A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity

Werner syndrome (WS) is an autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases. The gene responsible for WS encodes a member of the RecQ-like subfamily of DNA helicases. Here we show that its murine homologue maps to murine chromosome 8 in a region syntenic with the human WRN gene. We have deleted a segment of this gene and created Wrn-deficient embryonic stem (ES) cells and WS mice. While displaying reduced embryonic survival, live-born WS mice otherwise appear normal during their first year of life. Nonetheless, although several DNA repair systems are apparently intact in homozygous WS ES cells, such cells display a higher mutation rate and are significantly more sensitive to topoisomerase inhibitors (especially camptothecin) than are wild-type ES cells. Furthermore, mouse embryo fibroblasts derived from homozygous WS embryos show premature loss of proliferative capacity. At the molecular level, wild-type, but not mutant, WS protein copurifies through a series of centrifugation and chromatography steps with a multiprotein DNA replication complex.

Comparative mapping of the human 22q11 chromosomal region and the orthologous region in mice reveals complex changes in gene organization

The region of human chromosome 22q11 is prone to rearrangements. The resulting chromosomal abnormalities are involved in Velo-cardio-facial and DiGeorge syndromes (VCFS and DGS) (deletions), “cat eye” syndrome (duplications), and certain types of tumors (translocations). As a prelude to the development of mouse models for VCFS/DGS by generating targeted deletions in the mouse genome, we examined the organization of genes from human chromosome 22q11 in the mouse. Using genetic linkage analysis and detailed physical mapping, we show that genes from a relatively small region of human 22q11 are distributed on three mouse chromosomes (MMU6, MMU10, and MMU16). Furthermore, although the region corresponding to about 2.5 megabases of the VCFS/DGS critical region is located on mouse chromosome 16, the relative organization of the region is quite different from that in humans. Our results show that the instability of the 22q11 region is not restricted to humans but may have been present throughout evolution. The results also underscore the importance of detailed comparative mapping of genes in mice and humans as a prerequisite for the development of mouse models of human diseases involving chromosomal rearrangements.

Targeted gene deletion of heme oxygenase 2 reveals neural role for carbon monoxide

Neuronal nitric oxide synthase (nNOS) generates NO in neurons, and heme-oxygenase-2 (HO-2) synthesizes carbon monoxide (CO). We have evaluated the roles of NO and CO in intestinal neurotransmission using mice with targeted deletions of nNOS or HO-2. Immunohistochemical analysis demonstrated colocalization of nNOS and HO-2 in myenteric ganglia. Nonadrenergic noncholinergic relaxation and cyclic guanosine 3′,5′ monophosphate elevations evoked by electrical field stimulation were diminished markedly in both nNOSΔ/Δ and HO-2Δ/Δ mice. In wild-type mice, NOS inhibitors and HO inhibitors partially inhibited nonadrenergic noncholinergic relaxation. In nNOSΔ/Δ animals, NOS inhibitors selectively lost their efficacy, and HO inhibitors were inactive in HO-2Δ/Δ animals.

Normal cardiovascular development in mice deficient for 16 genes in 550 kb of the velocardiofacial/ DiGeorge syndrome region

Hemizygous interstitial deletions in human chromosome 22q11 are associated with velocardiofacial syndrome and DiGeorge syndrome and lead to multiple congenital abnormalities, including cardiovascular defects. The gene(s) responsible for these disorders is thought to reside in a 1.5-Mb region of 22q11 in which 27 genes have been identified. We have used Cre-mediated recombination of LoxP sites in embryonic stem cells and mice to generate a 550-kb deletion encompassing 16 of these genes in the corresponding region on mouse chromosome 16. Mice heterozygous for this deletion are normal and do not exhibit cardiovascular abnormalities. Because mice with a larger deletion on mouse chromosome 16 do have heart defects, the results allow us to exclude these 16 genes as being solely, or in combination among themselves, responsible for the cardiovascular abnormalities in velocardiofacial/DiGeorge syndrome. We also generated mice with a duplication of the 16 genes that may help dissect the genetic basis of “cat eye” and derivative 22 syndromes that are characterized by extra copies of portions of 22q11, including these 16 genes. We also describe a strategy for selecting cell lines with defined chromosomal rearrangements. The method is based on reconstitution of a dominant selection marker after Cre-mediated recombination of LoxP sites. Therefore it should be widely applicable to many cell lines.

Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome

Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 → Trp and Pro-253 → Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2) . These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2–FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 → Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 → Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients.

Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and 2B) was partially inhibited by rapamycin, a blocker of some of the signals mediated by IL-2R, but not by cyclosporin A, FK-506, and prednisone, which suppress signals mediated by the T-cell receptor. All the cell lines expressed on their surface the high-affinity IL-2R (alpha, beta, and gamma c chains). They showed basal, constitutive phosphorylation, and coassociation of Jak3, STAT5, and STAT3. Weak basal phosphorylation of IL-2R gamma c was also detected. In regard to SzS, peripheral blood mononuclear cells from 10 of 14 patients showed basal phosphorylation of Jak3, accompanied by phosphorylation of STAT5 in 9 patients, and STAT3 in 4 patients. However, in vitro overnight culture of SzS cells without exogenous cytokines resulted in markedly decreased Jak3 and STAT5 phosphorylation, which could be reversed by stimulation with IL-2. This indicates that the basal phosphorylation of Jak3 and STAT5 in freshly isolated SzS cells is induced rather than constitutive. The basal activation of the Jak/STAT pathway involved in IL-2R signal transduction in ALCL and SzS cells reported here suggests that this pathway may play a role in the pathogenesis of cutaneous T-cell lymphomas, although the mechanism (induced versus constitutive) may vary between different lymphoma types.

A region of consistent deletion in neuroblastoma maps within human chromosome 1p36.2-36.3.

Deletion of the short arm of human chromosome 1 is the most common cytogenetic abnormality observed in neuroblastoma. To characterize the region of consistent deletion, we performed loss of heterozygosity (LOH) studies on 122 neuroblastoma tumor samples with 30 distal chromosome 1p polymorphisms. LOH was detected in 32 of the 122 tumors (26%). A single region of LOH, marked distally by D1Z2 and proximally by D1S228, was detected in all tumors demonstrating loss. Also, cells from a patient with a constitutional deletion of 1p36, and from a neuroblastoma cell line with a small 1p36 deletion, were analyzed by fluorescence in situ hybridization. Cells from both sources had interstitial deletions of 1p36.2-36.3 which overlapped the consensus region of LOH defined by the tumors. Interstitial deletion in the constitutional case was confirmed by allelic loss studies using the panel of polymorphic markers. Four proposed candidate genes--DAN, ID3 (heir-1), CDC2L1 (p58), and TNFR2--were shown to lie outside of the consensus region of allelic loss, as defined by the above deletions. These results more precisely define the location of a neuroblastoma suppressor gene within 1p36.2-36.3, eliminating 33 centimorgans of proximal 1p36 from consideration. Furthermore, a consensus region of loss, which excludes the four leading candidate genes, was found in all tumors with 1p36 LOH.

Butyrylcholinesterase: Association with the metabolic syndrome and identification of 2 gene loci affecting activity

Background: Plasma cholinesterase activity is known to be correlated with plasma triglycerides, HDL- and LDL-cholesterol, and other features of the metabolic syndrome. A role in triglyceride metabolism has been proposed. Genetic variants that decrease activity have been studied extensively, but the factors contributing to overall variation in the population are poorly understood. We studied plasma cholinesterase activity in a sample of 2200 adult twins to assess covariation with cardiovascular risk factors and components of the metabolic syndrome, to determine the degree of genetic effects on enzyme activity, and to search for quantitative trait loci affecting activity. Methods and Results: Cholinesterase activity was lower in women than in men before the age of 50, but increased to activity values similar to those in males after that age. There were highly significant correlations with variables associated with the metabolic syndrome: plasma triglyceride, HDL- and LDL-cholesterol, apolipoprotein B and E, urate, and insulin concentrations; gamma-glutamyltransferase and aspartate and alanine aminotransferase activities; body mass index; and blood pressure. The heritability of plasma cholinesterase activity was 65%. Linkage analysis with data from the dizygotic twin pairs showed suggestive linkage on chromosome 3 at the location of the cholinesterase WHO gene and also on chromosome 5. Conclusions: Our results confirm and extend the connection between cholinesterase, cardiovascular risk factors, and metabolic syndrome. They establish a substantial heritability for plasma cholinesterase activity that might be attributable to variation near the structural gene and at an independent locus. (c) 2006 American Association for Clinical Chemistry.

