. The locus for IP has been linked genetically to the factor VIII gene in Xq28 (ref. 3). The gene for NEMO (NF-kappaB essential modulator)/IKKgamma (IkappaB kinase-gamma) has been mapped to a position 200 kilobases proximal to the factor VIII locus. NEMO is required for the activation of the transcription factor NF-kappaB and is therefore central to many immune, inflammatory and apoptotic pathways. Here we show that most cases of IP are due to mutations of this locus and that a new genomic rearrangement accounts for 80% of new mutations. As a consequence, NF-kappaB activation is defective in IP cells.

Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.

The neuronal glycine receptor is a ligand-gated chloride channel composed of ligand binding α and structural β polypeptides. Homology screening of a human fetal brain cDNA library resulted in the identification of two alternative splice variants of the glycine receptor α3 subunit. The amino acid sequence predicted for the α3L variant was largely identical to the corresponding rat subunit. In contrast, the novel splice variant α3K lacked the coding sequence for 15 amino acids located within the cytoplasmic loop connecting transmembrane spanning region 3 (TM3) and TM4. Using P1 artificial chromosome (PAC) clones, the structure of theGLRA3 gene was elucidated and its locus assigned to human chromosomal bands 4q33-q34 by fluorescence in situhybridization. Two transcripts of 2.4 and 9 kilobases, corresponding to α3L and α3K, respectively, were identified and found to be widely distributed throughout the human central nervous system. Structural analysis of the GLRA3 gene revealed that the α3K transcript resulted from a complex splice event where excision of the novel exon 8A comprising the alternative sequence of 45 base pairs coincides with the persistence of a large intronic sequence in the 3′-untranslated region. Functional expression in HEK 293 cells of α3L and α3K subunits resulted in the formation of glycine-gated chloride channels that differed significantly in desensitization behavior, thus defining the cytoplasmic loop as an important determinant of channel inactivation kinetics.

We have previously located the genes of the five human main type H1 genes and the gene encoding the testicular subtype H1t to the region 21.1 to 22.2 on the short arm of chromosome 6. To investigate the organization of the histone genes in this region, we isolated two YACs from a human YAC library by PCR screening with primers specific for histone H1.1. This screen revealed two YAC clones, YAC Y23 (corresponding to ICRFy901D1223) contains an insert of about 480 kb, whereas the smaller YAC 4A (corresponding to ICRFy900C104) spans about 340 kb and is completely covered by YAC Y23. We have subcloned the YAC inserts in cosmids, determined the linear orientation of the cosmids by cosmid walking, and constructed a restriction map of the entire region by mapping the individual cosmids using partial digests and hybridization with labeled oligonucleotides complementary to the cos site of the vector. Hybridization analysis, subcloning, restriction mapping, and sequencing revealed that most of the previously isolated phage and cosmid clones containing histone genes are part of this YAC including the clones containing the four human main type H1 histone genes H1.1 to H1.4, the H1t gene, and core histone genes. Thirty-five histone genes map within 260 kb of the YAC Y23 insert. All newly identified histone genes were sequenced, and the sequences were deposited with the EMBL nucleotide sequence database. The histone H1.5 gene is not part of this region, and we therefore conclude that the H1.5 gene and the associated core histone genes form a separate subcluster within this chromosomal region.

The X-linked myotubular myopathy locus (MTM1) has been assigned to the Xq28 region by linkage analysis. By observation of an interstitial deletion in a female patient, the candidate region could be further reduced to a region of 600 kb flanked by the markers DXS304 and DXS497. We describe here cosmid contigs covering a region of 900 kb, including the entire MTM1 candidate region. Cosmids from the region were used to construct an enriched cDNA library from this area. Filter grids carrying this library were then screened by hybridization with whole cosmid clones, with CpG island-containing fragments from linking clones located in the area, and with total exon trap products of cosmid clones from the candidate region. In this analysis, 10 new transcripts were identified and localized precisely within the map. Genes in this area are candidates for MTM1 and a number of other diseases localized by genetic linkage studies to the chromosomal band Xq28.

