SLC1A4 mutations cause a novel disorder of intellectual disability, progressive microcephaly, spasticity and thin corpus callosum.

Two unrelated patients, presenting with significant global developmental delay, severe progressive microcephaly, seizures, spasticity and thin corpus callosum (CC) underwent trio whole-exome sequencing. No candidate variant was found in any known genes related to the phenotype. However, crossing the data of the patients illustrated that they both manifested pathogenic variants in the SLC1A4 gene which codes the ASCT1 transporter of serine and other neutral amino acids. The Ashkenazi patient is homozygous for a deleterious missense c.766G>A, p.(E256K) mutation whereas the Ashkenazi-Iraqi patient is compound heterozygous for this mutation and a nonsense c.945delTT, p.(Leu315Hisfs*42) mutation. Structural prediction demonstrates truncation of significant portion of the protein by the nonsense mutation and speculates functional disruption by the missense mutation. Both mutations are extremely rare in general population databases, however, the missense mutation was found in heterozygous mode in 1:100 Jewish Ashkenazi controls suggesting a higher carrier rate among Ashkenazi Jews. We conclude that SLC1A4 is the disease causing gene of a novel neurologic disorder manifesting with significant intellectual disability, severe postnatal microcephaly, spasticity and thin CC. The role of SLC1A4 in the serine transport from astrocytes to neurons suggests a possible pathomechanism for this disease and implies a potential therapeutic approach.

Co-existence of clonal expanded autologous and transplacental-acquired maternal T cells in recombination activating gene-deficient severe combined immunodeficiency.

It is commonly accepted that the presence of high amounts of maternal T cells excludes Omenn syndrome (OS) in severe combined immunodeficiency (SCID). We report a SCID patient with a novel mutation in the recombination activating gene (RAG)1 gene (4-BP DEL.1406 TTGC) who presented with immunodeficiency and OS. Several assays, including representatives of specific T cell receptors (TCR), Vß families and TCR-γ rearrangements, were performed in order to understand more clearly the nature and origin of the patient's T cells. The patient had oligoclonal T cells which, based on the patient-mother human leucocyte antigen (HLA)-B50 mismatch, were either autologous or of maternal origin. These cell populations were different in their numbers of regulatory T cells (T(reg)) and the diversity of TCR repertoires. This is the first description of the co-existence of large amounts of clonal expanded autologous and transplacental-acquired maternal T cells in RAG1-deficient SCID.

Selective clinical and immune response of the oligoclonal autoreactive T cells in Omenn patients after cyclosporin A treatment.

The immunological hallmark of Omenn syndrome (OS) is the expansion and activation of an oligoclonal population of autoreactive T cells. These cells should be controlled rapidly by immunosuppressive agents, such as cyclosporin A (CsA), to avoid tissue infiltration and to improve the general outcome of the patients. Here we studied the clinical and the immune response to CsA in two Omenn patients and also examined the gene expression profile associated with good clinical response to such therapy. T cell receptor diversity was studied in cells obtained from OS patients during CsA therapy. Characterization of gene expression in these cells was carried out by using the TaqMan low-density array. One patient showed complete resolution of his symptoms after CsA therapy. The other patient showed selective response of his oligoclonal T cell population and combination therapy was required to control his symptoms. Transcriptional profile associated with good clinical response to CsA therapy revealed significant changes in 26·6% of the tested genes when compared with the transcriptional profile of the cells before treatment. Different clinical response to CsA in two OS patients is correlated with their immunological response. Varying clonal expansions in OS patients can cause autoimmune features and can respond differently to immunosuppressive therapy; therefore, additional treatment is sometimes indicated. CsA for OS patients causes regulation of genes that are involved closely with self-tolerance and autoimmunity.

P53 in blind subterranean mole rats--loss-of-function versus gain-of-function activities on newly cloned Spalax target genes.

A tumor suppressor gene, p53, controls cellular responses to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and/or apoptosis. Recently, we demonstrated that in blind subterranean mole rats, Spalax, a model organism for hypoxia tolerance, the p53 DNA-binding domain contains a specific Arg174Lys amino acid substitution. This substitution reduces the p53 effect on the transcription of apoptosis genes (apaf1, puma, pten and noxa) and enhances it on human cell cycle arrest and p53 stabilization/homeostasis genes (mdm2, pten, p21 and cycG). In the current study, we cloned Spalax apaf1 promoter and mdm2 intronic regions containing consensus p53-responsive elements. We compared the Spalax-responsive elements to those of human, mouse and rat and investigated the transcriptional activity of Spalax and human Arg174Lys-mutated p53 on target genes of both species. Spalax and human-mutated p53 lost induction of apaf1 transcription, and increased induction of mdm2 transcription. We conclude that Spalax evolved hypoxia-adaptive mechanisms, analogous to the alterations acquired by cancer cells during tumor development, with a bias against apoptosis while favoring cell arrest and DNA repair.