Alogliptin after acute coronary syndrome in patients with type 2 diabetes

Background - To assess potentially elevated cardiovascular risk related to new antihyperglycemic drugs in patients with type 2 diabetes, regulatory agencies require a comprehensive evaluation of the cardiovascular safety profile of new antidiabetic therapies. We assessed cardiovascular outcomes with alogliptin, a new inhibitor of dipeptidyl peptidase 4 (DPP-4), as compared with placebo in patients with type 2 diabetes who had had a recent acute coronary syndrome. Methods - We randomly assigned patients with type 2 diabetes and either an acute myocardial infarction or unstable angina requiring hospitalization within the previous 15 to 90 days to receive alogliptin or placebo in addition to existing antihyperglycemic and cardiovascular drug therapy. The study design was a double-blind, noninferiority trial with a prespecified noninferiority margin of 1.3 for the hazard ratio for the primary end point of a composite of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. Results - A total of 5380 patients underwent randomization and were followed for up to 40 months (median, 18 months). A primary end-point event occurred in 305 patients assigned to alogliptin (11.3%) and in 316 patients assigned to placebo (11.8%) (hazard ratio, 0.96; upper boundary of the one-sided repeated confidence interval, 1.16; P<0.001 for noninferiority). Glycated hemoglobin levels were significantly lower with alogliptin than with placebo (mean difference, -0.36 percentage points; P<0.001). Incidences of hypoglycemia, cancer, pancreatitis, and initiation of dialysis were similar with alogliptin and placebo. Conclusions - Among patients with type 2 diabetes who had had a recent acute coronary syndrome, the rates of major adverse cardiovascular events were not increased with the DPP-4 inhibitor alogliptin as compared with placebo. (Funded by Takeda Development Center Americas; EXAMINE ClinicalTrials.gov number, NCT00968708.)

Understanding Ten-Eleven Translocation-2 in Hematological and Nervous Systems

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

The clinical significance of 15q11.2 BP1-BP2 duplications: - Where do we stand?

The 15q11.2-q13 region has been well characterized, being associated with a range of syndromatic copy number variants (CNVs), and comprises five established break points sites (BP1 to BP5). While the clinical effect for BP1-BP3, BP2-BP3 and BP4-BP5 CNVs is well established, the same cannot be said for BP1-BP2 CNVs. Recently the 15q11.2 BP1-BP2 deletion has been reviewed, emerging as a microdeletion syndrome with low penetrance and variable expressivity being the CNV frequently inherited from a healthy parent. This microdeletion is considered to be a risk factor for several neurodevelopment disorders. For the reciprocal duplication the picture has been less conclusive. Aiming for a better understanding of the clinical significance of this CNV, we collected patients with intellectual disability and/or other clinical features, referred for microarray testing, gathering clinical details for the ones with the duplication. Data was collected from two genetic laboratories. With a total of 1545 patients, we identified eleven carrying the duplication at 15q11.2 BP1-BP2. It was possible to assess inheritance in only four cases, all inherited from a healthy parent. All patients presented intellectual disability,and facial dysmorphism was the second most common feature observed. Microcephaly, autism, congenital abnormalities, dystonia and cataplexy where reported individually. The magnitude of the effect of 15q11.2 duplication remains elusive, and the outcome unclear, posing a major challenge to genetic counseling. Nevertheless, we expect the collection of more of these cases will establish this gain, as it happened with the reciprocal deletion, as a microduplication syndrome with low penetrance and variable expressivity.

Molecular analysis of exons 8, 9 and 10 of the fibroblast growth factor receptor 2 (FGFR2) gene in two families with index cases of Apert Syndrome

Introduction: Apert syndrome (AS) is a craniosynostosis condition caused by mutations in the Fibroblast Growth Factor Receptor 2 (FGFR2) gene. Clinical features include cutaneous and osseous symmetric syndactily in hands and feet, with variable presentations in bones, brain, skin and other internal organs. Methods: Members of two families with an index case of Apert Syndrome were assessed to describe relevant clinical features and molecular analysis (sequencing and amplification) of exons 8, 9 and 10 of FGFR2 gen. Results: Family 1 consists of the mother, the index case and half -brother who has a cleft lip and palate. In this family we found a single FGFR2 mutation, S252W, in the sequence of exon 8. Although mutations were not found in the study of the patient affected with cleft lip and palate, it is known that these diseases share signaling pathways, allowing suspected alterations in shared genes. In the patient of family 2, we found a sequence variant T78.501A located near the splicing site, which could interfere in this process, and consequently with the protein function.

Understanding ten-eleven translocation-2 in hematological and nervous systems

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. ^ In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. ^ In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.^