Autofluorescence spectra, images, and decay kinetics of U251-MG glioblastoma cells prior and subse- quent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence inten- sity patterns of tumor (control) cells and less malignant cells are similar, differences can be deduced from autofluorescence spectra and decay kinetics. In particular, upon near UV excitation, the fluorescence ratio of the free and protein-bound coenzyme nicotinamid adenine dinucleotide depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. While larger numbers of fluorescence spectra are evaluated by principal component analysis, a multivariate data analysis method, addi- tional information on cell metabolism is obtained from spectral imaging and fluorescence lifetime imaging micro

Adrenoleukodystrophy (ALD), the most frequent peroxisomal disorder, is a severe neurodegenerative disease associated with an impairment of very long chain fatty acids β-oxidation. We have recently identified by positional cloning the gene responsible for ALD, located in Xq28. It encodes a new member of the "ABC" superfamily of membrane-associated transporters that shows, in particular, significant homology to the 70-kDa peroxisomal membrane protein (PMP70). We report here a detailed characterization of the ALD gene structure. It extends over 21 kb and consists of 10 exons. To facilitate the detection of mutations in ALD patients, we have determined the intronic sequences flanking the exons as well as the sequence of the 3′ untranslated region and of the immediate 5′ promoter region. Sequences present in distal exons cross-hybridize strongly to additional sequences in the human genome. The ALD gene has been positioned on a pulsed-field map between DXS15 and the L1CAM gene, about 650 kb upstream from the color pigment genes. The frequent occurrence of color vision anomalies observed in patients with adrenomyeloneuropathy (the adult onset form of ALD) thus does not represent a contiguous gene syndrome but a secondary manifestation of ALD.

An essential requirement in the analysis of genomes is the identification of functionally important sequence elements, which are often evolutionarily conserved. We describe here the development of a novel procedure for the selective isolation of conserved sequences which is based on hybridization of PCR-amplifiable DNA fragments from the whole genome of one species to biotinylated DNA from a genomic region of another species. The interspecies DNA hybrids are immobilized and the PCR-amplifiable DNA fragments are eluted, amplified and after further hybridization-amplification rounds cloned. This method was used for the generation of sublibraries of conserved sequences from mouse and pig DNA from regions corresponding to cosmids from the human Xq28 region. Mouse and pig homologs of sequences containing exons of known human genes, as well as exons from novel genes have been identified.

Endoglin is a membrane-inserted protein that is preferentially synthesized in angiogenic vascular endothelial and smooth muscle cells. Endoglin associates with members of the transforming growth factor-beta (TGF-beta) receptor family and has been identified as the gene involved in hereditary hemorrhagic telangiectasia. Although endoglin is known to affect cell responses to TGF-beta, its mode of action is largely unknown. We performed yeast two-hybrid screening of a human placental cDNA library and isolated a new endoglin-binding partner, a novel 221-amino acid member of the Tctex1/2 family of cytoplasmic dynein light chains named Tctex2beta, as the founder of a new Tctex1/2 subfamily. The interaction was localized exclusively to the cytoplasmic domain of endoglin. Reverse transcription-PCR showed expression of Tctex2beta in a wide range of tissues, including vascular endothelial and smooth muscle cells, placenta, and testis, as well as in several tumor cell lines. High expression levels were found in human umbilical vein endothelial cells and the large cell lung cancer cell line. Forced expression of Tctex2beta had a profound inhibitory effect on TGF-beta signaling. Additional Tctex2beta-interacting receptors were identified to be the TGF-beta type II receptor and most likely beta-glycan, but not ALK5, ALK1, or the bone morphogenetic protein type II receptor. Upon fluorescence tagging, co-localization of Tctex2beta and endoglin, as well as Tctex2beta, endoglin, and the TGF-beta type II receptor, was observed by different microscopy techniques. Our findings link endoglin for the first time to microtubule-based minus end-directed transport machinery, suggesting that some endoglin functions might be regulated and directed by its interaction with the cytoplasmic dynein light chain Tctex2beta.

Background Well known for its gene density and the large number of mapped diseases, the human sub-chromosomal region Xq28 has long been a focus of genome research. Over 40 of approximately 300 X-linked diseases map to this region, and systematic mapping, transcript identification, and mutation analysis has led to the identification of causative genes for 26 of these diseases, leaving another 17 diseases mapped to Xq28, where the causative gene is still unknown. To expedite disease gene identification, we have initiated the functional characterisation of all known Xq28 genes. Results By using a systematic approach, we describe the Xq28 genes by RNA in situ hybridisation and Northern blotting of the mouse orthologs, as well as subcellular localisation and data mining of the human genes. We have developed a relational web-accessible database with comprehensive query options integrating all experimental data. Using this database, we matched gene expression patterns with affected tissues for 16 of the 17 remaining Xq28 linked diseases, where the causative gene is unknown. Conclusion By using this systematic approach, we have prioritised genes in linkage regions of Xq28-mapped diseases to an amenable number for mutational screens. Our database can be queried by any researcher performing highly specified searches including diseases not listed in OMIM or diseases that might be linked to Xq28 in the future.