Nutrition-induced catch-up growth increases hypoxia inducible factor 1alpha RNA levels in the growth plate.

Although catch-up growth is a well-known phenomenon, the local pathways at the epiphyseal growth plate that govern this process remain poorly understood. To study the mechanisms governing catch-up growth in the growth plate, we subjected prepubertal rats to 10 days of 40% food restriction, followed by a renewal of the regular food supply to induce catch-up growth. The animals were weighed daily, and their humeral length was measured at sacrifice. The proximal tibial epiphyseal growth plates (EGPs) were studied, and findings were compared with EGPs from animals fed ad libitum and animals under food restriction. The gene expression profile in the growth plates was examined using DNA microarrays, and the expression levels of selected genes were validated by real-time polymerase chain reaction. To localize gene expression in different growth plate zones, microdissection was used. Protein levels and localization were examined using immunohistochemistry. We showed that the expression level of 550 genes decreased during food restriction and increased during catch-up growth, starting already one day after refeeding. HIF-1alpha, as well as several of its downstream targets, was found among these genes. Immunohistochemistry showed a similar pattern for HIF-1alpha protein abundance. Additionally, HIF-1alpha mRNA and protein levels were higher in the proliferating than in the hypertrophic zone, and this distribution was unaffected by nutritional status. These findings indicate that nutrition has a profound effect on gene expression level during growth plate growth, and suggest an important role for HIF-1alpha in the growth plate and its response to nutritional manipulation.

Age-dependent spatial memory loss can be partially restored by immune activation.

Aging is often associated with a decline in hippocampus-dependent spatial memory. Here, we show that functional cell-mediated immunity is required for the maintenance of hippocampus-dependent spatial memory. Sudden imposition of immune compromise in young mice caused spatial memory impairment, whereas immune reconstitution reversed memory deficit in immune-deficient mice. Analysis of hippocampal gene expression suggested that immune-dependent spatial memory performance was associated with the expression of insulin-like growth factor (Igf1) and of genes encoding proteins related to presynaptic activity (Syt10, Cplx2). We further showed that memory loss in aged mice could be attributed to age-related attenuation of the immune response and could be reversed by immune system activation. Homeostatic-driven proliferation of lymphocytes, which expands the existing T cell repertoire, restored spatial memory deficits in aged mice. Thus, our results identify a novel function of the immune system in the maintenance of spatial memory and suggest an original approach for arresting or reversing age-associated memory loss.

Parallel induction of ATM-dependent pro- and antiapoptotic signals in response to ionizing radiation in murine lymphoid tissue.

The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia, is a master regulator of the cellular network induced by DNA double-strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combined gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation in murine lymphoid tissue. Cluster analysis revealed a prominent pattern characterizing dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of NF-kappaB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and antiapoptotic signals were simultaneously induced, with the proapoptotic pathway mediated by p53 targets, and the prosurvival pathway by NF-kappaB targets. These findings further elucidate the molecular network induced by IR, point to novel putative NF-kappaB targets, and suggest a mechanistic model for cellular balancing between pro- and antiapoptotic signals induced by IR in lymphoid tissues, which has implications for cancer management. The emerging model suggests that restoring the p53-mediated apoptotic arm while blocking the NF-kappaB-mediated prosurvival arm could effectively increase the radiosensitivity of lymphoid tumors.

The UVB-induced gene expression profile of human epidermis in vivo is different from that of cultured keratinocytes.

In order to obtain a comprehensive picture of the molecular events regulating cutaneous photodamage of intact human epidermis, suction blister roofs obtained after a single dose of in vivo ultraviolet (UV)B exposure were used for microarray profiling. We found a changed expression of 619 genes. Half of the UVB-regulated genes had returned to pre-exposure baseline levels at 72 h, underscoring the transient character of the molecular cutaneous UVB response. Of special interest was our finding that several of the central p53 target genes remained unaffected following UVB exposure in spite of p53 protein accumulation. We next compared the in vivo expression profiles of epidermal sheets to that of cultured human epidermal keratinocytes exposed to UVB in vitro. We found 1931 genes that differed in their expression profiles between the two groups. The expression profile in intact epidemis was geared mainly towards DNA repair, whereas cultured keratinocytes responded predominantly by activating genes associated with cell-cycle arrest and apoptosis. These differences in expression profiles might reflect differences between mature differentiating keratinocytes in the suprabasal epidermal layers versus exponentially proliferating keratinocytes in cell culture. Our findings show that extreme care should be taken when extrapolating from findings based on keratinocyte cultures to changes in intact epidermis.

S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation.

The p53 paralogues p73, p63 and their respective truncated isoforms have been shown to be critical regulators of developmental and differentiation processes. Indeed, both p73- and p63-deficient mice exhibit severe developmental defects. Here, we show that S100A2 gene, whose transcript and protein are induced during keratinocyte differentiation of HaCaT cells, is a direct transcriptional target of p73beta and DeltaNp63alpha and is required for proper keratinocyte differentiation. Transactivation assays reveal that p73beta and DeltaNp63alpha exert opposite transcriptional effects on S100A2 gene. While DeltaNp63alpha is found in vivo onto S100A2 regulatory regions predominantly in proliferating cells, p73beta is recruited in differentiating cells. Silencing of p73 impairs the induction of S100A2 during the differentiation of HaCaT cells. Moreover, silencing of p73 or S100A2 impairs the proper expression of keratinocyte differentiation markers. Of note, p53 family members do not trigger S100A2 gene expression in response to apoptotic doses of cisplatin and doxorubicin.

Chromosomal aberrations and gene expression profiles in non-small cell lung cancer.

Alterations in genomic content and changes in gene expression levels are central characteristics of tumors and pivotal to the tumorigenic process. We analyzed 23 non-small cell lung cancer (NSCLC) tumors by array comparative genomic hybridization (array CGH). Aberrant regions identified included well-characterized chromosomal aberrations such as amplifications of 3q and 8q and deletions of 3p21.31. Less frequently identified aberrations such as amplifications of 7q22.3-31.31 and 12p11.23-13.2, and previously unidentified aberrations such as deletion of 11q12.3-13.3 were also detected. To enhance our ability to identify key acting genes residing in these regions, we combined array CGH results with gene expression profiling performed on the same tumor samples. We identified a set of genes with concordant changes in DNA copy number and expression levels, i.e. overexpressed genes located in amplified regions and underexpressed genes located in deleted regions. This set included members of the Wnt/beta-catenin pathway, genes involved in DNA replication, and matrix metalloproteases (MMPs). Functional enrichment analysis of the genes both overexpressed and amplified revealed a significant enrichment for DNA replication and repair, and extracellular matrix component gene ontology annotations. We verified the changes in expressions of MCM2, MCM6, RUVBL1, MMP1, MMP12 by real-time quantitative PCR. Our results provide a high resolution map of copy number changes in non-small cell lung cancer. The joint analysis of array CGH and gene expression analysis highlights genes with concordant changes in expression and copy number that may be critical to lung cancer development and progression.

P2X4 is up-regulated in gingival fibroblasts after periodontal surgery.

Several studies have shown that surgical detachment of marginal gingiva close to the cervical cementum of molar teeth in a rat mandible is a distinct stimulus for alveolar bone resorption. Recently, we found that P2X4, an ATP-receptor, is significantly up-regulated in marginal gingival cells soon after surgery. We hypothesized that local release of ATP signaling through P2X4 elicits activation of osteoclasts on the alveolar bone surface. In this study, we identified intense immunoreactivity of gingival fibroblasts to P2X4-specific antibodies and a 6.4-fold increase in expression by real-time RT-PCR. Moreover, a single local application, at the time of surgery, of Apyrase (which degrades ATP) or Coomassie Brilliant Blue (an antagonist of purinoreceptors) significantly reduced alveolar bone loss. We propose that ATP flowing from cells after surgery can directly activate P2X4 receptors in the sensor cells of marginal gingiva through Ca(2+) signaling, or by direct activation of osteoclasts on the bone surface.

Gene expression profiles of AML derived stem cells; similarity to hematopoietic stem cells.