During the course of a large-scale sequencing project in Xq28, a human creatine transporter (CRTR) gene was discovered. The gene is located approximately 36 kb centromeric to ALD. The gene contains 13 exons and spans about 8.5 kb of genomic DNA. Since the creatine transporter has a prominent function in muscular physiology, it is a candidate gene for Barth syndrome and infantile cardiomyopathy mapped to Xq28.

Mutations in activin receptor-like kinase 1 (ALK1), a transforming growth factor (TGF)-beta type I receptor, lead to the vascular disorder hereditary hemorrhagic telangiectasia caused by abnormal vascular remodeling. The underlying molecular cause of this disease is not well understood. Identifying binding partners for ALK1 will help to understand its cellular function. Using the two-hybrid system, we identified an ALK1-binding protein encoded by an ancient retroviral/retrotransposon element integrated as a single copy gene known as PEG10 on human chromosome 7q21. PEG10 contains two overlapping reading frames from which two proteins, PEG10-RF1 and PEG10-RF1/2, are translated by a typical retroviral -1 ribosomal frameshift mechanism. Reverse transcription-PCR and Northern blot analysis showed a broad range of PEG10 expression in different tissues and cell types, i.e. human placenta, brain, kidney, endothelial cells, lymphoblasts, and HepG2 and HEK293 cells. However, endogenous PEG10-RF1 and PEG10-RF1/2 proteins were only detected in HepG2 and HEK293 cells. PEG10-RF1, which is the major PEG10 protein product, represents a gag-like protein, and PEG10-RF1/2 represents a gag-pol-like protein. PEG10-RF1 also interacts with different members of TGF-beta superfamily type I and II receptors. PEG10-RF1 binding to ALK1 is mediated by a 200-amino acid domain with no recognized motif. PEG10-RF1 inhibits ALK1 as well as ALK5 signaling. Co-expression of ALK1 and PEG10-RF1 in different cell types induced morphological changes reminiscent of neuronal cells or sprouting cells. This is the first report of a human retroviral-like protein interacting with members of the TGF-beta receptor family.

Background The requirement of a large amount of high-quality RNA is a major limiting factor for microarray experiments using biopsies. An average microarray experiment requires 10–100 μg of RNA. However, due to their small size, most biopsies do not yield this amount. Several different approaches for RNA amplification in vitro have been described and applied for microarray studies. In most of these, systematic analyses of the potential bias introduced by the enzymatic modifications are lacking. Results We examined the sources of error introduced by the T7 RNA polymerase based RNA amplification method through hybridisation studies on microarrays and performed statistical analysis of the parameters that need to be evaluated prior to routine laboratory use. The results demonstrate that amplification of the RNA has no systematic influence on the outcome of the microarray experiment. Although variations in differential expression between amplified and total RNA hybridisations can be observed, RNA amplification is reproducible, and there is no evidence that it introduces a large systematic bias. Conclusions Our results underline the utility of the T7 based RNA amplification for use in microarray experiments provided that all samples under study are equally treated.

Drug resistance of tumor cells leads to major drawbacks in the treatment of cancer. To identify candidate genes for drug resistance, we compared the expression patterns of the drug-sensitive human malignant melanoma cell line MeWo and three derived sublines with acquired resistance to the DNA-damaging agents cisplatin, etoposide, and fotemustine. Subarray analyses confirmed 57 candidate genes recovered from a genome-wide scan for differential expression. By specifically addressing cancer genes we retrieved another set of 209 candidates. Exemplary Northern blot studies indicated qualitative concordance for 110 of 135 (81.4%) data points. Whereas the etoposide-resistant line showed constant expression patterns over a period of approximately 2.5 years, the fotemustine- and cisplatin-resistant sublines exhibited considerable variability. Initially representing distinct entities, these two sublines finally converged in their expression patterns. A total of 110 genes was transiently or permanently deregulated in at least two resistant sublines. Fourteen genes displayed differential expression in all three of the sublines. We hypothesize that the variations in fotemustine and cisplatin resistance are based on progressive optimization and/or polyclonality. This, in addition to genomic alterations investigated by comparative genomic hybridization and evaluation of short-term response genes, can be used as a criterion for the selection of promising candidates. Among these are CYR61, AHCYL1, and MPP1, as well as several apoptosis-related genes, in particular STK17A and CRYAB. As MPP1 and CRYAB are also among the 14 genes differentially expressed in all three of the drug-resistant sublines, they represent the strongest candidates for resistance against DNA-damaging drugs.

Udgivelsesdato: 1999-Nov-4 Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3-q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.

MTM1 is responsible for X-linked recessive myotubular myopathy, which is a congenital muscle disorder linked to Xq28. MTM1 is highly conserved from yeast to humans. A number of related genes also exist. The MTM1 gene family contains a consensus sequence consisting of the active enzyme site of protein tyrosine phosphatases (PTPs), suggesting that they belong to a new family of PTPs. Database searches revealed homology of myotubularin and all related peptides to the cisplatin resistance-associated alpha protein, which implicates an as yet unknown function. In addition, homology to the Sbf1 protein (SET binding factor 1), involved in the oncogenic transformation of fibroblasts and differentiation of myoblasts, was also evident. We describe 225 kb of genomic sequence containing MTM1 and the related gene, MTMR1, which lies 20 kb distal to MTM1. Although there is only moderate conservation of the exons, the striking similarity in the gene structures indicates that these two genes arose by duplication. Calculations suggest that this event occurred early in evolution long before separation of the human and mouse lineages. So far, mutations have been identified in the coding sequence of only 65% of the patients analyzed, indicating that the remaining mutations may lie in noncoding regions of MTM1 or possibly in MTMR1. Knowledge of the genomic sequence will facilitate mutation analyses of the coding and noncoding sequences of MTM1 and MTMR1.

Construction of a transcript map in the DXS52 region in Xq28 had previously led to the isolation of a cDNA with a LIM zinc finger domain in the carboxyl terminus. In parallel, the orthologous murine transcript was isolated from the syntenic region. The human and mouse cDNAs have been designated ZNF185 andZfp185,respectively. By integrating the cDNA sequence with the cosmid-derived genomic sequence the exon–intron structure of the 3′ end of the ZNF185 gene was resolved. Comparative sequence analyses of the human genomic sequence with the full-length murine cDNA facilitated prediction of the 5′ end of the gene. The selective expression of three transcripts corresponding to the ZNF185 gene and a related gene was shown by Northern and Southern blots.In situhybridizations revealed a nonubiquitous and stage-specific expression ofZfp185,especially in differentiating connective tissue. Since LIM proteins regulate cellular proliferation and/or differentiation by diverse mechanisms, and some have directly been associated with disease, conceivably ZNF185 may represent a candidate for a disease-causing gene linked to Xq28. Knowledge of the genomic structure will permit detailed mutation analyses.

We have identified a novel human gene that is entirely deleted in two boys with abnormal genital development and myotubular myopathy (MTM1). The gene, F18, is located in proximal Xq28, approximately 80 kb centromeric to the recently isolated MTM1 gene. Northern analysis of mRNA showed a ubiquitous pattern and suggested high levels of expression in skeletal muscle, brain, and heart. A transcript of 4.6 kb was detected in a range of tissues, and additional alternate forms of 3.8 and 2.6 kb were present in placenta and pancreas, respectively. The gene extends over 100 kb and is composed of at least seven exons, of which two are noncoding. Sequence analysis of a 4.6-kb cDNA contig revealed two overlapping open reading frames (ORFs) that encode putative proteins of 701 and 424 amino acids, respectively. Two alternative spliced transcripts affecting the large open reading frame were identified that, together with the Northern blot results, suggest that distinct proteins are derived from the gene. No significant homology to other known proteins was detected, but segments of the first ORF encode polyglutamine tracts and proline-rich domains, which are frequently observed in DNA-binding proteins. The F18 gene is a strong candidate for being implicated in the intersexual genitalia present in the two MTM1-deleted patients. The gene also serves as a candidate for other disorders that map to proximal Xq28.

We have recently refined the localization of the myotubular myopathy (MTM1) gene to a 430-kb region between DXS304 and DXS1345 in proximal Xq28. We report two new polymorphic microsatellite markers, DXS8377 and DXS7423, that were physically mapped within the critical interval. A recombination event in a family segregating for MTM1 placed the disease gene telomeric to the trinucleotide polymorphism DXS8377. Together with the recent mapping of two microdeletions associated with MTM1, the recombination refines the critical region to 280 kb. A second recombination event was observed distal to the tetranucleotide repeat DXS7423. This recombination has occurred in the off-spring of a female with a more than 67% probability of being a carrier and very likely restricts the MTM1 gene to a 130-kb region. This physical refinement is significant for positional cloning of the disease gene. The highly polymorphic markers and the precise localization of the MTM1 gene will facilitate genetic diagnosis of the disorder.