Tumors contain a fraction of cancer stem cells that maintain the propagation of the disease. The CD34(+)CD38(-) cells, isolated from acute myeloid leukemia (AML), were shown to be enriched leukemic stem cells (LSC). We isolated the CD34(+)CD38(-) cell fraction from AML and compared their gene expression profiles to the CD34(+)CD38(+) cell fraction, using microarrays. We found 409 genes that were at least twofold over- or underexpressed between the two cell populations. These include underexpression of DNA repair, signal transduction and cell cycle genes, consistent with the relative quiescence of stem cells, and chromosomal aberrations and mutations of leukemic cells. Comparison of the LSC expression data to that of normal hematopoietic stem cells (HSC) revealed that 34% of the modulated genes are shared by both LSC and HSC, supporting the suggestion that the LSC originated within the HSC progenitors. We focused on the Notch pathway since Jagged-2, a Notch ligand was found to be overexpressed in the LSC samples. We show that DAPT, an inhibitor of gamma-secretase, a protease that is involved in Jagged and Notch signaling, inhibits LSC growth in colony formation assays. Identification of additional genes that regulate LSC self-renewal may provide new targets for therapy.

The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non-syndromic mental retardation.

BACKGROUND: The molecular basis of autosomal recessive non-syndromic mental retardation (NSMR) is poorly understood, mostly owing to heterogeneity and absence of clinical criteria for grouping families for linkage analysis. Only two autosomal genes, the PRSS12 gene on chromosome 4q26 and the CRBN on chromosome 3p26, have been shown to cause autosomal recessive NSMR, each gene in only one family. OBJECTIVE: To identify the gene causing autosomal recessive NSMR on chromosome 19p13.12. RESULTS: The candidate region established by homozygosity mapping was narrowed down from 2.4 Mb to 0.9 Mb on chromosome 19p13.12. A protein truncating mutation was identified in the gene CC2D1A in nine consanguineous families with severe autosomal recessive NSMR. The absence of the wild type protein in the lymphoblastoid cells of the patients was confirmed. CC2D1A is a member of a previously uncharacterised gene family that carries two conserved motifs, a C2 domain and a DM14 domain. The C2 domain is found in proteins which function in calcium dependent phospholipid binding; the DM14 domain is unique to the CC2D1A protein family and its role is unknown. CC2D1A is a putative signal transducer participating in positive regulation of I-kappaB kinase/NFkappaB cascade. Expression of CC2D1A mRNA was shown in the embryonic ventricular zone and developing cortical plate in staged mouse embryos, persisting into adulthood, with highest expression in the cerebral cortex and hippocampus. CONCLUSIONS: A previously unknown signal transduction pathway is important in human cognitive development.

Structural organization of the murine c-kit proto-oncogene.

The murine Kit receptor gene on chromosome 5 has been found to be frequently involved in germline mutations and rearrangements, leading to a characteristic set of severe developmental defects, known as the W phenotype. Here we describe the structure of the murine c-kit gene, based on restriction analysis of genomic phage clones and sequence determination of exon-intron boundaries. The Kit-coding region is distributed over 21 exons, most of which have sizes that range between 100 and 200 base pairs. The 3' non-translated sequence and the 3' end of the coding region form a single large exon, which encompasses 2.3 kb and is flanked by polyadenylation signals. The entire region spans a genomic distance of at least 70 kb. Though the exonic demarcations of c-kit show remarkable similarity to those of the human c-fms gene (which encodes the highly related colony-stimulating factor 1 receptor), no correlation could be found between the sizes of introns that separate homologous exon pairs. The data suggest that evolutionary pressures were confined to the conservation of structures of coding exons, whereas flanking regions were subject to large changes, owing to insertions and deletions. Finally, the analysis of the Kit genomic structure reveals that the inherited mutations of the Kit gene that have been reported thus far occur at various dispersed positions within the gene. Hence, the entire gene appears to have as yet unknown features which cause it to be frequently subject to mutations in murine germline tissues.

Common origin of transmissible venereal tumors (TVT) in dogs.

We determined the sequence of the 1.5-kb insert upstream to c-myc in the transmissible venereal tumor (TVT) of dogs. The sequence is highly homologous to the 3' region of the mammalian repetitive LINE element. The insert is bound by a 10-bp repeat indicating DNA transposition by a mechanism involving reverse transcriptase. We analyzed DNA of four TVT tumors from various geographical locations as well as normal canine DNA for the presence of the LINE insert. The results indicate that in all TVT tumors, but not in normal tissues, the same LINE insert was present upstream to c-myc. This result suggests that TVT tumors in various dogs may have a common cellular origin.