The chromosomal band Xq28 has been a focus of interest in human genetics because > 20 hereditary diseases have been mapped to this region. However, about two-thirds of the disease genes remain uncloned. The region around the polymorphic DXS52 locus (ST14) within Xq28 lies in the candidate regions for several as-yet-uncloned disease genes. So far, only four melanoma antigen genes (MAGE) and the human biglycan (BGN) gene, have been mapped within the 700-kb stretch around DXS52, suggesting that more genes may reside in this region. By combining exon trapping and direct cDNA selection methods, we sought to identify novel transcripts around the DXS52 locus. In addition to recovering the MAGE and BGN genes, we isolated and mapped six putative novel genes (XAP103-XAP108), the caltractin gene, and a gene encoding a novel Ca(2+)-transporting ATPase isoform (hPMCA5). The newly isolated sequences were considered as representing parts of putative genes if they contained at least one unique exon-trap product and/or at least one expressed sequence tag (EST) from sequence data bases and if, in addition, they showed evidence of expressed RT-OCT and/or Northern blot analysis. Our data facilitated the integration of the transcription map with the physical map around the DXS52 locus. Future analysis of the novel genes as candidates for Barth syndrome (BTHS) and chondrodysplasia punctata (CDPX2) is in progress.

We have recently described a female patient with myotubular myopathy (MTM1) and an interstitial deletion at Xq28. Characterisation of the deletion allowed us to position the MTM1 gene to a 600 kb region between DXS304 and DXS497. In order to further restrict the region we screened for deletions in a set of 38 patients. We found two overlapping deletions in boys that in addition to MTM1 showed an unexpected abnormal genital development. As the latter phenotype is not found in the other non-deleted MTM1 patients, our observations are best explained by a contiguous gene syndrome. The deletions define a 430 kb region that contains the MTM1 gene and most likely a gene implicated in male sexual development. A high resolution physical map of this region is presented.

Glioblastoma is the most common and malign form of brain cancer that is highly resistant to therapy and particularly hard to cure since the blood-barrier is not very permeable to drugs. Moreover, a surgery is always highly risky. Thus, there is a real need to develop technique enabling accurate identification of potentially tumor cells at an early stage. It is getting well established that cancer cells are usually softer than their normal homologues and Atomic Force Microscopy (AFM) has proven itself over the last decade to be a tool of choice to characterize cells mechanical properties. Among the various AFM techniques, Force Spectroscopy (FS), especially Force Volume (FV) is the most commonly used. In the present study, AFM has been used to successfully characterize malignant and modified less malignant forms of glioblastoma U-251MG isogenic cells, using FV and Peak Force Tapping (PFT), a newly released AFM mode. Although both modes are quantitative and easy to use, PFT appears as the most relevant. Benefits and drawbacks of both techniques are discussed.

Adrenoleukodystrophy (ALD) is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloneuropathy (AMN) in adults. Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age. Central nervous system demyelination progresses rapidly and death occurs within a few years. AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course. Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD. The principal biochemical abnormality of ALD is the accumulation of very-long-chain fatty acids (VLCFA) because of impaired beta-oxidation in peroxisomes. The normal oxidation of VLCFA-CoA in patients' fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD. Here we use positional cloning to identify a gene partially deleted in 6 of 85 independent patients with ALD. In familial cases, the deletions segregated with the disease. An identical deletion was detected in two brothers presenting with different clinical ALD phenotypes. Candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary DNAs by exon connection and screening of cDNA libraries. The deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein of M(r) 70K that is involved in peroxisome biogenesis and belongs to the 'ATP-binding cassette' superfamily of transporters.

Balanced translocations affecting the paternal copy of 15q11--q13 are a rare cause of Prader-Willi syndrome (PWS) or PWS-like features. Here we report on the cytogenetic and molecular characterization of a de novo balanced reciprocal translocation t(X;15)(q28;q12) in a female patient with atypical PWS. The translocation breakpoints in this patient and two previously reported patients map 70-80 kb distal to the SNURF-SNRPN gene and define a breakpoint cluster region. The breakpoints disrupt one of several hitherto unknown 3' exons of this gene. Using RT--PCR we demonstrate that sequences distal to the breakpoint, including the recently identified C/D box small nucleolar RNA (snoRNA) gene cluster HBII-85 as well as IPW and PAR1, are not expressed in the patient. Our data suggest that lack of expression of these sequences contributes to the PWS phenotype.