DNA microarray analysis of genes involved in p53 mediated apoptosis: activation of Apaf-1.

The transcription regulation activity of p53 controls cellular response to a variety of stress conditions, leading to growth arrest and apoptosis. Despite major progress in the understanding of the global effects of p53 on cellular function the pathways by which p53 activates apoptosis are not well defined. To study genes activated in the p53 induced apoptotic process, we used a mouse myeloid leukemic cell line (LTR6) expressing the temperature-sensitive p53 (val135) that undergoes apoptosis upon shifting the temperature to 32 degrees C. We analysed the gene expression profile at different time points after p53 activation using oligonucleotide microarray capable of detecting approximately 11,000 mRNA species. Cluster analysis of the p53-regulated genes indicate a pattern of early and late induced sets of genes. We show that 91 and 44 genes were substantially up and down regulated, respectively, by p53. Functional classification of these genes reveals that they are involved in many aspects of cell function, in addition to growth arrest and apoptosis. Comparison of p53 regulated gene expression profile in LTR6 cells to that of a human lung cancer cell line (H1299) that undergoes growth arrest but not apoptosis demonstrates that only 15% of the genes are common to both systems. This observation supports the presence of two distinct transcriptional programs in response to p53 signaling, one leading to growth arrest and the other to apoptosis. The proapoptotic genes induced only in LTR6 cells like Apaf-1, Sumo-1 and gelsolin among others may suggest a possible explanation for apoptosis in LTR6 cells.

DNA microarrays identification of primary and secondary target genes regulated by p53.

The transcriptional program regulated by the tumor suppressor p53 was analysed using oligonucleotide microarrays. A human lung cancer cell line that expresses the temperature sensitive murine p53 was utilized to quantitate mRNA levels of various genes at different time points after shifting the temperature to 32 degrees C. Inhibition of protein synthesis by cycloheximide (CHX) was used to distinguish between primary and secondary target genes regulated by p53. In the absence of CHX, 259 and 125 genes were up or down-regulated respectively; only 38 and 24 of these genes were up and down-regulated by p53 also in the presence of CHX and are considered primary targets in this cell line. Cluster analysis of these data using the super paramagnetic clustering (SPC) algorithm demonstrate that the primary genes can be distinguished as a single cluster among a large pool of p53 regulated genes. This procedure identified additional genes that co-cluster with the primary targets and can also be classified as such genes. In addition to cell cycle (e.g. p21, TGF-beta, Cyclin E) and apoptosis (e.g. Fas, Bak, IAP) related genes, the primary targets of p53 include genes involved in many aspects of cell function, including cell adhesion (e.g. Thymosin, Smoothelin), signaling (e.g. H-Ras, Diacylglycerol kinase), transcription (e.g. ATF3, LISCH7), neuronal growth (e.g. Ninjurin, NSCL2) and DNA repair (e.g. BTG2, DDB2). The results suggest that p53 activates concerted opposing signals and exerts its effect through a diverse network of transcriptional changes that collectively alter the cell phenotype in response to stress.

More than two immunoglobulin heavy chain J region genes in the majority of infant leukemia.

Congenital and infant leukemia are rare conditions associated with a very poor prognosis due to the high frequency of adverse clinical and laboratory parameters. As the occurrence of multiple immunoglobulin heavy chain hybridization band in childhood leukemia has been associated with poor prognosis, we studied whether it was present in this type of leukemia as well. Seven cases were examined, 4 of them less than 7 months of age. The immunophenotype was lymphoid in 5 and hybrid in 2. Most had abnormal karyotypes. In 5 of the 7, including all with congenital leukemia, an immunoglobulin heavy chain J region multiband pattern was found by Southern blot. The multiband pattern, whether primary or due to clonal evolution, seems to be associated with poor prognosis.

A search for bcl1, bcl2, and c-myc oncogene rearrangements in chronic lymphocytic leukemia.

Three cellular or putative oncogenes: c-myc, bcl1, and bcl2 were previously found to be rearranged in some B cell malignancies due to chromosomal translocations. Data concerning the role of such genetic rearrangements in B-CLL are very scanty and limited to few cases in which bcl1 rearrangements were found. We studied DNA samples from 38 cases of B-CLL by Southern blot technique in order to find out the existence and frequency of such events. No bcl1 or bcl2 rearrangements were found in any of the studied cases; thus, involvement of these genes in CLL must be rare. In one patient who had an aggressive and resistant disease, c-myc rearrangement was found.